RESEARCH ARTICLE https://doi.org/10.1158/2767-9764.CRC-21-0103 OPEN ACCESS MYC Inhibition Halts Metastatic Breast Cancer Check for updates Progression by Blocking Growth, Invasion, and Seeding Daniel Massó-Vallés1,2,3 , Marie-Eve Beaulieu1,2 , Toni Jauset1,2,3 , Fabio Giuntini1 , Mariano F. Zacarías-Fluck1 , Laia Foradada2 , Sandra Martínez-Martín1,3 , Erika Serrano1 , Génesis Martín-Fernández1 , Sílvia Casacuberta-Serra2 , Virginia Castillo Cano2 , Jastrinjan Kaur1 , Sergio López-Estévez2 , Miguel Ángel Morcillo4 , Mohammad Alzrigat5 , Loay Mahmoud5 , Antonio Luque-García1 , Marta Escorihuela1 , Marta Guzman1 , Joaquín Arribas1,3,6 , Violeta Serra1 , Lars-Gunnar Larsson5 , Jonathan R. Whitfield1 , and Laura Soucek1,2,3,6 ABSTRACT MYC’s role in promoting tumorigenesis is beyond doubt, but its function recombinantly produced Omomyc miniprotein, recently entering a clini- in the metastatic process is still controversial. Omomyc is a MYC domi- cal trial in solid tumors, recapitulates several key features of expression of nant negative that has shown potent antitumor activity in multiple cancer the Omomyc transgene, confirming its clinical applicability to metastatic cell lines and mouse models, regardless of their tissue of origin or driver breast cancer, including advanced triple-negative breast cancer, a disease in mutations, by impacting on several of the hallmarks of cancer. However, its urgent need of better therapeutic options. therapeutic efficacy against metastasis has not been elucidated yet. Here we Significance: While MYC role in metastasis has been long controversial, demonstrate for the first time that MYC inhibition by transgenic Omomyc this manuscript demonstrates that MYC inhibition by either transgenic ex- is efficacious against all breast cancer molecular subtypes, including triple- pression or pharmacologic use of the recombinantly produced Omomyc negative breast cancer, where it displays potent antimetastatic properties miniprotein exerts antitumor and antimetastatic activity in breast cancer both in vitro and in vivo. Importantly, pharmacologic treatment with the models in vitro and in vivo, suggesting its clinical applicability. Introduction divided into histologic and molecular subtypes (2). In clinical practice, patients are classified according to the expression of estrogen receptor (ER), proges- Breast cancer is the most common cancer in women and the second lead- terone receptor (PR), amplification of human epidermal growth factor receptor ing cause of cancer mortality, with more than 2.2 million annual cases and 2 (HER2), and proliferation rate (Ki67 positivity). These molecular subtypes 685,000 estimated deaths globally (1). It is a highly heterogeneous disease differ in incidence, prognosis, and treatment options (3–5). Triple-negative breast cancers (TNBC) are a heterogeneous group of tumors, devoid of expres- 1 sion of ER, PR, and HER2, with one common feature: a distinctly aggressive Preclinical & Translational Research Program, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, C/ Natzaret, Barcelona, Spain. nature with higher rates of relapse and shorter overall survival in the metastatic 2 Peptomyc S.L., Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain. setting compared with other subtypes of breast cancer (6). Hence, their prog- 3 Department of Biochemistry and Molecular Biology, Universitat Autònoma de nosis remains dire, with an average median survival of 1 year for metastatic Barcelona, Bellaterra, Spain. 4 Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain. 5 Department of disease (7). Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden. 6 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Despite major improvements in prevention, diagnosis and treatment, many pa- Spain. tients still develop metastatic breast cancer (mBC). Because of the selective Corresponding Author: Laura Soucek, Vall d’Hebron Institute of Oncology (VHIO), pressure imposed on the primary tumors, metastases evolve and differentiate, C/ Natzaret, 115-117, CELLEX Centre, Barcelona 08035, Spain. Phone: sometimes acquiring characteristics of more aggressive subtypes (8). Therefore, 349-3254-3450; E-mail: lsoucek@vhio.net most metastatic patients become resistant to therapy and eventually succumb doi: 10.1158/2767-9764.CRC-21-0103 to their disease. Regardless of the molecular subtype, mBC is essentially incur- This open access article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. able, with only 1% to 3% of patients with chemotherapy-naïve mBC remaining © 2022 The Authors; Published by the American Association for Cancer Research disease-free over the long term after systemic therapy (9). AACRJournals.org Cancer Res Commun; 2(2) February 2022 110 MYC Inhibition Is Antimetastatic in Breast Cancer The MYC oncogene is amplified or deregulated in the majority of human (ATCC catalog no. CRL-10317, RRID:CVCL_0598)], Joaquín Arribas [BT-474 cancers (10). In breast cancer, amplification and overexpression of MYC are (ATCC catalog no. HTB-20, RRID:CVCL_0179), MDA-MB-231 (ATCC frequent in high grade and invasive malignancies and are consistently cor- catalog no. HTB-26, RRID:CVCL_0062), and SK-BR-3 (ATCC catalog no. related with poor outcome and early recurrence (11–14). Even distant lethal HTB-30, RRID:CVCL_0033)], and Josep Villanueva [BT-549 (ATCC catalog breast cancer metastases from MYC-unamplified primary tumors often ac- no. HTB-122, RRID:CVCL_1092) and Hs 578T (ATCC catalog no. CRL-7849, quire MYC amplification (15). Interestingly, MYC is disproportionately elevated RRID:CVCL_0332)] throughout 2013. The MDA-MB-231 cells used for isPLA in TNBC compared with ER/PR+ and HER2-amplified breast tumors (16). It were purchased from the ATCC repository in March 2021. CAL-51, Hs 578T, drives multiple aspects of tumor progression and metastasis by controlling the JIMT-1, and MCF7 cells were grown in DMEM (Life Technologies). BT-474, transcription of genes promoting cell proliferation and survival, genetic insta- MDA-MB-231, SK-BR-3, and MCF 10A cells were grown in DMEM/F12 bility, differentiation block, cell invasion, and migration (13, 17). In addition, medium (Life Technologies). BT-549, T-47D, and HCC1954 cells were grown MYC maintains the tumor microenvironment by instructing tissue remodeling, in RPMI medium (Life Technologies). MDA-MB-361 and MDA-MB-453 cells angiogenesis, and inflammation, even when it is not overexpressed (18). In this were grown in L-15 medium (Life Technologies). MCF 10A cells were supple- context, MYC also controls the activation of tumor-associated macrophages, mented with 5% horse serum, 20 ng/mL EGF, 0.5 μg/mL hydrocortisone, 100 which increase cancer’s aggressiveness (19), and regulates the expression of ng/mL cholera toxin, 10 μg/mL insulin, 15 mmol/L HEPES, and 1% glutamine. epithelial-to-mesenchymal transition (EMT) effectors (13). While most reports All other cell lines were supplemented with 10% FBS and 1% glutamine. All indicate that MYC is a prometastatic gene (20–27), some describe it as a cells were grown at 37°C and 5% CO2 , except MDA-MB-361 and MDA-MB- metastatic suppressor instead (28–31), even suggesting that caution would be 453, which were grown without CO2 . Cells were used for a maximum of 10 needed when designing clinical approaches against MYC in a metastatic setting. passages after collection or thawing. All cell lines were periodically checked for Mycoplasma contamination with the MycoAlert Assay Control Set (Lonza), Omomyc is a MYC dominant negative designed by our group, which com- following the manufacturer’s instructions, and used only if the result was prises the MYC helix–loop–helix leucine zipper domain and harbors 4 amino negative. MDA-MB-231 and BT-549 cell lines were authenticated at Instituto acid substitutions in the leucine zipper that alter its dimerization specificity de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM) by microsatellite (32). Through extensive characterization, we have demonstrated that Omomyc analysis. prevents MYC’s interaction with its natural partner MAX, sequestering it away from target promoters as Omomyc/MYC dimers and occupying the E- Cell lines were infected with the pSLIK-Hygro lentiviral vector, a gift from Iain box consensus DNA-binding site in the form of inactive MAX/Omomyc and Fraser (RRID:Addgene_25737) containing a GFP or Omomyc cassette, which Omomyc/Omomyc dimers (33). Omomyc’s antitumor activity as a genetically were inserted by gateway technology. For infections, HEK 293T cells (ATCC engineered transgene has been extensively described by our group and oth- catalog no. CRL-3216, RRID:CVCL_0063) were seeded at 30% confluence, ers against multiple tumor types (18, 34–40). Recently, the demonstration of and the next morning 25 mmol/L chloroquine added. Two hours later, HEK the remarkable therapeutic impact elicited by the recombinantly produced 293T cells were transfected with pSLIK-Hygro-Omomyc or pSLIK-Hygro- Omomyc miniprotein in lung adenocarcinoma models constituted a paradigm GFP plus the lentiviral vectors pMD2.G (RRID:Addgene_12259) and psPAX2 shift, demonstrating that Omomyc could be used as a clinically viable drug (RRID:Addgene_12260) by the CaPO4 method. The medium was changed the (33, 41, 42). However, its therapeutic impact on metastatic disease has not been next day and sodium butyrate added at 5 mmol/L. Viral supernatants were elucidated yet. Here we made use of a panel of breast cancer cell lines, repre- harvested on the subsequent 2 days, filtered with 0.45-μm polyvinylidene di- sentative of all molecular subtypes of the disease, to evaluate Omomyc’s efficacy fluoride (PVDF) filters (Millipore) and added to target cells with polybrene against breast cancer and metastasis. By inducible expression of the Omomyc (0.8 mg/mL). Transduced cells were selected with 1 mg/mL hygromycin. transgene, we studied the effect of MYC inhibition on proliferation, angiogen- For most in vivo experiments, MDA-MB-231 containing a pSLIK-Hygro- esis, migration, and invasion. We also assessed Omomyc’s impact in vivo on Omomyc vector obtained by lentiviral infection and a triple-fusion protein primary mammary tumor growth and, for the first time, on lung colonization reporter construct encoding herpes simplex virus thymidine kinase 1, GFP, and metastatic growth. In addition, to be able to translate these results to a and firefly luciferase obtained by retroviral infection (43, 44) were used. In the pharmacologically meaningful context, we studied how exogenous administra- biodistribution experiment, MDA-MB-231 cells containing a pQCXIH-firefly tion of the Omomyc miniprotein compares with the expression of the Omomyc luciferase vector obtained by retroviral infection were used. In the experiment transgene, assessing its therapeutic effect in TNBC cell line- and patient-derived to study the effect of intravenous administration of the Omomyc miniprotein models. on primary tumor growth, wild-type MDA-MB-231 cells were used. Materials and Methods Western Blots Cell Lines and Lentiviral Infections For Western blots (WB), cells were cultured in the presence or absence of 0.6 μg/mL doxycycline for 3 days. Then, culture media was discarded and cells All cell lines were kindly provided by the laboratories of Violeta Serra were placed on ice. PBS + 1 mmol/L EDTA was added to the plate and cells [CAL-51 (DSMZ catalog no. ACC-302, RRID:CVCL_1110), JIMT-1 (DSMZ were scraped, centrifuged, and washed twice. Cell pellets were frozen and kept catalog no. ACC-589, RRID:CVCL_2077), MCF7 (ATCC catalog no. at −80°C. HTB-22, RRID:CVCL_0031), MDA-MB-361 (ATCC catalog no. HTB- 27, RRID:CVCL_0620), MDA-MB-453 (ATCC catalog no. HTB-131, Cells were lysed with RIPA buffer supplemented with protease and phos- RRID:CVCL_0418), T-47D (ATCC catalog no. HTB-133, RRID:CVCL_0553), phatase inhibitors (Roche) and the protein fraction collected. Proteins were HCC1954 (ATCC catalog no. CRL-2338, RRID:CVCL_1259), and MCF 10A quantified by the DC Protein Assay (Bio-Rad) and absorbance at 560 nm AACRJournals.org Cancer Res Commun; 2(2) February 2022 111 Massó-Vallés et al. was measured with a spectrophotometer (Victor3, PerkinElmer or TECAN Clonogenic Assays Spark, Life Sciences). 10 to 30 μg of protein extract in Laemmli buffer +15% 500 cells/well were plated in 6-well plates in the presence or absence of 0.6 β-mercaptoethanol were run on 10% or 12% precast gels (Life Technologies). μg/mL doxycycline. Cells were fixed with 4% formaldehyde between 2 and 7 Proteins were transferred to nitrocellulose or PVDF membranes by the iBlot weeks later, depending on the doubling time of each cell line. After 20 min- 2 Dry Blotting System (Life Technologies) and membranes were stained with utes of fixation, colonies were stained with 0.05% crystal violet for 1 hour and Ponceau red. After washing with PBS + 0.1% Tween and blocking with 2% the plates washed with distilled water and air dried. The bottom of the plate milk, membranes were incubated overnight at 4°C with the following pri- was scanned in high resolution and the images obtained were analyzed with mary antibodies: rabbit polyclonal anti-Omomyc (affinity purified and selected the ColonyArea plugin for ImageJ (47) to determine, for each well, the area against recognition of the Myc B-HLH-LZ epitope, 1:5,000 or 1:12,000) rab- percentage occupied with cells and the intensity of the staining, as described bit monoclonal anti-CDK4 (D9G3E; Cell Signaling Technology, catalog no. previously (47). 42749, RRID:AB_2799229, 1:1,000), rabbit polyclonal anti-Histone H3 (Abcam, catalog no. ab70550, RRID:AB_1209471, 1:500), and mouse monoclonal anti-β- actin (AC-15; Sigma-Aldrich, catalog no. A1978, RRID:AB_476692, 1:50,000). Detection of Lentivirally Expressed Omomyc by Membranes were washed with PBS-Tween and incubated for 1 hour at room Flow Cytometry temperature with the following secondary antibodies: ECL anti-rabbit IgG- For the detection of lentivirally expressed Omomyc, MDA-MB-231-Omomyc HRP (GE Healthcare, catalog no. NA934, RRID:AB_772206, 1:5,000) and ECL cells were treated for 3 days with or without 0.6 μg/mL doxycycline, har- anti-mouse IgG-HRP (GE Healthcare, catalog no. NA931, RRID:AB_772210, vested by trypsinization and fixed/permeabilized in absolute ethanol overnight 1:5,000). Membranes were washed twice with PBS-Tween and once with PBS, at 4°C. Cells were washed with PBS-Azide (PBS with 0.1% sodium azide and and then incubated for 5 minutes with SuperSignal West Pico Chemilumi- 1% BSA) and blocked with 2.5% BSA in PBS for 30 minutes at room temper- nescent Substrate (Thermo Fisher Scientific) before revealing. To quantify the ature. Cells were stained with rabbit polyclonal anti-Omomyc antibody (1:50) Omomyc levels expressed in each cell line, the intensity of the Omomyc and β- for 1 hour at room temperature. After washing, cells were incubated with goat actin bands was quantified with ImageJ (RRID:SCR_003070; ref. 45) and a ratio anti-rabbit AlexaFluor647 (1:1,000, Life Technologies) for 45 minutes at room Omomyc/β-actin established. This ratio was then correlated with the relative temperature. Navios Flow Cytometer (Beckman Coulter) was used for data cell number obtained in the colony formation assays. acquisition and FlowJo V10 software (RRID:SCR_008520) was used for data analysis. Subcellular Fractionation Subcellular fractionation was performed as previously described in ref. 46. Angiogenesis Assay 10 μg of protein from the core nuclear and cytoplasmic fractions were loaded 1 million MDA-MB-231-GFP cells ± 0.6 μg/mL doxycycline, 1 million and a WB performed. Therefore, only the lanes from the same fraction can be MDA-MB-231-Omomyc cells without doxycycline, and 1.5 million MDA-MB- compared (nuclear vs. nuclear and cytoplasmic vs. cytoplasmic, but not nuclear 231-Omomyc cells + 0.6 μg/mL doxycycline were plated and their conditioned vs. cytoplasmic), as the protein content does not reflect the relative amount ob- media collected after 3 days. Cells expressing Omomyc were plated at a higher tained for each fraction. The perinuclear fraction is not shown due to the very density so that at the time of medium collection, the number of cells was the low protein yield obtained. same as the nonexpressing ones. Conditioned media was centrifuged and fil- tered with 0.45-μm PVDF filters (Millipore). 75,000 human umbilical vein BrdU Incorporation and Cell-Cycle Analysis endothelial cells (HUVEC) were mixed with 300 μL of each conditioned media For bromodeoxyuridine (BrdU) incorporation and cell-cycle analysis, MDA- and 100 μL of the mix containing 25,000 cells was plated per well in a 96-well MB-231-GFP, MDA-MB-231-Omomyc, or MDA-MB-231 cells were treated for plate, on top of a Matrigel layer to allow tube formation. After 6 hours, rep- 3 days with either vehicle, 0.6 μg/mL doxycycline or 20 μmol/L Omomyc, then resentative images were acquired with an IX71 Olympus inverted microscope. labeled with 10 μmol/L BrdU for 2 hours, harvested by trypsinization, and Tube formation was assessed by the Angiogenesis Analyzer plugin for ImageJ fixed/permeabilized in absolute ethanol overnight at 4°C. After denaturation in as described previously (48). 2 mol/L HCl for 30 minutes at room temperature, they were neutralized with 0.5 mol/L sodium borate and stained with anti-BrdU-FITC antibody (BD Bio- Wound Healing Assay sciences, catalog no. 556028, RRID:AB_396304, 1:5) diluted in PBS + 0.5% BSA and 0.5% Tween. Cells were incubated in 25 mg/L propidium iodide (PI) + 100 MDA-MB-231-GFP and MDA-MB-231-Omomyc cells were plated with or μg/mL RNAse A and 0.3 μmol/L Triton X-100 for 30 minutes. Navios Flow without 0.6 μg/mL doxycycline. After 3 days, cells were trypsinized, counted, Cytometer (Beckman Coulter) was used for data acquisition and FCS Express and 400,000 cells/well were seeded in triplicates in a 12-well plate ± doxy- 4 software was used for data analysis. cycline. Seven hours later, when cells were attached and forming a confluent monolayer, a p1000 pipette tip was used to scratch the surface of the well, forming a wound. An Olympus CellR microscope equipped with a Hama- Proliferation Assay matsu C9100 camera was used to follow the closure of the wound. Pictures 20,000 MDA-MB-231-GFP or MDA-MB-231-Omomyc cells/well were plated were taken automatically every 30 minutes and the wound area was measured in 6-well plates with or without 0.6 μg/mL doxycycline. After 6 days, cells at the endpoint with ImageJ. Cells were then trypsinized, counted with the Tali were trypsinized and counted with the Tali Image-based Cytometer (Life Image-based Cytometer (Life Technologies) and the difference in wound area Technologies). was corrected for the total number of cells. 112 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer Boyden Chamber Assays Omomyc Miniprotein Cell Entry by Flow Cytometry Migration Assay MDA-MB-231 and MCF7 cells were treated with buffer (25 mmol/L NaAc MDA-MB-231-GFP, MDA-MB-231-Omomyc, BT-549-GFP, BT-549-Omomyc, 2 mol/L urea), 0.5 μmol/L, 1 μmol/L, or 5 μmol/L Omomyc-AlexaFluor 488 CAL-51-GFP, CAL-51-Omomyc, MCF7-GFP, and MCF7-Omomyc cells were for 15 minutes. Then, they were trypsinized with 0.25% Trypsin-EDTA (Gibco) plated in complete medium (+10% FBS) with or without 0.6 μg/mL doxycy- for 30 minutes at 37°C to remove membrane-bound peptide and trypsin was cline. After 3 days, cells were washed with PBS, refed with medium containing deactivated with 10% FBS. Cells were washed and resuspended in PBS and im- 0.5% FBS ± doxycycline, and left overnight. Then, cells were counted with mediately analyzed. CytoFlex Flow Cytometer (Beckman Coulter) was used for the Tali Image-based Cytometer (Life Technologies) and 30,000 cells/well data acquisition and CytExpert software (Beckman Coulter) was used for data were seeded on top of Corning FluoroBlok inserts in a 24-well plate. The in- analysis. serts consisted of a chamber with a dark porous membrane (pore diameter: 8 μm). Cells were seeded in triplicates, ± doxycycline, in medium containing In Situ Proximity Ligation Assay 0.5% FBS, and the bottom of the well was filled with complete medium ± MDA-MB-231 cells were seeded in 8-chamber tissue culture–treated glass slides doxycycline. After 24 or 48 hours, depending on the migratory capacity of (Falcon 354118) at cell density of 20,000 cells per chamber for 48 hours. Cells each cell line, migrated cells were stained with the fluorescent CellTracker were then treated with either 20 μmol/L Omomyc or DMSO for 24 hours, Green CMFDA dye (Life Technologies), fixed with 4% formaldehyde, and rep- after which culture medium was removed and cells were washed with 1× resentative images were taken with an IX71 Olympus inverted microscope. PBS at room temperature. Cells were fixed with 4% formaldehyde at room In parallel, 30,000 cells/well were seeded in a 24-well control plate under temperature for 10 minutes, then washed 2 times for 10 minutes with cold the same conditions, with the exception of the inserts. After 24 or 48 hours, 1× PBS. Permeabilization was performed at room temperature using 1× PBS they were harvested by trypsinization and counted with the Tali Image-based containing 0.2% Triton X100, 1% DMSO, and 1% blocking solution provided Cytometer. Migrated cells from the acquired images were analyzed with Fiji by the in situ proximity ligation assay (isPLA) Kit. Slides were washed three (RRID:SCR_002285), a distribution of the ImageJ software (49). The values of times for 5 minutes with 1× PBS at room temperature and incubated in the images from the same condition were averaged, normalized, and corrected blocking buffer (provided by the isPLA kit) for 1 hour at 37°C in a pre- for the difference in cell number obtained from the control plate, if any was heated humidity chamber. Incubation with primary antibodies was performed observed. at 4°C overnight in the humidity chamber. The following antibodies were used: mouse monoclonal anti-MYC (G-4; Santa Cruz Biotechnology, cata- log no. sc-373712, RRID:AB_10916994), rabbit polyclonal anti-MAX (Abcam, Invasion Assay catalog no. ab101271, RRID:AB_10673343), rabbit polyclonal anti-Omomyc MDA-MB-231-GFP and MDA-MB-231-Omomyc cells were plated under the (affinity purified and selected against recognition of the Myc B-HLH-LZ epi- same conditions as the migration assay. The only difference was the use of Corn- tope) and mouse monoclonal anti-Omomyc (clone 21–1-3, Ximbio 153657). ing BioCoat invasion chambers, in which the porous membrane is coated with isPLA was performed using the Duolink In Situ Detection Reagents Red a layer of Matrigel to mimic an ECM. Cells were allowed to invade through the (Sigma-Aldrich DUO92008) following the manufacturer’s protocol using anti- coated membrane for 24 hours and they were fixed, stained, and counted as ex- rabbit plus (Sigma-Aldrich DUO92002) and anti-mouse minus (Sigma-Aldrich plained for the migration assay. A control plate under the same conditions was DUO92004) probes. DNA was stained with DAPI. Image acquisition was per- also plated, and the differences in invasive capacity of the cells were corrected formed using ZEISS LSM 980 with Airyscan 2 confocal microscope (Carl for the difference in cell number. Zeiss Microscopy GmbH) using the 20× objective. All image stacks were acquired with comparable settings, at a resolution of 2048 × 2048 pixels, z- stack size of 1 μm. Fluorescent dots were quantified using CellProfiler 4.2.1 Immunofluorescence software (RRID:SCR_007358; ref. 50). All data were analyzed using Rstu- Sections from paraffin-embedded tumors or lungs were cut at 3.5-μm thick. dio (RRID:SCR_000432) version 4.1.0 with the following packages: tidyverse Antigen retrieval was performed by heating 20 minutes at 400 W in a mi- (RRID:SCR_019186) and ggplot2 (RRID:SCR_014601). crowave in a sodium citrate buffer (10 mmol/L sodium citrate, 0.05% Tween 20, pH 6.0). After blocking for 1 hour with serum-free Dako Protein Block Dose Response to the Omomyc Miniprotein (#X0909), slides were incubated overnight at 4°C with mouse monoclonal anti- 250–10,000 cells/well, depending on their size and growth rate, were seeded in Omomyc antibody (clone 21–1-3, Ximbio 153657, 1:100 dilution) combined with 96-well plates and the next day treated with a single dose of increasing con- rabbit polyclonal anti-Ki67 (Abcam, catalog no. ab15580, RRID:AB_443209, centrations of Omomyc (0, 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L). 1:100) or with rabbit anti-cleaved caspase-3 (Cell Signaling Technology, After 5 days, alamarBlue (Thermo Fisher Scientific) was added to the wells catalog no. 9661, RRID:AB_2341188, 1:200) in Dako Ready-to-use diluent and 4 hours later, fluorescence at 590 nm was measured with a Spark mi- (S2022). After three times, TBS-Tween 20 washes, slides were incubated for 1 croplate reader (Tecan). GI50 values were calculated using GraphPad Prism 9 hour at room temperature with goat anti-mouse IgG (H+L)–AlexaFluor488 (RRID:SCR_002798). conjugate (Thermo Fisher Scientific A11001, 1:200), goat anti-rabbit IgG (H+L)–AlexaFluor594 conjugate (Thermo Fisher Scientific A11012, 1:200) and Combination with Paclitaxel 5 μg/mL DAPI (Life Technologies D1306), washed three times with distilled 2,500 MDA-MB-231 and 1,000 BT-549 cells were seeded in 96-well plates and water and mounted with fluorescence mounting medium (Dako S3023). Images the next day treated with increasing concentrations of Omomyc (0, 2.5, 5, 10, 20 were obtained with a Nikon Eclipse TI inverted epifluorescence microscope and 40 μmol/L) and/or paclitaxel (0, 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and equipped with a Nikon DS-Qi2 camera. 25 nmol/L). After 5 days, AlamarBlue (Thermo Fisher Scientific) was added to AACRJournals.org Cancer Res Commun; 2(2) February 2022 113 Massó-Vallés et al. the wells and 2 hours later, fluorescence at 590 nm was measured with a Spark in at least 1 of the 7 seven genes were designated as “Altered” whereas the rest microplate reader (Tecan). ZIP synergy scores were calculated and plotted using were considered “Unaltered.” We investigated differences in overall survival of SynergyFinder.org (51). When the synergy score ≤10, the interaction between “altered” versus “unaltered” patients and the frequency of genomic alterations two drugs is likely to be antagonistic; from −10 to 10, it is likely to be additive; in patients with amplification of at least 1 of the 7 genes (“amplified”) or without and when >10, it is likely to be synergistic. amplification (“unamplified”). Microarray Analysis Relapse-free Survival Plots MDA-MB-231-Omomyc cells were seeded into 15 × 6 cm dishes, and 5 of them Relapse-free survival (RFS) plots were generated with the Kaplan–Meier Plotter treated with 1 μg/mL doxycycline to induce the expression of Omomyc. The for breast cancer tool (https://kmplot.com/), which contains clinical data from next day, 5 plates were treated with 20 μmol/L Omomyc and the remaining 5 7,830 patients with breast cancer (61). with an equivalent volume of vehicle. After 3 days, plates were washed twice with PBS and frozen at −80°C until processing. The Omomyc miniprotein Animal Studies was added 1 day after doxycycline because our time-course experiments indi- All the animal studies were performed in accordance with the ARRIVE guide- cate that full expression of transgenic Omomyc takes around 12–24 hours. A lines and the 3 Rs rule of Replacement, Reduction and Refinement principles. higher number of cells were seeded on the +Omomyc plates compared with Mouse weights were recorded for every experiment. Mice were housed and the control ones so that at collection, their number was equivalent. RNA was treated following the protocols approved by the Ethical Committee for the Use extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s in- of Experimental Animals (CEEA) at VHIR, Barcelona, Spain. The tumor sam- structions. The quality of RNA was confirmed with Agilent 2100 Bioanalyzer. ple from a patient used to establish the TNBC PDX was collected following Clariom S Human HT microarray plate (Applied Biosystems) was processed an Institutional Research Board approved protocol and the associated written at Vall d’Hebron Institute of Research (VHIR)’s High Technology Unit. The informed consent. microarray data were analyzed with Partek Genomics Suite software, v7.18. All the schematics were created with Biorender.com (RRID:SCR_018361). Gene-set enrichment analysis (GSEA) was performed using publicly available software provided by the Broad Institute (RRID:SCR_003199, version 3.0) with Cell Line–Derived Orthotopic Model (Efficacy Studies) the Hallmarks, Curated, Motif, GO, Oncogenic Signatures, and Immunolog- Effect of Omomyc Expression on Primary Tumor Growth: a suspension of ical Signatures gene sets from the Molecular Signature Database (MSigDB; luciferase-expressing MDA-MB-231-Omomyc cells was mixed with Matrigel www.broad.mit.edu/gsea). We acknowledge our use of the GSEA software and (1:1) and, after a small incision, 1.5 million cells/mouse were injected between MSigDB (www.broad.mit.edu/gsea; ref. 52). The number of permutations was the fourth and fifth right mammary fat pads of 22 6-week-old BALB/c nude set to 1,000, and the genes were ranked according to Signal2Noise. female mice (Janvier RRID:IMSR_JCL:JCL:mID-0001). The wound was su- tured with nonabsorbable thread (6–0). Before surgery, mice were anesthetized qRT-PCR with 2% isoflurane and buprenorphine (0.75 mg/kg) was administered subcuta- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was neously. Tumor size was evaluated three times a week by caliper measurements performed in MDA-MB-231-Omomyc cells treated with 1 μg/mL doxycycline and tumor volume calculated using the following formula: volume = (length × for 4 days, 20 μmol/L Omomyc or vehicle for 3 days to mimic the microarray width)2 /2. When tumors reached 100 mm3 , mice were randomized into two conditions. RNA was then extracted using RNeasy kit (QIAGEN) and quan- groups (n = 11). One group was given 2 g/L doxycycline in 5% sucrose in the tified using NanoDrop. Equal amounts of RNA were reverse transcribed to drinking water to induce expression of the Omomyc transgene. The control generate cDNA using iScript Reverse Transcription Supermix for qRT-PCR group was given 5% sucrose. Sucrose is used to counteract the bitter taste of (Bio-Rad). SYBR Green qRT-PCR analysis was then performed on these cDNA doxycycline so that mice drink enough water. Mice were treated for 4 weeks samples with PerfeCTa SYBR Green FastMix, Low Rox (Quantabio) using and euthanized by CO2 inhalation. Tumors were then excised, weighed, pho- QuantStudio 6 FLEX system (Applied Biosystems). The data thus obtained were tographed, fixed for 48 hours in buffered 4% formaldehyde, transferred to 70% analyzed following the comparative (Ct ) method described in by Livak and ethanol, and embedded in paraffin. Schmittgen (53). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β- Tubulin were used as housekeeping genes. Sequences of primers used are listed Effect of Omomyc Expression on Metastatic Growth: a suspension of in Supplementary Table S2. luciferase-expressing MDA-MB-231-Omomyc cells were inoculated orthotopi- cally into 50 six-week-old BALB/c nude females and tumor growth monitored cBioPortal for Cancer Genomics Data as described above. Tumors were also followed by weekly IVIS imaging. We gathered genomic and clinical data from cBioPortal (54, 55) by performing Between 8 and 10 weeks after inoculation, mice were anesthetized by an a combined study on patient samples from four breast cancer datasets: intraperitoneal injection of a ketamine/xylazine mix (80 mg/kg ketamine, 10 mg/kg xylazine) and primary tumors were surgically resected. After resec- Breast Invasive Carcinoma (TCGA, PanCancer Atlas; ref. 56) tion, the 12 mice that presented metastases were randomized into two groups Breast Cancer (METABRIC ; refs. 57, 58) (n = 6) and treated with either 2 g/L doxycycline and 5% sucrose in the drink- Breast Cancer (MSK 2018; refs. 59) ing water or with 5% sucrose only. The evolution of metastases was followed Breast Cancer (MSK 2020; ref. 60) weekly by IVIS imaging, and mice were treated for a maximum of 12 weeks. Two control mice from the sucrose-treated group had to be euthanized 3 and We classified these samples according to the status of the seven selected genes 6 weeks after treatment onset due to metastatic burden. Mice were euthanized significantly downregulated by Omomyc. Tumors bearing genetic alterations by CO2 inhalation, and an ex vivo IVIS scan was performed. 114 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer Effect of Intravenous Administration of the Omomyc Miniprotein on Pri- with vehicle only and served as controls. Lungs and mammary fat pads were mary Tumor Growth: a suspension of MDA-MB-231 cells was mixed with analyzed ex vivo by IVIS imaging and the signal emitted by labeled Omomyc Matrigel (1:1) and 1.5 million cells/mouse were injected between the fourth and was quantified. fifth right mammary fat pads of 13 six-week-old BALB/c nude females (Janvier). Before surgery, mice were anesthetized with 2% isoflurane and buprenorphine Survival Study in a Patient-Derived Xenograft (0.75 mg/kg) was administered subcutaneously. Tumor size was evaluated once A tumor biopsy obtained from a metastatic TNBC patient of the Vall a week by caliper measurements, and tumor volume calculated using the follow- d’Hebron University Hospital was expanded in NMRI nude female mice (Jan- ing formula: volume = (length × width)2 /2. When tumors were established, vier RRID:IMSR_TAC:nmrinu), whose drinking water was supplemented with mice were sent to the Centro de Investigaciones Energéticas, Medioambien- 1 μmol/L 17-β-estradiol. To obtain experimental mice, a tumor was excised, di- tales y Tecnológicas (CIEMAT), their tumor volume analyzed by PET-CT, and vided into 30 pieces of equal size, and each one inoculated subcutaneously into randomized into two treatment groups. One group (n = 7) was treated intra- NMRI nude female mice. Briefly, mice were anesthetized with intraperitoneal venously with 50 mg/kg Omomyc twice a week, and the other group (n = 6) was ketamine/xylazine (80/10 mg/kg respectively). Methocel was administered for treated with an equivalent amount of vehicle. Mice were treated for 4 weeks, protection of eyes. After a small dorsolateral incision on the flank, a tumor piece a final PET-CT scan performed, and euthanized by CO2 inhalation. Caliper was implanted and the wound closed with Histoacryl. Meloxicam (5 mg/kg) measures were also taken throughout the course of the treatment period. was administered subcutaneously on the day of the surgery and for 3 consec- utive days. Tumor size was evaluated twice a week by caliper measurements Cell Line–Derived Lung Colonization Model (Efficacy Studies) and tumor volume calculated using the following formula: volume = (length × Effect of Omomyc Expression on Lung Colonization: a suspension of width)2 /2. When tumors reached 100 mm3 , mice were randomized into two 500,000 luciferase-expressing MDA-MB-231-Omomyc cells in PBS were inoc- groups. One group (n = 14) was given 50 mg/kg Omomyc intravenously twice ulated through the lateral tail vein of 20 BALB/c nude female mice (Janvier). a week and the other one (n = 15) was given an equal amount of vehicle twice a Starting the day after inoculation, lung colonization and growth of tumor cells week. A survival study was performed and mice were treated until they showed was monitored weekly by IVIS imaging. One week after inoculation, mice were excessive bodyweight loss (>20%), excessive tumor volume (>1,750 mm3 ) or randomized into two groups and treated with either 2 g/L doxycycline and 5% tumor ulceration, and then were euthanized by CO2 inhalation. sucrose in the drinking water (n = 10) or with 5% sucrose only (n = 10). Five weeks later, a final in vivo bioluminescence detection was performed. Moreover, IVIS, μCT, and PET-CT Imaging micro-CT (μCT) scans were performed to allow visualization of individual IVIS studies were performed with a Xenogen IVIS Spectrum (Perkin Elmer). lung lesions. Mice were euthanized, and their lungs excised, fixed for 48 hours in For imaging, mice were injected intraperitoneally with a d-luciferin solution buffered 4% formaldehyde, transferred to 70% ethanol and embedded in paraf- (150 mg/kg in PBS) 5 to 10 minutes prior to acquisition. Mice were anesthetized fin. Sections were cut 3.5-μm thick and stained with hematoxylin and eosin with isoflurane (5% for induction and 2% during acquisition) and air flow (H&E). Metastatic foci were counted and their area measured with ImageJ. was set at 0.8 L/minute. IVIS data were analyzed with Living Image software (Perkin Elmer). Study analysis consisted of a light radiance quantification. Sig- Effect of Pretreatment with the Omomyc Miniprotein on Lung Colo- nals from the light sources were detected and characterized. Working units were nization: MDA-MB-231 cells were treated with 20 μmol/L Omomyc or an photons/sec/cm2 /sr, which allow comparison between images obtained by dif- equivalent volume of vehicle and, after 3 days, trypsinized for 30 minutes with ferent acquisition parameters. Acquisition and analysis were carried out by the 0.25% trypsin to remove any potential membrane-bound Omomyc. 500,000 Preclinical Imaging Platform staff at VHIR. cells in PBS previously treated with vehicle were inoculated through the lat- eral tail vein of 15 BALB/c nude female mice (Janvier), and 500,000 cells in μCT studies were performed with a Quantum GX microCT Imaging Sys- PBS previously treated with Omomyc were inoculated into 15 other female tem (PerkinElmer). Mice were anesthetized with isoflurane (5% for induction mice. After 24 days, mice were euthanized and their lungs excised, fixed for 48 and 2% during acquisition) and air flow was set at 0.8 L/minute. μCT re- hours in buffered 4% formaldehyde, transferred to 70% ethanol, and embed- constructions were performed with the Quantum GX microCT software ded in paraffin. Sections were cut 3.5-μm thick and stained with H&E. Whole and AMIDE software was used for the analysis (http://amide.sourceforge. lung sections were scanned with a digital slide scanner (Hamamatsu Photonics) net, RRID:SCR_005940). Acquisition and analysis were carried out by the and metastatic foci were counted and their area measured with the NDP.view2 Preclinical Imaging Platform staff at VHIR. viewing software. PET-CT studies were performed using a small-animal PET scanner (SuperAr- Biodistribution Study gus PET-CT 4R, SEDECAL). Mice were fasted overnight prior to the PET-CT scan. PET (energy window 425–700 keV and 20-minute static acquisition in Omomyc was labeled with BDP-650/665 maleimide (Lumiprobe #58480) with head-prone position) and CT studies (voltage 45 kV, current 150 μA, 8 shots, an efficiency of 70%. 360 projections, and standard resolution) were performed at 45 min after intra- Twenty BALB/c nude female mice (Janvier) were used for the study. 10 mice venous injection of 18 FDG (8.6±0.9 MBq) via the tail vein in mice anesthetized received 0.5 million cells intravenously and 10 mice received 1.5 million cells or- by inhalation of 2%–2.5% isoflurane in 100% oxygen at a flow rate of 5% oxygen thotopically as described before to induce the formation of lung and mammary using a Fluovac System (Harvard Bioscience). Throughout the imaging ses- tumors, respectively. Lung tumor growth was followed by weekly IVIS imag- sion, body temperature was constantly kept at 38°C with a heating pad. PET ing and mammary tumor growth was followed by caliper measurements. Seven images were corrected for random events and scatter with and without at- weeks after inoculation, mice were treated with 50 mg/kg of Omomyc-BDP- tenuation correction and reconstructed using the 3D–OSEM (Ordered Subset 650/665 and euthanized after 1 hour. Two mice from each model were treated Expectation Maximization) algorithm (16 subsets and 3 iterations). Free-hand AACRJournals.org Cancer Res Commun; 2(2) February 2022 115 Massó-Vallés et al. regions-of-interest (ROI) were drawn along the tumors in the CT images; number after 6 days of Omomyc treatment, while cells expressing GFP showed 18 FDG PET images were used to help locate and delimit the tumor in the CT. only a slight reduction (Fig. 1D). Images were analyzed using the image analysis software AMIDE, version 1.0.4. To confirm whether the impact of MYC inhibition was conserved across differ- Acquisition and analysis were carried out at CIEMAT. ent genetic backgrounds and breast cancer subtypes, our panel of cell lines was seeded at low density and treated for periods of 2 to 7 weeks (depending on their Statistical Analysis proliferation rate), and then stained with crystal violet. While cells expressing For in vitro experiments, a minimum of three biological replicates with 1–3 tech- GFP showed no impairment in their colony formation capacity, all Omomyc- nical replicates was used. For in vivo experiments, the number of mice used per expressing cells presented fewer and smaller colonies (Fig. 1E). Quantification treatment group is indicated in the text. of the crystal violet area and intensity (a readout of cell number) revealed Statistical analysis and representation of the data was performed using Graph- significant reductions when Omomyc (Fig. 1F), but not GFP (Supplementary Pad Prism 9. For histograms, mean + SD or mean – SD is shown if the mean is Fig. S1C), was expressed in each cell line. The degree of sensitivity to Omomyc positive or negative, respectively. For scattered dot plots, mean ± SD is shown. expression was variable among cell lines and did not correlate with molec- For X-Y graphs, mean ± SD, mean + SD or mean − SD is shown depend- ular subtype, MYC levels, or the extent of MYC downregulation, whereas it ing on the overlap between groups to help data visualization, unless otherwise showed an inverse correlation with Omomyc expression levels (Supplementary specified. Fig. S1D). Hence, the variability in the response to Omomyc is not due to intrin- sic genetic differences among cell lines but rather to the amount of transgenic To determine statistical significance among two groups, a two-tailed unpaired Omomyc that the cells are expressing (Fig. 1F). t test (parametric) or a two-tailed Mann–Whitney test (nonparametric) was used. Outliers were identified with the ROUT method and discarded from the analysis and graphical representation. Omomyc Impairs the Capacity of MDA-MB-231 Cells to Induce Angiogenesis In Vitro For all tests, an alpha level of 0.05 was established. In all graphs, *, **, *** and **** are used to describe P values below 0.05, 0.01, 0.001, and 0.0001, Given that MYC is a recognized proangiogenic factor (62) and that angiogenesis respectively. For all histograms and scattered dot plots, asterisks above one is a key aspect in tumor growth, survival, and metastasis (63), we analyzed the bar/dot plot indicate statistical significance between that group and the control capacity of MDA-MB-231 cells to induce angiogenesis after 3 days of Omomyc group. expression and compared it with that of untreated or GFP-expressing cells. HU- VECs exposed to conditioned media from Omomyc-expressing TNBC cells Data Availability Statement presented a clear impairment in tube formation after only 6 hours (Fig. 2A and B; Supplementary Fig. S2A–S2F), demonstrating that Omomyc expression is The data generated in this study are available within the article and its Supple- antiangiogenic in vitro. mentary Data files. Microarray data are publicly available in Gene Expression Omnibus (GEO) (RRID:SCR_005012) at GSE174447. Omomyc Reduces the Metastatic Potential of Breast Cancer Cells In Vitro Results To test the capacity of Omomyc to affect the metastatic phenotype of TNBC Expression of Omomyc in Breast Cancer Cells Alters cells, we conducted migration and invasion assays in MDA-MB-231-GFP and Their Cell Cycle and Reduces Proliferation MDA-MB-231-Omomyc cells. In a wound-healing assay, 12 hours of Omomyc To test the hypothesis that Omomyc is an effective therapy against breast can- expression led to a significant delay in the gap closure (Fig. 2C and D). Further- cer, we infected a panel of human breast cancer cell lines with lentiviral vectors more, in a Boyden chamber migration assay, Omomyc reduced cell migration containing a doxycycline-inducible Omomyc cassette or a GFP control. Eleven toward nutrients by 50% after 24 hours (Fig. 2E and F). Finally, to test the in- stable cell lines representative of the four intrinsic molecular subtypes of breast vasive capacity of cells to migrate through an extracellular matrix (ECM), we cancer were generated. We specifically focused on TNBC, the subtype with made use of a Boyden chamber assay where cells have to migrate through a layer fewer therapeutic options. In particular, we made use of the well-characterized of Matrigel that mimics the ECM (64) and the process by which metastatic cells TP-, BRAF- and CDKNA-mutated MDA-MB-231 cell line as a paradigm reach the bloodstream or the lymphatic system in the invasion process in vivo. metastatic TNBC model for most in vitro and in vivo studies. Doxycycline- Under these conditions, Omomyc-expressing cells showed a dramatic (80%) dependent expression of Omomyc in most cell lines was confirmed by WB reduction in their invasive capacity (Fig. 2G and H). Taken together, these re- after a 3-day doxycycline treatment (Supplementary Fig. S1A), and, in the sults demonstrate that Omomyc can impair several aspects of the metastatic case of MDA-MB-231, confirmed by flow cytometry as well (Supplementary phenotype of MDA-MB-231 cells. Fig. S1B). To verify whether this was also true for other breast cancer cell lines besides We then characterized the cell cycle of MDA-MB-231 cells expressing GFP MDA-MB-231, Boyden chamber migration experiments were performed in (MDA-MB-231-GFP) or Omomyc (MDA-MB-231-Omomyc) upon 3-day doxy- an additional highly metastatic cell line (BT-549), and two poorly metastatic cycline treatment (Fig. 1A). In these assays, Omomyc expression clearly reduced cell lines (MCF7 and CAL-51). Even though the intrinsic ability of the cells DNA synthesis during the S-phase of the cell cycle (Fig. 1A and B) and caused to migrate was very different among cell lines, in all cases, Omomyc expres- accumulation of cells in the G0 –G1 phase, while control GFP had no detectable sion reduced their migratory capacity by approximately 50% (Supplementary effect (Fig. 1A-C). This alteration in cell cycle dramatically decreased the cell Fig. S2G–S2I). 116 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer FIGURE 1 Expression of Omomyc in breast cancer cells alters their cell cycle and reduces proliferation. A, Representative density plots of MDA-MB-231-GFP untreated cells (−GFP) or treated with 0.6 μg/mL doxycycline (+GFP) and MDA-MB-231-Omomyc untreated cells (−Omomyc) or treated with 0.6 μg/mL doxycycline (+Omomyc) for 3 days measured by flow cytometry. BrdU-FITC: Bromodeoxyuridine-Fluorescein isothiocyanate. PI, propidium iodide. B, Percent of BrdU-positive MDA-MB-231 cells from A. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. C, Percent of G0 –G1 MDA-MB-231 cells from A. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. D, Cell counts of MDA-MB-231-GFP and MDA-MB-231-Omomyc cells treated with doxycycline for 6 days. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. E, Representative images of wells stained with crystal violet after Omomyc expression in a panel of breast cancer cell lines. An example of a GFP-expressing cell line is also shown. Expression of Omomyc or GFP was induced by addition 0.6 μg/mL doxycycline in the culture media. F, Quantification of relative cell number after Omomyc expression compared with control cell without doxycycline. Graph shows mean + SD; statistical significance was determined by two-tailed Mann–Whitney test. Omomyc Expression Reduces the Growth of Primary BALB/c nude mice. When tumors reached 100 mm3 , mice were treated with 5% Mammary Tumors In Vivo in a Cell Line–Derived sucrose or 2 g/L doxycycline diluted in 5% sucrose added to their drinking wa- Orthotopic Model ter for 4 weeks (schematic in Fig. 3A). Tumors from Omomyc-expressing mice To test the effect of Omomyc in TNBC in vivo, luciferase-expressing MDA-MB- showed a much slower growth rate than controls (Fig. 3B) that translated into a 231–Omomyc cells were inoculated orthotopically in the mammary fat pad of very significant difference in tumor volume and tumor weight at experimental AACRJournals.org Cancer Res Commun; 2(2) February 2022 117 Massó-Vallés et al. FIGURE 2 Omomyc impairs the capacity of MDA-MB-231 cells to induce angiogenesis and reduces their metastatic potential in vitro. A, Representative images of HUVECs 6 hours after addition of conditioned media from MDA-MB-231-Omomyc cells untreated (−Omomyc) or treated for 3 days with 0.6 μg/mL doxycycline (+Omomyc). B, Quantification of total tube length in HUVECs from A and in HUVEC cells treated with conditioned media from control MDA-MB-231-GFP cells under the same conditions. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. C, Representative images of a wound-healing assay of MDA-MB-231-Omomyc cells at (Continued on the following page.) 118 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer (Continued) 0 hours (onset) and 12 hours (endpoint) after treatment with 0.6 μg/mL doxycycline (+Omomyc) or left untreated (−Omomyc). The approximate area of the wound is delimited by a blue line. D, Quantification of the relative wound area from C at endpoint and from control MDA-MB-231-GFP cells under the same conditions, corrected for the total cell number. Graph shows mean + SD; statistical significance was determined by two-tailed Mann–Whitney test. E, Representative images of fluorescently labeled MDA-MB-231-Omomyc cells after 24 hours of migration through and FBS gradient. Cells were plated on top of a Boyden chamber in 0.5% FBS with (+Omomyc) or without (−Omomyc) 0.6 μg/mL doxycycline and migrated through a porous membrane toward wells containing 10% FBS. F, Quantification of migrated cells from E and from control MDA-MB-231-GFP cells under the same conditions, corrected for the total cell number. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. G, Representative images of fluorescently labeled MDA-MB-231-Omomyc cells after 24 hours of invasion through and FBS gradient. Cells were plated on top of a Matrigel-coated Boyden chamber in 0.5% FBS with (+Omomyc) or without (−Omomyc) 0.6 μg/mL doxycycline and migrated through a porous membrane toward wells containing 10% FBS. H, Quantification of invaded cells from G and from control MDA-MB-231-GFP cells under the same conditions, corrected for the total cell number. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. endpoint (Fig. 3C-E). At that stage, most of the control animals were close to the the bloodstream of BALB/c nude mice through the lateral tail vein, and, 1 ethical endpoint in terms of tumor volume, while none of the treated ones had week later, animals received sucrose or doxycycline for five consecutive weeks reached that stage yet (Fig. 3B). Notably, Omomyc expression did not cause any (schematic in Fig. 4A). Strikingly, lung lesions in doxycycline-treated mice weight loss in the animals throughout the experiment, except for a drop during presented hardly any growth compared with control mice, showing statistical the first 2 days that was reverted at 4 days and was probably due to the change differences in BLI intensity already 1 week after treatment onset, which were to water supplemented with doxycycline. In fact, differences observed in mouse maintained until the endpoint (Fig. 4B and C). To analyze lung lesions indi- weight from 9 days onwards were mainly due to differences in tumor size (Sup- vidually, μCT images of the thoracic cavity were acquired for each mouse after plementary Fig. S3A). To check for Omomyc’s effect at the cellular level, double 5 weeks of treatment. Lung tumors were counted and their volume calculated, immunofluorescence (IF) for Omomyc and the proliferation marker Ki67 or the showing that Omomyc expression significantly reduced both their number and apoptotic marker cleaved caspase-3 (CC3) was performed in tumor sections of volume, resulting in a significant decrease of the total tumor burden per mouse control and treated mice. As expected, Omomyc expression impaired tumor (Fig. 4D). As in primary tumors, Omomyc expression significantly reduced cell growth by both decreasing cell proliferation (Fig. 3F) and enhancing apoptosis proliferation (Supplementary Fig. S4A). (Fig. 3G). In addition, given that the resolution of μCT does not allow the detection of micrometastases, lung sections from untreated and treated mice were stained Omomyc Expression Causes Regression of Established Metastases After Primary Tumor Resection with hematoxylin and eosin (H&E) and their number and area calculated. As expected, lungs from Omomyc-treated mice presented smaller tumors Following these encouraging results, we further tested Omomyc’s effect against (Supplementary Fig. S4B and S4C), but also a lower number of them (in- metastasis in vivo, in a model in which cells metastasize from the primary tu- cluding micrometastases; Supplementary Fig. S4D), suggesting that Omomyc mor site. Luciferase-expressing MDA-MB-231–Omomyc cells were inoculated is not only affecting their growth but also the successful establishment of orthotopically in the mammary fat pad of BALB/c nude mice until primary tu- micrometastases after seeding. mors established. Then, these were surgically resected to mimic the standard clinical procedure for human patients, which, unfortunately, is often followed by metastatic growth in secondary sites. After surgery, metastasis-bearing mice The Omomyc Miniprotein as a Pharmacologic Approach were treated with sucrose or doxycycline, and metastatic growth followed for to Inhibit MYC in Breast Cancer 12 weeks (schematic in Fig. 3H). The majority of sucrose-treated mice showed To translate the encouraging data obtained with the Omomyc transgene into sustained growth of established metastases, while most of doxycycline-treated a viable clinical option for patients with breast cancer, we made use of the mice actually showed metastatic regression and even complete eradication recombinantly produced Omomyc miniprotein, an 11-kDa drug that displays (Fig. 3I and J). Indeed, the difference in bioluminescence intensity (BLI) from cell-penetrating properties, reaches the nuclei, and has already shown remark- treatment onset until experimental endpoint was calculated for both groups and able antitumor activity in non–small cell lung cancer (NSCLC) models in vitro was significantly different. The mean BLI change was positive in the control and in vivo (41). This protein is currently being tested in clinical trials in solid group, indicating metastatic growth, and negative in the treated one, indicat- tumors (ClinicalTrials.gov identifier NCT04808362). ing metastatic regression (Fig. 3J, right). Most tellingly, after ex vivo analysis of the mice, control animals presented an average of 4.3 organs with metastasis, We treated MDA-MB-231 and MCF7 cells with increasing concentrations compared with 0.3 in treated mice (Fig. 3K). of fluorescently labeled Omomyc and observed that, within 15 minutes, the miniprotein was internalized in a dose-dependent manner, demonstrating for Omomyc Expression Reduces Metastatic Lung the first time its spontaneous cell-penetrating capacity in breast cancer (Sup- Colonization plementary Fig. S5). To confirm that the Omomyc miniprotein was functional After demonstrating how Omomyc impacts established metastases, we won- inside the cells, MDA-MB-231 cells were treated with vehicle or 20 μmol/L dered whether its expression would also prevent them in the context of Omomyc and, after 24 hours, the Omomyc-MYC, Omomyc-MAX, and MYC– lung colonization, an earlier stage related to metastatic seeding and initial MAX interactions were imaged by isPLA (Fig. 5A). As expected, Omomyc growth. Luciferase-expressing MDA-MB-231-Omomyc cells were injected into interacts with both MYC and MAX, and causes a reduction in the number of AACRJournals.org Cancer Res Commun; 2(2) February 2022 119 Massó-Vallés et al. FIGURE 3 Omomyc expression reduces primary mammary tumor growth and causes regression of established metastases in a cell line–derived orthotopic model. A, Schematic representation of the mouse model used to assess primary tumor growth. Luciferase-expressing MDA-MB-231-Omomyc cells were inoculated into BALB/c nude females. When tumors reached 100 mm3 , mice were treated with 5% sucrose (−Omomyc) or with 2 g/L doxycycline in 5% sucrose (+Omomyc) for 4 weeks. B, Tumor volume of sucrose- and doxycycline-treated tumors for 4 weeks. C, Image of three representative tumors from sucrose- and doxycycline-treated mice. D, Tumor volume at experimental endpoint. Graph shows mean ± SD; statistical significance was determined by two-tailed Mann–Whitney test. E, Ex vivo tumor weight at experimental endpoint. Graph shows mean ± SD; statistical significance was determined by two-tailed unpaired t test. F, Representative images of double immunofluorescence for Omomyc (Omo) and Ki67 in control and treated tumors with its quantification. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. G, Representative images of double immunofluorescence for Omomyc (Omo) and cleaved caspase-3 (CC3) in control and treated tumors with its quantification. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. H, Schematic representation of the mouse model used to assess metastatic growth. Luciferase-expressing MDA-MB-231-Omomyc cells were inoculated into BALB/c nude females. After 8–10 weeks, primary tumors were surgically resected and mice were treated with 5% sucrose (−Omomyc) or with 2 g/L doxycycline in 5% sucrose (+Omomyc) for 12 weeks. I, The percentage of mice from each treatment group showing sustained growth, regression, and eradication of metastasis, as well as the percentage of mice that required euthanasia due to excessive metastatic growth. J, IVIS-acquired images of a representative sucrose-treated and a doxycycline-treated mouse at treatment onset and endpoint, and quantification of BLI. Graph shows mean + SD (−Omomyc) and mean − SD (+Omomyc); statistical significance was determined by two-tailed unpaired t test. K, Number of organs with metastasis per mouse in each treatment group. Graph shows mean + SD; statistical significance was determined by two-tailed unpaired t test. 120 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer FIGURE 4 Omomyc expression reduces tumor appearance and growth in a cell line–derived model of lung colonization. A, Schematic representation of the mouse model. Luciferase-expressing MDA-MB-231 cells were inoculated through the lateral tail vein into BALB/c nude females. 1 week later, they were treated with 5% sucrose (−Omomyc) or with 2 g/L doxycycline in 5% sucrose (+Omomyc) for 5 weeks. B, Quantification of luciferase activity measured weekly by IVIS imaging as BLI intensity over the 5-week treatment. Graph shows mean + SD (−Omomyc) and mean − SD (+Omomyc); statistical significance was determined via two-tailed Mann–Whitney test. C, IVIS-acquired images of two representative sucrose-treated and doxycycline-treated mice and quantification of BLI intensity at experimental endpoint. Graph shows mean ± SD; statistical significance was determined via two-tailed Mann–Whitney test. D, Representative μCT images of the thoracic cavity of 3 mice treated with sucrose and 3 mice treated with doxycycline at experimental endpoint, with lung tumors circled in red (left), and quantification of the number of tumors, mean tumor volume and total tumor volume per mouse (right). Graphs show mean ± SD; statistical significance was determined via two-tailed unpaired t test (number of tumors and mean tumor volume) or via two-tailed Mann–Whitney test (total tumor volume per mouse). MYC–MAX dimers (Fig. 5A and B). Interestingly, most Omomyc-MAX dimers with paclitaxel in TNBC. To do so, we treated metastatic TNBC cell lines MDA- colocalize with DNA, while most Omomyc-MYC dimers do not. MB-231 and BT-549 with increasing concentrations of paclitaxel and Omomyc, alone or in combination, for 5 days. In both cell lines, the combination was more To characterize the effect of the Omomyc miniprotein on TNBC proliferation, potent than either standalone therapy and showed synergy particularly at lower MDA-MB-231 cells were incubated with 20 μmol/L Omomyc or an equiva- concentrations (Fig. 5G), indicating that the combination could show increased lent volume of vehicle for 3 days, and their cell cycle analyzed. The cell-cycle efficacy in vivo, or that the concentration of both drugs could be reduced to profile was similar to the one previously shown with the Omomyc transgene obtain the same efficacy with potential lower toxicity. (Fig. 1B-D), with a reduction of DNA incorporation during the S-phase and an increase in the percentage of cells in G0 –G1 (Fig. 5C and D). In addition, To get more insight into Omomyc’s mechanism of action against all breast Omomyc-treated cells also showed an increase in the sub-G1 population, indi- cancer cell lines tested, we performed microarray analysis. For this analysis, cating that the miniprotein is capable of inducing cell death (Fig. 5E). We then we made use of MDA-MB-231-Omomyc cells treated with either doxycycline tested increasing concentrations of the Omomyc miniprotein in other breast for 4 days to activate transgenic Omomyc expression or with 20 μmol/L of cancer cell lines, observing a dose-dependent reduction in viability for all of Omomyc miniprotein for 3 days. The analysis showed that the transgene and them, with GI50 values in the low micromolar range (Fig. 5F). Interestingly, the miniprotein significantly modulated gene sets that confirm shutdown of the nontransformed mammary MCF 10A cells were comparatively more resistant MYC fingerprint (downregulation of MYC targets) and can explain its antitu- to the treatment, showing only a slight reduction in cell number at very high mor and antimetastatic activity observed in vitro and in vivo (downregulation concentrations of Omomyc (Fig. 5F). This is indicative of MYC dependence of cell-cycle progression, EMT and breast cancer grade, and upregulation of exclusively in aggressive tumor cell lines. tumor rejection genes, among many others; Fig. 6A and B). Interestingly, the TNBC is currently treated mainly with chemotherapy (e.g., anthracyclines and Omomyc miniprotein regulated more genes and gene sets than its transgenic taxanes) in the neoadjuvant and adjuvant setting (65). Importantly, aberrant counterpart (Supplementary Fig. S6A and S6B), probably due to higher nuclear MYC expression has been described to induce resistance to chemotherapy in levels (Supplementary Fig. S6C). At the individual gene level, we observed a cer- general, and paclitaxel in particular, in breast and other cancers (66, 67). For tain overlap between the transgene and the miniprotein. In particular, 22.7% of this reason, we explored the therapeutic effect of the combination of Omomyc the downregulated and 15.5% of the upregulated genes by the transgene were AACRJournals.org Cancer Res Commun; 2(2) February 2022 121 Massó-Vallés et al. FIGURE 5 The Omomyc miniprotein binds to MYC and MAX, reduces the number of MYC–MAX interactions, has an impact on the cell cycle and reduces cell viability, alone and in combination with paclitaxel. A, isPLA of Omomyc-MYC, Omomyc-MAX, and MYC–MAX interactions after 24 hours of treatment with vehicle (DMSO) or 20 μmol/L Omomyc. Each red dot corresponds to an interaction. B, Number of MYC–MAX interactions from A. Graph shows mean + SD; statistical significance was determined via two-tailed unpaired t test. C–E, Percent of MDA-MB-231 cells positive for BrdU (C), in the sub-G1 phase of the cell cycle (D), or in the G0 –G1 phase (E) after treatment with PBS (vehicle) or 20 μmol/L Omomyc for 3 days, measured by flow cytometry. Graphs show mean + SD; statistical significance was determined via two-tailed unpaired t test. F, Cell viability in a panel of human breast cancer cell lines and in the nontransformed mammary MCF 10A cells treated with increasing concentrations of Omomyc for 5 days. Graph shows mean ± SD. G, Graphical representations of the synergy score of MDA-MB-231 and BT-549 TNBC cells treated with increasing concentrations of paclitaxel and Omomyc, alone and in combination, for 5 days. also regulated by the miniprotein. At the gene set level, the overlap was much tein only (YEATS, TGFBI; Fig. 6D). Notably, making use of available clinical more evident, with 72% of the gene sets downregulated and 56% upregulated data, we observed that all the aforementioned genes except SCNA, increas- by the transgene also being regulated by the miniprotein. ing or decreasing the mRNA level in the same direction as Omomyc, could confer a significant survival advantage in patients with breast cancer (Supple- To identify potential mediators of Omomyc effect, we selected 8 genes signif- mentary Fig. S6D). On the same line, patients with breast cancer harboring icantly regulated by both conditions (7 downregulated and 1 upregulated; Fig. alterations in at least 1 of the 7 downregulated genes — mainly gene amplifica- 6C). These genes belong to the MYC network, regulate cell cycle, proliferation, tions (Supplementary Fig. S6E) — have a significantly shorter overall survival apoptosis, migration, invasion, and metastasis, and/or are prognostic in breast (Fig. 6E). Strikingly, MYC is the top altered gene among such patients, fol- cancer. We then validated their regulation by Omomyc at the mRNA level by lowed by long noncoding RNA (lncRNA) genes from the 8q24 gene desert qRT-PCR. In these conditions, the expression of five of them was clearly mod- region that have been shown to cooperate with MYC and to exert protumori- ulated by both the Omomyc transgene and the miniprotein (SKP, CHAFB, genic functions in breast and other cancers (Fig. 6F; Supplementary Table S1; MADL, DEPDCB, and EGR, although EGR was not statistically significant refs. 68, 69). for the transgene), one by the transgene only (SCNA), and two by the minipro- 122 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer FIGURE 6 Lentiviral expression of an Omomyc construct (1 μg/mL doxycycline for 4 days) or treatment with the Omomyc miniprotein (20 μmol/L for 3 days) has a profound impact on MDA-MB-231 gene expression. A, Gene set enrichment analysis (GSEA) of selected cancer-related gene sets in MDA-MB-231-Omomyc cells that are significantly regulated by both conditions. Normalized Enrichment score (NES) and FDR q value (q-val) are shown. PBS, vehicle; DOX, Omomyc transgene; CPP, Omomyc miniprotein. B, NES of a selection of gene sets implicated in several hallmarks of breast cancer, that are significantly regulated by both conditions. The cutoff FDR q-val was set at 0.25. C, Relative expression by microarray analysis of a selection of genes significantly regulated by both conditions. D, Quantification of the mRNA expression by qRT-PCR from genes shown in C under the same treatment conditions. Graph shows mean + SD; statistical significance was determined via two-tailed unpaired t test. E, Kaplan–Meier overall survival plot of patients with breast cancer harboring genomic alterations in at least 1 out of the 7 genes downregulated by Omomyc shown in C (TGFBI, SKP2, YEATS4, MAD2L1, CHAF1B, SCN5A, and DEPDC1B). Source: cBioPortal. F, Top 10 altered genes of patients with breast cancer harboring genomic amplifications in at least 1 of the 7 genes downregulated by Omomyc shown in C (TGFBI, SKP2, YEATS4, MAD2L1, CHAF1B, SCN5A, and DEPDC1B). Source: cBioPortal. AACRJournals.org Cancer Res Commun; 2(2) February 2022 123 Massó-Vallés et al. Encouraged by these in vitro results, we then assessed the antimetastatic and We previously demonstrated that many, perhaps all, primary tumors are ad- antitumor function of the Omomyc miniprotein in vivo. To do so, we initially dicted to MYC (18, 34–41), but we had not explored the possibility that MYC pretreated MDA-MB-231 cells with 20 μmol/L of the Omomyc miniprotein or dependency is still conserved in metastases. Hence, in this study, we sought to with vehicle for 3 days, and inoculated them through the lateral tail vein of establish the therapeutic utility of inhibiting MYC in mBC models in vitro and BALB/c nude mice. 3.5 weeks later, lung sections were stained with H&E and in vivo, with a special focus on TNBC. the number and area of lung tumors calculated (schematic in Fig. 7A). Strik- In this context, we hypothesized that targeting MYC, a nonredundant node in ingly, mice inoculated with cells pretreated with Omomyc presented a dramatic cancer, would be effective against metastatic disease for at least two reasons: reduction in the number of micrometastases (Fig. 7B and C), indicating that a short pretreatment with Omomyc was able to reduce the seeding of these cells (i) Metastases are genetically unstable, meaning that information from a into the lung. In addition, despite the fact that the area of each of the few mi- patient’s primary tumor may not accurately reflect the metastasis, and crometastases present in the lungs of mice inoculated with Omomyc-treated one metastasis may vary from another, hindering the benefits of targeted cells was no different from the one in control mice (to be expected, since these therapies (70). Given our data suggesting that MYC inhibition would be mice did not receive Omomyc after inoculation; Fig. 7D), the impairment in effective independently of the mutational profile of the tumor (33), using the seeding caused by Omomyc was clearly sufficient to significantly reduce the this approach could overcome this heterogeneity barrier. total tumor area in these mice (Fig. 7E). (ii) MYC promotes EMT and dedifferentiation, two key aspects of metas- Then, to determine whether the Omomyc miniprotein could be used as a drug tasis (13), suggesting that its inhibition could revert these features and against established metastatic TNBC, we performed both a biodistribution and impair the metastatic process from its very inception. an efficacy study. In the first case, we inoculated MDA-MB-231 cells either With all these data in mind and the intention of shedding some light on the mat- orthotopically or intravenously into BALB/c nude mice, to generate primary ter, we first treated a panel of breast cancer cell lines and showed that Omomyc tumors and lung metastases, respectively. We then treated both models with was able to reduce the clonogenic capacity of all of them, independently of a single intravenous injection of 50 mg/kg of fluorescently labeled Omomyc their molecular subtype. Actually, the degree of response, which differed sig- and assessed its localization in the target organs after 1 hour (schematic in nificantly among cell lines, positively correlated with the levels of transgenic Fig. 7F). Interestingly, at this time point, Omomyc localized in the mammary Omomyc expressed in each of them, and did not correlate with MYC lev- tumors (more than in the healthy mammary fat pad) and in tumor-bearing els or the extent of MYC downregulation. These observations suggest that all lungs (although it should be noted that micrometastases represent a very small breast cancer cell lines tested are, in principle, equally sensitive to Omomyc. proportion of the total lung; Fig. 7G). This is in line with previous observations in other cancer types (38, 41) and In the efficacy study, instead, we treated the orthotopic model with twice weekly with the idea that MYC is indispensable for the proliferation of all cancer intravenous administrations of 50 mg/kg Omomyc for 4 weeks (schematic in cells. In fact, lack of correlation with MYC levels is not surprising, if we con- Fig. 7F). Importantly, this Omomyc treatment significantly impaired tumor sider that cancer is more often dependent on tonic expression of MYC, rather growth, as demonstrated by both caliper measurements (Fig. 7H) and CT scan than on its overexpression (71). It should also be noticed that MYC downreg- (Fig. 7I). ulation is not necessarily expected as a consequence of Omomyc treatment, because Omomyc inhibits MYC-dependent transcription by both sequestra- Finally, to further demonstrate Omomyc’s potential in a more clinically rele- tion of MYC away from the DNA and binding to E-boxes in the form of vant model, we made use of a subcutaneous TNBC patient-derived xenograft transcriptionally inactive dimers (Omomyc/Omomyc or Omomyc/MAX; ref. (PDX). Again, we treated tumor-bearing mice with the same dose and regi- 33), both mechanisms that would not necessarily cause any downregulation of men (50 mg/kg twice-weekly) and this time, we performed a survival study MYC levels, as opposed to other MYC-inhibitory strategies such as siRNA or (schematic in Fig. 8A). Encouragingly, treatment with Omomyc significantly degraders. reduced tumor volume (Fig. 8B and C) and conferred a survival advantage to the mice (Fig. 8D). Remarkably, treatment with intravenous Omomyc for up To then challenge Omomyc with the worst-case scenario in breast cancer, we to 70 days did not cause any significant weight change when compared with used the well-characterized metastatic TNBC MDA-MB-231 cells to study the vehicle treatment (Fig. 8E), and no indications of toxicity were observed be- phenotypic changes induced by Omomyc expression in vitro and in vivo and sides the ones caused by excessive tumor burden at the experimental endpoint, its potential therapeutic impact. In this context, we showed that Omomyc consistent with the previously reported safety profile described in ref. 41. induces an antiproliferative effect, blocking cell-cycle progression, consistent with MYC’s well-established functions in controlling several cell cycle–related factors (72). Discussion Then, to study Omomyc’s impact on the metastatic phenotype in vitro, we fo- The literature is populated with contradictory reports on the impact of MYC cused on direct and indirect contributors to metastasis, such as migration and inhibition in metastasis, because there are data pointing at MYC as both in- invasion of tumor cells, and promotion of angiogenesis. Regarding the latter, hibiting and enhancing it. Several publications associate MYC with metastasis we observed that conditioned medium from cells expressing Omomyc heavily in breast and other cancers, sometimes as a repressor (28–31), but mostly as impaired tube formation in endothelial cells compared with control medium, an inducer (20–27). For instance, repression of integrin expression by MYC in consistent with MYC’s role in promoting vasculogenesis and angiogenesis breast cancer cells has been linked to suppression of cell migration and metasta- through regulation of angiogenic factors such as VEGF, thrombospondin- sis (31), while another report suggests that Skp2 cooperates with MYC to induce 1, and angiopoietin-1 (62, 73). We also demonstrated that MYC inhibition RhoA transcription, thereby promoting metastasis (20). by Omomyc caused a profound reduction in directional migration of 124 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer FIGURE 7 Pretreatment of MDA-MB-231 cells with the Omomyc miniprotein in vitro prevents subsequent lung colonization in vivo. Intravenous administration of Omomyc in vivo reaches mammary tumors and tumor-bearing lungs and reduces mammary tumor growth. A, Schematic representation of the lung colonization prevention experiment. Luciferase-expressing MDA-MB-231 cells were pretreated with 20 μmol/L Omomyc or vehicle for 3 days and inoculated through the lateral tail vein of BALB/c nude females. 3.5 weeks later, lung colonization was assessed. B, Representative images of hematoxylin and eosin (H&E)-stained lung sections from mice inoculated with vehicle- or Omomyc-treated cells as described in A. C, Quantification of the number of tumors present in the lungs of mice inoculated with vehicle- or Omomyc-treated cells from A. Graph shows mean ± SD; statistical significance was determined via two-tailed unpaired t test. D, Quantification of the mean tumor area of tumors present in the lungs of mice inoculated with vehicle- or Omomyc-treated cells from A. Graph shows mean ± SD; statistical significance was determined via two-tailed unpaired t test. E, Quantification of the total tumor area in the lungs of mice inoculated with vehicle- or Omomyc-treated cells from A. Graph shows mean ± SD; statistical significance was determined via two-tailed unpaired t test. F, Schematic representation of the biodistribution and efficacy experiment. Luciferase-expressing MDA-MB-231 cells were inoculated orthotopically or through the lateral tail vein of BALB/c nude females. Seven weeks later, mice received a single intravenous injection of vehicle or 50 mg/kg Omomyc-BDP-650/665. Mice bearing orthotopic tumors were also treated twice a week for 4 weeks with vehicle or 50 mg/kg Omomyc. G, Quantification of total fluorescence per organ in the mammary fat pad, mammary tumor, healthy lungs, or tumor-bearing lungs of mice treated with vehicle or with 50 mg/kg Omomyc-BDP-650/665, 1 hour after intravenous administration. Graph shows mean + SD. H, Relative tumor volume calculated from caliper measurements in mice bearing mammary tumors treated twice a week for 4 weeks with vehicle or with 50 mg/kg Omomyc. Graph shows mean + SD (vehicle) and mean − SD (Omomyc); statistical significance was determined via two-tailed unpaired t test. I, Relative tumor volume obtained by CT scans of the same mice in H. Graph shows mean ± SD; statistical significance was determined via two-tailed unpaired t test. AACRJournals.org Cancer Res Commun; 2(2) February 2022 125 Massó-Vallés et al. FIGURE 8 Omomyc reduces mammary tumor growth and extends survival in a TNBC PDX. A, Schematic representation of the PDX mouse model. Pieces from a TNBC tumor biopsy were inoculated subcutaneously into NMRI nude female mice. When tumors reached 100 mm3 , mice were treated intravenously with vehicle or with 50 mg/kg Omomyc twice a week until they had to be euthanized (survival study). B, Tumor volume from vehicle- and Omomyc-treated mice during the first 24 days of treatment (when most of the animals were still under treatment). Graph shows mean + SD (vehicle) and mean − SD (Omomyc); statistical significance was determined via two-tailed unpaired t test. C, Relative tumor volume from vehicle- and Omomyc-treated mice after 20 days of treatment. Graph shows mean ± SD; statistical significance was determined via two-tailed unpaired t test. D, Kaplan–Meier survival plot from vehicle- and Omomyc-treated mice. Statistical significance was determined via log-rank (Mantel–Cox) test. E, Percent weight change throughout the survival study. Graph shows mean ± SD. MDA-MB-231 cells in a wound-healing assay. This effect was more pronounced and with previous reports showing that indirect inhibition of MYC is extremely in the presence of chemoattractants (Boyden chamber assay), and, above all, in effective in the treatment of TNBC models (82). the presence of ECM (Matrigel-coated Boyden chamber assay). This implies However, as mentioned previously, some reports claim that MYC, while pro- that MYC is able to promote cell motility itself, as well as directional movement moting primary tumor growth, impairs metastasis, suggesting that MYC in a gradient of chemoattractant factors, and that the expression of Omomyc inhibition reduces primary tumor growth but could enhance invasiveness (31). can block these prometastatic features. The results in the presence of ECM are Therefore, an overarching goal of this project was to clarify this important as- consistent with the previously described role of MYC in the release of ECM pect of MYC biology and demonstrate the utility of inhibiting MYC in the proteases (74–76). Furthermore, it is known that MYC inhibits the expression metastatic setting in vivo. To this end, we made use of two different metastatic of E-cadherin in epithelial cells and controls the expression of many other EMT models. In the lung colonization model, we assessed the capacity of Omomyc regulators, including N-cadherin and Snail (77, 78). Indeed, two independent to prevent the seeding of breast cancer cells in the lung, mimicking incipient publications confirmed this by showing the effect of Omomyc on cadherins, metastases found in patients with breast cancer. In this context, we showed that thus reenforcing our data. In one, Omomyc expression in lung epithelial cells Omomyc significantly reduced both the establishment of micrometastases after harboring knock down of p53, mutant KrasG12V , and MYC was found to sup- seeding and also the growth of the fewer established lung secondary tumors. In press EMT, decreasing expression of the metastasis-promoting ZEB gene at the another model, we treated established metastases after resection of the primary RNA level, combined with an increase in CDH, the gene encoding E-cadherin tumors. In this case, Omomyc unveiled its full potential by making established (79). In the other, Omomyc downregulates GLI1, a transcription factor respon- lesions shrink and, in some cases, even eradicating them completely. sible for inducing metastatic and stem-like phenotypes in colon carcinoma cell lines (80). Although Omomyc has proven efficacious against multiple mouse models of cancer, and we have shown here that it could also be employed against Here we show that Omomyc behaved similarly in vivo. When challenged against metastasis, its value as a viable pharmacologic approach was, until recently, primary mammary tumors, transgenic Omomyc dramatically reduced tumor still questionable. However, we recently showed that the purified Omomyc expansion by reducing cell proliferation and enhancing apoptosis, in line with miniprotein displays cell-penetrating properties and has the innate capacity to what had been shown in the MMTV-Wnt mouse model of breast cancer (81), enter NSCLC cells, reach their nuclei and interfere with MYC transcriptional 126 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer activity, causing a therapeutic impact in mouse models of lung cancer (41). Other altered genes in these patients are FRS, LYZ, and CPSF, all associated Here we have shown that these features are recapitulated in mouse models of to breast cancer pathogenesis (103–105). For these reasons, we believe that the breast cancer as well. More in detail, in vitro, we have demonstrated that the identification of these 8 genes is a first step towards understanding the antitu- Omomyc miniprotein penetrates into breast cancer cells in a dose-dependent mor and antimetastatic properties of Omomyc in mBC, but further validation manner, interfering with cell proliferation, phenocopying the effects of lentivi- and functional studies will be required to fully elucidate Omomyc’s mechanism rally expressed Omomyc, and promoting cell death. We have also shown that of action. combination with paclitaxel is very effective against TNBC cells, pointing to a To translate the potential of MYC inhibition in breast cancer to a relevant clin- potential combination therapy in the clinic. ical setting, we made use of three models of TNBC to validate the Omomyc Importantly, microarray analysis of MDA-MB-231 cells treated with either miniprotein therapeutic impact in vivo. Lung colonization was heavily impaired transgenic Omomyc or the Omomyc miniprotein demonstrated a high degree by pretreatment of TNBC cells with a single dose of Omomyc, demonstrating of overlap, especially at the gene set level. In a nutshell, treatment with either that the miniprotein interferes with the seeding of circulating cells, a key as- form of Omomyc switches off MYC targets and impacts on protumorigenic pect of the metastatic process. In addition, upon intravenous administration, and prometastatic gene sets, downregulating cell-cycle progression, EMT, and Omomyc was found localized in both mammary tumors and metastasis- genes related to breast cancer grade, and upregulating genes involved in tu- bearing lungs and exerted a significant therapeutic impact in cell line–derived mor rejection, among many others. In these gene sets, we identified a panel of (CDX) and PDX models, without causing any relevant side effect to the animals, eight candidates among the top genes regulated by both conditions that can at demonstrating that Omomyc is a safe and effective drug against the disease. least partially explain the therapeutic effect exerted by Omomyc: 7 downregu- These data have contributed to the design of the MYCure clinical trial, a phase lated (SKP, YEATS, SCNA, CHAFB, TGFBI, MADL, and DEPDCB) and I/II study to evaluate the safety, pharmacokinetics, and efficacy of intravenous 1 upregulated (EGR). SKP is a recognized direct MYC target and has been re- Omomyc in solid tumors (NCT04808362). ported to interact with MYC to promote gene transcription and regulate RhoA, In conclusion, this work demonstrates that MYC inhibition by Omomyc is an and its downregulation by shRNA inhibits lung metastasis of TNBC in vivo (20, effective therapeutic option against mBC, by impairing cell proliferation, angio- 83, 84). YEATS has been described as an oncogene in several cancer types, in- genesis, migration and invasion in vitro, dramatically reducing both primary teracts directly with MYC and has been implicated in migration and invasion in tumor and metastatic growth, and, in some cases, even eradicating established breast cancer through regulation of miRNAs (85, 86). SCNA codes for a sub- metastases. Thus, we have demonstrated for the first time the applicability of unit of a voltage-gated Na+ -channel that is elevated in aggressive breast cancer Omomyc against metastasis, challenging the controversial notion that MYC and enhances breast cancer growth and metastatic dissemination; its silencing inhibition could potentiate – rather than inhibit – invasion. Finally, we have by siRNA in MDA-MB-231 cells reduces invasion (87, 88). CHAFB is a subunit validated the Omomyc miniprotein as the first directly deliverable Omomyc- of the chromatin assembly factor-1 (CAF-1), which plays a role in DNA repli- based drug for the treatment of metastatic TNBC, providing a new therapeutic cation and repair; it is a proliferation marker in breast cancer and has been opportunity for patients suffering from this dreadful and incurable disease. associated with histologic grade in breast and other cancers; its knockdown inhibits tumor growth and migration in a model of hepatocellular carcinoma (89, 90). TGFBI codes for a matrix protein that modulates cell–collagen in- Authors’ Disclosures teractions and has a controversial role in cancer, with studies describing both D. Massó-Vallés reports grants from Departament d’Empresa i Coneixement tumor-suppressing and tumor-promoting roles (91); in breast cancer in partic- de la Generalitat de Catalunya, Spanish Ministry of Science and Innovation, ular, in one study, TGFBI expression was shown to reduce cellular growth and European Research Council, and FERO Foundation during the conduct of the tumorigenicity (92), in contrast with a more recent study in which TGFBI was study; other from Peptomyc S.L. outside the submitted work. M. Beaulieu is related to cancer stem cells and metastasis (93). MADL is a component of a cofounder and shareholder of Peptomyc, a company focused on developing the mitotic spindle assembly checkpoint that has been linked to early metasta- Myc inhibitors for cancer treatment. M.F. Zacarias Fluck reports other from sis in breast cancer and is associated with BRCA/ pathogenic mutations (94, Peptomyc S.L. outside the submitted work. L. Foradada reports personal fees 95). DEPDCB is upregulated in lung cancer, shows a negative correlation with from Peptomyc S.L. during the conduct of the study; personal fees from Pepto- patient survival and has been linked to a metastasis-related malignant phe- myc S.L. outside the submitted work. S. Martínez-Martín reports personal fees notype (96); it has also been described as tumor- or metastasis-promoting in from Peptomyc S.L. during the conduct of the study; personal fees from Pepto- various other cancer types, such as glioblastoma, bladder, prostate, and pancre- myc S.L. outside the submitted work. S. Casacuberta-Serra reports personal fees atic cancer (97–100). Finally, EGR is a tumor suppressor that is a direct target from Peptomyc S.L. during the conduct of the study; personal fees from Pep- of MYC through a noncanonical promoter; MYC is recruited to the promoter tomyc S.L. outside the submitted work; in addition, S. Casacuberta-Serra has a together with ARF, which is necessary for transcriptional induction of EGR, patent to EP19382194 pending. V. Castillo Cano reports other from Peptomyc and potentiates p53-independent, MYC-induced apoptosis (101, 102). S.L outside the submitted work. S. López-Estévez reports personal fees from In addition to their involvement in breast cancer pathogenesis, our analysis Peptomyc SL during the conduct of the study; personal fees from Peptomyc S.L. of clinical data has also shown that these genes have a prognostic value. Fur- outside the submitted work. M. Morcillo reports other from Peptomyc during thermore, MYC is the most altered gene among patients harboring alterations the conduct of the study. J. Arribas reports grants from Roche, grants from Syn- in at least 1 of the 7 downregulated genes, followed by the lncRNAs CASC, thon/Byondis, grants from Molecular Partners, and grants and personal fees POUFB, CCAT, PVT, PCAT, and PRNCR, which not only belong the 8q24 from Menarini during the conduct of the study; in addition, J. Arribas has a gene locus (that contains MYC), commonly amplified in breast cancer, but also patent to P200801652 with royalties paid and a patent to EP20382457.8 with roy- have been associated with promoting MYC-induced tumorigenesis (68, 69). alties paid. J.R. Whitfield reports Shareholder in Peptomyc. L. Soucek reports AACRJournals.org Cancer Res Commun; 2(2) February 2022 127 Massó-Vallés et al. personal fees from Peptomyc S.L.; grants from FERO Foundation (FERO/1284), resources. J.R. Whitfield: Funding acquisition, writing-review and editing. European Research Council (ERC-PoC II / 3079 / SYST-iMYC [813132]), Span- L. Soucek: Conceptualization, resources, funding acquisition, methodology, ish Ministry of Science and Innovation (FI20/00274), Spanish Ministry of writing-original draft, writing-review and editing. Science and Innovation RTC2019-007067-1), Spanish Ministry of Science and Innovation (RTC-2016-5079-1), and grants from Secretaria d’Universitats i Re- cerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya Acknowledgments (FI-DGR-2013) during the conduct of the study; personal fees from Pepto- This project was funded by grants from Secretaria d’Universitats i Re- myc S.L. outside the submitted work; in addition, L. Soucek has a patent to cerca del Departament d’Empresa i Coneixement de la Generalitat de EP13382167.8 issued. No other disclosures were reported. Catalunya (FI-DGR-2013), the Spanish Ministry of Science and Innovation (RTC-2016–5079–1, RTC2019–007067–1, PTQ-17–09299 and FI20/00274), the European Research Council (ERC-PoC II / 3079 / SYST-iMYC [813132]), the Authors’ Contributions FERO Foundation (FERO/1284), Cancerfonden (19 0561 Pj) and the Novo Nordisk Foundation (NNF19OC0059256). We thank Dr. Cristina Saura and D. Massó-Vallés: Conceptualization, supervision, funding acquisition, investi- VHIO’s Breast Cancer Group for providing study materials. We acknowl- gation, methodology, writing-original draft, writing-review and editing. M.-E. edge VHIO and the Cellex Foundation for providing research facilities and Beaulieu: Methodology, writing-review and editing. T. Jauset: Investigation, equipment. methodology. F. Giuntini: Investigation; M.F. Zacarías-Fluck: Methodology. L. Foradada: Investigation. S. Martínez-Martín: Investigation. E. Serrano: Investigation. G. Martín-Fernández: Investigation. S. Casacuberta-Serra: Note Investigation. V. Castillo Cano: Investigation. J. Kaur: Investigation, writing- Supplementary data for this article are available at Cancer Research Comm- review and editing. S. López-Estévez: Investigation. M. Ángel Morcillo: unications Online (https://aacrjournals.org/cancerrescommun/). Investigation. M. Alzrigat: Investigation. L. Mahmoud: Investigation; A. Luque-García: Investigation. M. Escorihuela: Investigation. A. Luque-García: Received October 26, 2021; revised December 31, 2021; accepted February 01, Investigation. J. Arribas: Resources. V. Serra: Resources. L.-G. Larsson: 2022; published first February 21, 2022. References 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 12. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An Global cancer statistics 2020: GLOBOCAN estimates of incidence and mor- embryonic stem cell-like gene expression signature in poorly differentiated tality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71: aggressive human tumors. Nat Genet 2008;40: 499-507. 209-49. 13. Wolfer A, Ramaswamy S. MYC and metastasis. Cancer Res 2011;71: 2034-7. 2. Weigelt B, Reis-Filho JS. Histological and molecular types of breast cancer: is 14. Xu J, Chen Y, Olopade OI. MYC and Breast Cancer. Genes Cancer 2010;1: 629- there a unifying taxonomy? Nat Rev Clin Oncol 2009;6: 718-30. 40. 3. Park S, Koo JS, Kim MS, Park HS, Lee JS, Kim SI, et al. Characteristics and 15. Singhi AD, Cimino-Mathews A, Jenkins RB, Lan F, Fink SR, Nassar H, et al. MYC outcomes according to molecular subtypes of breast cancer as classified gene amplification is often acquired in lethal distant breast cancer metastases by a panel of four biomarkers using immunohistochemistry. Breast 2012;21: of unamplified primary tumors. Mod Pathol 2012;25: 378-87. 50-7. 16. Fallah Y, Brundage J, Allegakoen P, Shajahan-Haq AN. MYC-driven pathways 4. Ooe A, Takahara S, Sumiyoshi K, Yamamoto H, Kawai J, Shiba E. Relationship in breast cancer subtypes. Biomolecules 2017;7: 53. between intrinsic subtypes and tumor responses to neoadjuvant chemother- 17. Whitfield JR, Soucek L. Tumor microenvironment: becoming sick of Myc. Cell apy in patients with locally advanced breast cancer. Breast Dis 2012;34: Mol Life Sci 2012;69: 931-4. 9-17. 18. Sodir NM, Swigart LB, Karnezis AN, Hanahan D, Evan GI, Soucek L. Endogenous 5. Goldhirsch A, Winer EP, Coates AS, Gelber RD, Piccart-Gebhart M, Thurlimann Myc maintains the tumor microenvironment. Genes Dev 2011;25: 907-16. B, et al. Personalizing the treatment of women with early breast cancer: high- lights of the St Gallen International Expert Consensus on the Primary Therapy 19. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev of Early Breast Cancer 2013. Ann Oncol 2013;24: 2206-23. Cancer 2009;9: 239-52. 6. Abramson VG, Lehmann BD, Ballinger TJ, Pietenpol JA. Subtyping of triple- 20. Chan CH, Lee SW, Li CF, Wang J, Yang WL, Wu CY, et al. Deciphering the tran- scriptional complex critical for RhoA gene expression and cancer metastasis. negative breast cancer: implications for therapy. Cancer 2015;121: 8-16. Nat Cell Biol 2010;12: 457-67. 7. McArthur HL. Innovations and challenges in the treatment of metastatic triple- negative breast cancer. J Oncol Pract 2018;14: 290-1. 21. Wolfer A, Wittner BS, Irimia D, Flavin RJ, Lupien M, Gunawardane RN, et al. MYC regulation of a “poor-prognosis" metastatic cancer cell state. Proc Natl 8. Cejalvo JM, Martinez de Duenas E, Galvan P, Garcia-Recio S, Gasion OB, Pare L, Acad Sci U S A 2010;107: 3698-703. et al. Intrinsic subtypes and gene expression profiles in primary and metastatic breast cancer. Cancer Res 2017;77: 2213-21. 22. Li X, Liu X, Xu W, Zhou P, Gao P, Jiang S, et al. c-MYC-regulated miR- 23a/24–2/27a cluster promotes mammary carcinoma cell invasion and hepatic 9. Lu J, Steeg PS, Price JE, Krishnamurthy S, Mani SA, Reuben J, et al. Breast metastasis by targeting Sprouty2. J Biol Chem 2013;288: 18121-33. cancer metastasis: challenges and opportunities. Cancer Res 2009;69: 4951-3. 23. Yeh PY, Lu YS, Ou DL, Cheng AL. IkappaB kinases increase Myc protein stability 10. Swami M. MYC matters. Nat Rev Cancer 2010;10: 813. and enhance progression of breast cancer cells. Mol Cancer 2011;10: 53. 11. Aulmann S, Adler N, Rom J, Helmchen B, Schirmacher P, Sinn HP. c-myc ampli- 24. Talamillo A, Grande L, Ruiz-Ontanon P, Velasquez C, Mollinedo P, Torices fications in primary breast carcinomas and their local recurrences. J Clin Pathol S, et al. ODZ1 allows glioblastoma to sustain invasiveness through a 2006;59: 424-8. 128 Cancer Res Commun; 2(2) February 2022 https://doi.org/10.1158/2767-9764.CRC-21-0103 | CANCER RESEARCH COMMUNICATIONS MYC Inhibition Is Antimetastatic in Breast Cancer Myc-dependent transcriptional upregulation of RhoA. Oncogene 2017;36: 48. Lee E, Lee SJ, Koskimaki JE, Han Z, Pandey NB, Popel AS. Inhibition of 1733-44. breast cancer growth and metastasis by a biomimetic peptide. Sci Rep 2014;4: 25. Lin X, Sun R, Zhao X, Zhu D, Gu Q, Dong X, et al. C-myc overexpression drives 7139. melanoma metastasis by promoting vasculogenic mimicry via c-myc/snail/Bax 49. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, signaling. J Mol Med (Berl) 2017;95: 53-67. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 26. Andrews FH, Singh AR, Joshi S, Smith CA, Morales GA, Garlich JR, et al. Dual- 2012;9: 676-82. activity PI3K-BRD4 inhibitor for the orthogonal inhibition of MYC to block 50. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, et al. tumor growth and metastasis. Proc Nat Acad Sci USA 2017;114: E1072-E80. CellProfiler: image analysis software for identifying and quantifying cell 27. Rapp UR, Korn C, Ceteci F, Karreman C, Luetkenhaus K, Serafin V, et al. MYC is phenotypes. Genome Biol 2006;7: R100. a metastasis gene for non-small-cell lung cancer. PLoS One 2009;4: e6029. 51. Yadav B, Wennerberg K, Aittokallio T, Tang J. Searching for drug synergy 28. Cichon MA, Moruzzi ME, Shqau TA, Miller E, Mehner C, Ethier SP, et al. MYC Is a in complex dose-response landscapes using an interaction potency model. crucial mediator of TGFβ-induced invasion in basal breast cancer. Cancer Res Comput Struct Biotechnol J 2015;13: 504-13. 2016;76: 3520-30. 52. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. 29. Lee M, Beggs SM, Gildea D, Bupp S, Lichtenberg J, Trivedi NS, et al. Necdin is a Gene set enrichment analysis: a knowledge-based approach for interpreting breast cancer metastasis suppressor that regulates the transcription of c-Myc. genome-wide expression profiles. Proc Natl Acad Sci U S A 2005;102: 15545- Oncotarget 2015;6: 31557-68. 50. 30. Elkon R, Loayza-Puch F, Korkmaz G, Lopes R, van Breugel PC, Bleijerveld OB, 53. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- et al. Myc coordinates transcription and translation to enhance transformation time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25: and suppress invasiveness. EMBO Rep 2015;16: 1723-36. 402-8. 31. Liu H, Radisky DC, Yang D, Xu R, Radisky ES, Bissell MJ, et al. MYC sup- 54. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The presses cancer metastasis by direct transcriptional silencing of alphav and cBio cancer genomics portal: an open platform for exploring multidimensional beta3 integrin subunits. Nat Cell Biol 2012;14: 567-74. cancer genomics data. Cancer Discov 2012;2: 401-4. 32. Soucek L, Helmer-Citterich M, Sacco A, Jucker R, Cesareni G, Nasi S. Design 55. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative and properties of a Myc derivative that efficiently homodimerizes. Oncogene analysis of complex cancer genomics and clinical profiles using the cBioPortal. 1998;17: 2463-72. Sci Signal 2013;6: pl1. 33. Masso-Valles D, Soucek L. Blocking Myc to treat cancer: reflecting on two 56. Berger AC, Korkut A, Kanchi RS, Hegde AM, Lenoir W, Liu W, et al. A compre- decades of Omomyc. Cells 2020;9: 883. hensive pan-cancer molecular study of gynecologic and breast cancers. Cancer Cell 2018;33: 690–705 e9. 34. Soucek L, Nasi S, Evan GI. Omomyc expression in skin prevents Myc-induced papillomatosis. Cell Death Differ 2004;11: 1038-45. 57. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The ge- nomic and transcriptomic architecture of 2,000 breast tumours reveals novel 35. Soucek L, Whitfield J, Martins CP, Finch AJ, Murphy DJ, Sodir NM, et al. subgroups. Nature 2012;486: 346-52. Modelling Myc inhibition as a cancer therapy. Nature 2008;455: 679-83. 58. Pereira B, Chin SF, Rueda OM, Vollan HK, Provenzano E, Bardwell HA, et al. 36. Soucek L, Whitfield JR, Sodir NM, Masso-Valles D, Serrano E, Karnezis AN, et al. The somatic mutation profiles of 2,433 breast cancers refines their genomic Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice. and transcriptomic landscapes. Nat Commun 2016;7: 11479. Genes Dev 2013;27: 504-13. 59. Razavi P, Chang MT, Xu G, Bandlamudi C, Ross DS, Vasan N, et al. The ge- 37. Annibali D, Whitfield JR, Favuzzi E, Jauset T, Serrano E, Cuartas I, et al. Myc nomic landscape of endocrine-resistant advanced breast cancers. Cancer Cell inhibition is effective against glioma and reveals a role for Myc in proficient 2018;34: 427-38.e6. mitosis. Nat Commun 2014;5: 4632. 60. Razavi P, Dickler MN, Shah PD, Toy W, Brown DN, Won HH, et al. Alter- 38. Fiorentino FP, Tokgun E, Sole-Sanchez S, Giampaolo S, Tokgun O, Jauset T, ations in PTEN and ESR1 promote clinical resistance to alpelisib plus aromatase et al. Growth suppression by MYC inhibition in small cell lung cancer cells with inhibitors. Nat Cancer 2020;1: 382-93. TP53 and RB1 inactivation. Oncotarget 2016;7: 31014-28. 61. Gyorffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online 39. Alimova I, Pierce A, Danis E, Donson A, Birks DK, Griesinger A, et al. Inhi- survival analysis tool to rapidly assess the effect of 22,277 genes on breast bition of MYC attenuates tumor cell self-renewal and promotes senescence cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res in SMARCB1-deficient Group 2 atypical teratoid rhabdoid tumors to suppress Treat 2010;123: 725-31. tumor growth in vivo. Int J Cancer 2019;144: 1983-95. 62. Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White 40. Sodir NM, Kortlever RM, Barthet VJA, Campos T, Pellegrinet L, Kupczak S, et al. EL, et al. c-Myc is essential for vasculogenesis and angiogenesis during MYC instructs and maintains pancreatic adenocarcinoma phenotype. Cancer development and tumor progression. Genes Dev 2002;16: 2530-43. Discov 2020;10: 588-607. 63. Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc 41. Beaulieu ME, Jauset T, Masso-Valles D, Martinez-Martin S, Rahl P, Maltais L, Health Risk Manag 2006;2: 213-9. et al. Intrinsic cell-penetrating activity propels Omomyc from proof of concept to viable anti-MYC therapy. Sci Transl Med 2019;11: eaar5012. 64. Benton G, Arnaoutova I, George J, Kleinman HK, Koblinski J. Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Adv 42. Beaulieu ME, Soucek L. Finding MYCure. Mol Cell Oncol 2019;6: e1618178. Drug Deliv Rev 2014;79-80: 3-18. 43. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate 65. Furlanetto J, Loibl S. Optimal systemic treatment for early triple-negative breast cancer metastasis to lung. Nature 2005;436: 518-24. breast cancer. Breast Care (Basel) 2020;15: 217-26. 44. Ponomarev V, Doubrovin M, Serganova I, Vider J, Shavrin A, Beresten T, et al. 66. Lee KM, Giltnane JM, Balko JM, Schwarz LJ, Guerrero-Zotano AL, Hutchinson A novel triple-modality reporter gene for whole-body fluorescent, biolumines- KE, et al. MYC and MCL1 cooperatively promote chemotherapy-resistant breast cent, and nuclear noninvasive imaging. Eur J Nucl Med Mol Imaging 2004;31: cancer stem cells via regulation of mitochondrial oxidative phosphorylation. 740-51. Cell Metab 2017;26: 633-47.e7. 45. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of 67. Parasido E, Avetian GS, Naeem A, Graham G, Pishvaian M, Glasgow E, et al. image analysis. Nat Methods 2012;9: 671-5. The sustained induction of c-MYC drives nab-paclitaxel resistance in primary 46. Shaiken TE, Opekun AR. Dissecting the cell to nucleus, perinucleus and cytosol. pancreatic ductal carcinoma cells. Mol Cancer Res 2019;17: 1815-27. Sci Rep 2014;4: 4923. 68. Homer-Bouthiette C, Zhao Y, Shunkwiler LB, Van Peel B, Garrett-Mayer E, Baird 47. Guzman C, Bagga M, Kaur A, Westermarck J, Abankwa D. ColonyArea: an Im- RC, et al. Deletion of the murine ortholog of the 8q24 gene desert has anti- ageJ plugin to automatically quantify colony formation in clonogenic assays. cancer effects in transgenic mammary cancer models. BMC Cancer 2018;18: PLoS One 2014;9: e92444. 1233. AACRJournals.org Cancer Res Commun; 2(2) February 2022 129 Massó-Vallés et al. 69. Gu Y, Lin X, Kapoor A, Chow MJ, Jiang Y, Zhao K, et al. The oncogenic potential 87. Nelson M, Yang M, Millican-Slater R, Brackenbury WJ. Nav1.5 regulates breast of the centromeric border protein FAM84B of the 8q24.21 gene desert. Genes tumor growth and metastatic dissemination in vivo. Oncotarget 2015;6: 32914- (Basel) 2020;11: 312. 29. 70. Marino N, Woditschka S, Reed LT, Nakayama J, Mayer M, Wetzel M, et al. 88. Brackenbury WJ. Voltage-gated sodium channels and metastatic disease. Breast cancer metastasis: issues for the personalization of its prevention and Channels (Austin) 2012;6: 352-61. treatment. Am J Pathol 2013;183: 1084-95. 89. Polo SE, Theocharis SE, Grandin L, Gambotti L, Antoni G, Savignoni A, et al. 71. Murphy DJ, Junttila MR, Pouyet L, Karnezis A, Shchors K, Bui DA, et al. Distinct Clinical significance and prognostic value of chromatin assembly factor-1 thresholds govern Myc’s biological output in vivo. Cancer Cell 2008;14: 447- overexpression in human solid tumours. Histopathology 2010;57: 716-24. 57. 90. Peng X, Fu H, Yin J, Zhao Q. CHAF1B knockdown blocks migration in a 72. Bretones G, Delgado MD, Leon J. Myc and cell cycle control. Biochim Biophys hepatocellular carcinoma model. Oncol Rep 2018;40: 405-13. Acta 2015;1849: 506-16. 91. Ween MP, Oehler MK, Ricciardelli C. Transforming growth Factor-Beta-Induced 73. Shchors K, Shchors E, Rostker F, Lawlor ER, Brown-Swigart L, Evan GI. The Protein (TGFBI)/(betaig-H3): a matrix protein with dual functions in ovarian Myc-dependent angiogenic switch in tumors is mediated by interleukin 1beta. cancer. Int J Mol Sci 2012;13: 10461-77. Genes Dev 2006;20: 2527-38. 92. Li B, Wen G, Zhao Y, Tong J, Hei TK. The role of TGFBI in mesothelioma and 74. Duffy MJ. The role of proteolytic enzymes in cancer invasion and metastasis. breast cancer: association with tumor suppression. BMC Cancer 2012;12: 239. Clin Exp Metastasis 1992;10: 145-55. 93. Fico F, Santamaria-Martinez A. TGFBI modulates tumour hypoxia and 75. Hu T, Lu YR. BCYRN1, a c-MYC-activated long non-coding RNA, regulates cell promotes breast cancer metastasis. Mol Oncol 2020;14: 3198-210. metastasis of non-small-cell lung cancer. Cancer Cell Int 2015;15: 36. 94. Tsantoulis P, Migliorini D, Martin-Lluesma S, Durigova A, Bodmer A, Dietrich P, 76. Noujaim D, van Golen CM, van Golen KL, Grauman A, Feldman EL. N-Myc et al. 283P – Mad2L1 overexpression leads to early metastasis in breast cancer. and Bcl-2 coexpression induces MMP-2 secretion and activation in human Ann Oncol 2014;25: iv95. neuroblastoma cells. Oncogene 2002;21: 4549-57. 95. Ben-Hamo R, Berger AJ, Gavert N, Miller M, Pines G, Oren R, et al. Predicting 77. Cho KB, Cho MK, Lee WY, Kang KW. Overexpression of c-myc induces epithe- and affecting response to cancer therapy based on pathway-level biomarkers. lial mesenchymal transition in mammary epithelial cells. Cancer Lett 2010;293: Nat Commun 2020;11: 3296. 230-9. 96. Yang Y, Liu L, Cai J, Wu J, Guan H, Zhu X, et al. DEPDC1B enhances 78. Smith AP, Verrecchia A, Faga G, Doni M, Perna D, Martinato F, et al. A migration and invasion of non-small cell lung cancer cells via activat- positive role for Myc in TGFbeta-induced Snail transcription and epithelial- ing Wnt/beta-catenin signaling. Biochem Biophys Res Commun 2014;450: to-mesenchymal transition. Oncogene 2009;28: 422-30. 899-905. 79. Larsen JE, Nathan V, Osborne JK, Farrow RK, Deb D, Sullivan JP, et al. 97. Lai CH, Xu K, Zhou J, Wang M, Zhang W, Liu X, et al. DEPDC1B is a tumor pro- ZEB1 drives epithelial-to-mesenchymal transition in lung cancer. J Clin Invest motor in development of bladder cancer through targeting SHC1. Cell Death 2016;126: 3219-35. Dis 2020;11: 986. 80. Varnat F, Siegl-Cachedenier I, Malerba M, Gervaz P, Ruiz i Altaba A. Loss 98. Li Z, Wang Q, Peng S, Yao K, Chen J, Tao Y, et al. The metastatic promoter of WNT-TCF addiction and enhancement of HH-GLI1 signalling define the DEPDC1B induces epithelial-mesenchymal transition and promotes prostate metastatic transition of human colon carcinomas. EMBO Mol Med 2010;2: cancer cell proliferation via Rac1-PAK1 signaling. Clin Transl Med 2020;10: e191. 440-57. 99. Chen X, Guo ZQ, Cao D, Chen Y, Chen J. Knockdown of DEPDC1B inhibits the 81. von Eyss B, Jaenicke LA, Kortlever RM, Royla N, Wiese KE, Letschert S, development of glioblastoma. Cancer Cell Int 2020;20: 310. et al. A MYC-driven change in mitochondrial dynamics limits YAP/TAZ func- 100. Liu X, Li T, Huang X, Wu W, Li J, Wei L, et al. DEPDC1B promotes mi- tion in mammary epithelial cells and breast cancer. Cancer Cell 2015;28: 743- gration and invasion in pancreatic ductal adenocarcinoma by activating the 57. Akt/GSK3beta/Snail pathway. Oncol Lett 2020;20: 146. 82. Horiuchi D, Camarda R, Zhou AY, Yau C, Momcilovic O, Balakrishnan S, et al. 101. Boone DN, Hann SR. The Myc-ARF-Egr1 pathway: unleashing the apoptotic PIM1 kinase inhibition as a targeted therapy against triple-negative breast power of c-Myc. Cell Cycle 2011;10: 2043-4. tumors with elevated MYC expression. Nat Med 2016;22: 1321-9. 102. Boone DN, Qi Y, Li Z, Hann SR. Egr1 mediates p53-independent c-Myc-induced 83. Hydbring P, Castell A, Larsson LG. MYC Modulation around the CDK2/p27/SKP2 apoptosis via a noncanonical ARF-dependent transcriptional mechanism. Proc Axis. Genes (Basel) 2017;8: 174. Natl Acad Sci U S A 2011;108: 632-7. 84. Bretones G, Acosta JC, Caraballo JM, Ferrandiz N, Gomez-Casares MT, Albajar 103. Sung VYC, Knight JF, Johnson RM, Stern YE, Saleh SM, Savage P, et al. Co- M, et al. SKP2 oncogene is a direct MYC target gene and MYC down-regulates dependency for MET and FGFR1 in basal triple-negative breast cancers. NPJ p27(KIP1) through SKP2 in human leukemia cells. J Biol Chem 2011;286: 9815- Breast Cancer 2021;7: 36. 25. 104. Serra C, Vizoso F, Alonso L, Rodriguez JC, Gonzalez LO, Fernandez M, et al. Ex- 85. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis pression and prognostic significance of lysozyme in male breast cancer. Breast initiated by microRNA-10b in breast cancer. Nature 2007;449: 682-8. Cancer Res 2002;4: R16. 86. Tao K, Yang J, Hu Y, Deng A. Knockdown of YEATS4 inhibits colorectal can- 105. Binothman N, Hachim IY, Lebrun JJ, Ali S. CPSF6 is a clinically relevant cer cell proliferation and induces apoptosis. Am J Transl Res 2015;7: 616- breast cancer vulnerability target: role of CPSF6 in breast cancer. 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