ORIGINAL RESEARCH published: 09 September 2021 doi: 10.3389/fcell.2021.695225 MCL-1 Inhibition Overcomes Anti-apoptotic Adaptation to Targeted Therapies in B-Cell Precursor Acute Lymphoblastic Leukemia Albert Manzano-Muñoz 1 , Clara Alcon 1 , Pablo Menéndez 2,3 , Manuel Ramírez 4 , Felix Seyfried 5 , Klaus-Michael Debatin 5 , Lüder H. Meyer 5 , Josep Samitier 1,6,7 and Joan Montero 1* 1 Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain, 2 Stem Cell Biology, Developmental Leukemia and Immunotherapy, Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain, 3 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Salud Carlos III (ISCIII), Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 4 Department of Pediatric Hematology and Oncology, Niño Jesús University Children’s Hospital, Madrid, Spain, 5 Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany, 6 Edited by: Department of Electronics and Biomedical Engineering, Faculty of Physics, University of Barcelona, Barcelona, Spain, 7 Tugba Bagci-Onder, Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain Koç University, Turkey Reviewed by: Multiple targeted therapies are currently explored for pediatric and young adult B-cell Elodie Lafont, precursor acute lymphoblastic leukemia (BCP-ALL) treatment. However, this new INSERM U1242 Laboratoire COSS, France armamentarium of therapies faces an old problem: choosing the right treatment for each Triona Ni Chonghaile, patient. The lack of predictive biomarkers is particularly worrying for pediatric patients Royal College of Surgeons in Ireland, Ireland since it impairs the implementation of new treatments in the clinic. In this study, we used *Correspondence: the functional assay dynamic BH3 profiling (DBP) to evaluate two new treatments for Joan Montero BCP-ALL that could improve clinical outcome, especially for relapsed patients. We found jmontero@ibecbarcelona.eu that the MEK inhibitor trametinib and the multi-target tyrosine kinase inhibitor sunitinib Specialty section: exquisitely increased apoptotic priming in an NRAS-mutant and in a KMT2A-rearranged This article was submitted to cell line presenting a high expression of FLT3, respectively. Following these observations, Cell Death and Survival, a section of the journal we sought to study potential adaptations to these treatments. Indeed, we identified with Frontiers in Cell and Developmental DBP anti-apoptotic changes in the BCL-2 family after treatment, particularly involving Biology MCL-1 – a pro-survival strategy previously observed in adult cancers. To overcome this Received: 14 April 2021 adaptation, we employed the BH3 mimetic S63845, a specific MCL-1 inhibitor, and Accepted: 20 August 2021 Published: 09 September 2021 evaluated its sequential addition to both kinase inhibitors to overcome resistance. We Citation: observed that the metronomic combination of both drugs with S63845 was synergistic Manzano-Muñoz A, Alcon C, and showed an increased efficacy compared to single agents. Similar observations were Menéndez P, Ramírez M, Seyfried F, Debatin K-M, Meyer LH, Samitier J made in BCP-ALL KMT2A-rearranged PDX cells in response to sunitinib, showing an and Montero J (2021) MCL-1 analogous DBP profile to the SEM cell line. These findings demonstrate that rational Inhibition Overcomes Anti-apoptotic sequences of targeted agents with BH3 mimetics, now extensively explored in clinical Adaptation to Targeted Therapies in B-Cell Precursor Acute trials, may improve treatment effectiveness by overcoming anti-apoptotic adaptations in Lymphoblastic Leukemia. BCP-ALL. Front. Cell Dev. Biol. 9:695225. doi: 10.3389/fcell.2021.695225 Keywords: pediatric leukemia, targeted therapies, resistance, apoptosis, BH3 mimetics Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance INTRODUCTION biomarkers (Savary et al., 2020). In contrast, functional assays overcome these drawbacks by directly exposing patient-isolated Acute lymphoblastic leukemia (ALL) is characterized by tumor cells to different treatment options and measuring their uncontrolled growth of lymphoid cells and it accounts for effectiveness (Howard et al., 2017). Yet, this approach presents a 25% of all pediatric cancers (Howlader et al., 2015). Three major limitation: primary cells rapidly decay, lose their viability out of four cases of pediatric ALL are caused by B-cell and experience phenotypic changes, preventing their use in precursors, also named BCP-ALL (Schwab and Harrison, prolonged assays (Meijer et al., 2017). In this regard, the 2018). BCP-ALL diagnosed patients are typically treated with functional assay dynamic BH3 profiling (DBP) (Montero et al., a chemotherapeutic combination of vincristine, asparaginase, 2015) can identify effective anti-cancer treatments in less than a corticosteroid (prednisone or dexamethasone) and an 24 h directly on patient samples. anthracycline (doxorubicin or daunorubicin), and most patients Most anti-cancer agents engage cell death by apoptosis, achieve a complete remission (PDQ Pediatric Treatment a process regulated by the BCL-2 family of proteins. Inside Editorial Board, 2002). Despite this outstanding treatment this family, BAX and BAK are considered effector members effectiveness, around 15–20% of patients relapse (Locatelli et al., and once activated, oligomerize to form pores and induce 2012) causing an overall survival rate of 90% (O’Brien et al., the Mitochondrial Outer Membrane Permeabilization (MOMP) 2018). And because of its high incidence this type of tumor which represents the point of no return for the apoptotic is still the deadliest pediatric cancer. Relapsed and refractory process (Wei et al., 2001) and the cell commitment to die patients (R/R) that fail to achieve a complete remission, present (Letai, 2011). MOMP induces the release of cytochrome c (and highly resistant tumors forcing clinicians to use more aggressive other proteins) from the mitochondria into the cytosol, and its and highly toxic treatments (Oskarsson et al., 2018). Even those binding to APAF-1 and caspase-9 form the apoptosome, which patients that are cured face long-term secondary effects including activates downstream effector caspases and executes apoptosis. mental problems, functional impairment, cardiotoxicity and Effector proteins are activated by proteins presenting a unique increased morbidity (Mody et al., 2008). There is a clear need for BCL-2 homology (BH) domain, known as BH3-only activator new treatments to enhance tumor elimination while reducing proteins (BIM, BID, and PUMA). The anti-apoptotic BCL-2 lasting toxicity. family proteins (BCL-2, BCL-xL, MCL-1, BCL-w, and BFL-1) can Important efforts have been made to identify and characterize inhibit both effectors and activator members, hence protecting oncogenic molecular targets to block them and impair tumor cells from apoptosis. A fourth subgroup of BCL-2 family proteins, growth and progression. Since the first successes treating the sensitizers – that include BAD, HRK and NOXA among pediatric ALL patients using folic acid antagonists achieved by others – exert a pro-apoptotic effect by specifically inhibiting the Sydney Farber in the late 1940s (Miller, 2006), a myriad of anti-apoptotic proteins (Letai et al., 2002). compounds targeting multiple proteins have been explored in Dynamic BH3 profiling uses synthetic peptides that mimic clinical trials. The first and most successful targeted therapy the BH3 domain of the pro-apoptotic BH3-only, with a similar approved for hematological malignancies was imatinib for adult effect as the full-length protein. It uses these peptides to measure chronic myelogenous leukemia (Deininger, 2008). Interestingly, how close cells are to commit apoptosis, or how “primed” are for imatinib targets the BCR-ABL fusion protein that is also death, after a short incubation with the treatment. DBP has been present in the Philadelphia chromosome-positive (Ph +) B-cell extensively used to identify effective anti-cancer treatments in cell acute lymphoblastic leukemia (B-ALL) subtype. Clinical trials lines, patient-derived xenografts (PDX) and directly on patient- evaluating imatinib and dasatinib for pediatric and young adult isolated cells from different types of cancer (Montero et al., 2015, Ph + B-ALL showed improvement in treatment response (Biondi 2017, 2019; Wu et al., 2015; Townsend et al., 2016; Deng et al., et al., 2012; Gore et al., 2018). Besides BCR-ABL fusion protein, 2017; Alcon et al., 2020) with an excellent predictive capacity. JAK/STAT pathway proteins, FLT3 receptor, MAPK pathway Interestingly, using sensitizer-analog BH3 peptides, DBP can proteins, precursor-B-cell receptor (pre-BCR) or the ubiquitin- identify the anti-apoptotic protein used by cancer cells to survive proteasome system have been proposed as potential targets for therapy (Frenzel et al., 2009; Montero and Letai, 2018). In fact, different B-ALL subtypes, increasing the armamentarium of most cytogenetic abnormalities found in BCP-ALL regulate anti- potential targeted therapies for this disease (Kuhlen et al., 2019). apoptotic BCL-2 proteins. For example, the BCR-ABL fusion Identifying new effective treatments for pediatric cancer is protein upregulates BCL-2 and ETV6/RUNX1 promotes BCL-xL challenging. If these therapies are not correctly assigned, there is overexpression (Brown et al., 2017). Hereof, BH3 mimetics, anti- a risk to provoke undesired secondary effects. Precision medicine apoptotic inhibitors widely explored in clinical trials, can be used aims to correctly assign effective treatments to every patient to overcome this therapy-acquired resistance. In fact, it has been based on molecular characterization of the tumor (Jameson and demonstrated in ALL that combining standard-of-care treatment Longo, 2015). Its successful implementation will lead to more with BH3 mimetics can greatly increase efficacy (Ni Chonghaile effective therapeutic regimes and reduce side effects (Mathur et al., 2014; Iacovelli et al., 2015; Khaw et al., 2016; Seyfried et al., and Sutton, 2017). Genetic analyses are the most common 2019). But the key question remains unsolved: how and when to strategy used to identify cancer patients that would benefit from better utilize them in the clinic. targeted therapies (Malone et al., 2020). However, the pediatric Here, we use DBP to identify new effective therapeutic population presents a low number of mutations compared strategies for pediatric and adolescent BCP-ALL. We tested to adult population, making difficult to identify predictive two promising targeted therapies, trametinib (MEK inhibitor) Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance and sunitinib (multi-target tyrosine kinase inhibitor), currently Corning, Madrid, Spain) and incubated at room temperature for explored in pre-clinical studies. We found that both caused an 1 h. After this incubation, cells were fixed with 25 µL of an 8% MCL-1 dependence to protect BCP-ALL cells from apoptotic cell paraformaldehyde solution for 15 min and neutralized with 50 µL death and that its inhibition with a BH3 mimetic significantly of N2 buffer (1.7 M tris base, 1.25 M glycine at pH 9.1). Finally, enhanced leukemia cell death. Finally, we were able to observe 25 µL of intracellular staining buffer (1% Tween20, 5% BSA in similar anti-apoptotic adaptations in a pediatric BCP-ALL PDX, PBS) with 1:1,000 dilution of the cytochrome C antibody (Alexa demonstrating its potential use in the clinic. Fluor 647 anti-Cytochrome c—6H2.B4, 612310, BioLegend) R was added and plates were incubated overnight at 4◦ C. Results were analyzed using a Sony flow cytometer (SONY SA3800, MATERIALS AND METHODS Surrey, United Kingdom) and processed with FlowJo to quantify cytochrome c release (%priming). 1%priming stands for the Cell Lines and Treatments difference of %priming between non-treated cells and treated NALM-6 and SEM cell lines were kindly provided by Prof. PM cells for every specific peptide. All results are represented as the laboratory at the Josep Carreras Leukaemia Research Institute. mean of at least three independent experiments. Both cell lines were cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) with 10% of heat- Protein Extraction inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), After treatment, cells were collected and washed with PBS. 1% of L-glutamine (25030, Thermo Fisher, Gibco) and 1% of Then, cells were resuspended in RIPA buffer [150 mM NaCl, penicillin/streptomycin (15140, Thermo Fisher, Gibco). Cells 5 mM EDTA, 50 mM Tris–HCl pH = 8, 1% Triton X-100, 0.1% were maintained inside a humidified incubator at 37◦ C and 5% SDS, EDTA-free Protease Inhibitor Cocktail (4693159001, Roche, of CO2 . Imatinib, dasatinib, sunitinib, trametinib, ibrutinib and MannKind, Germany)] and lysed for 30 min on ice following ruxolitinib were purchased at SelleckChem (Munich, Germany) centrifugation at 4◦ C for 10 min at 16,000g. Protein in the and ABT-199, A-1331852 and S63845 were purchased at supernatant was quantified using PierceTM BCA Protein Assay MedChemExpress (Monmouth Junction, NJ, United States). Kit (23227, Thermo Fisher) and stored at −20◦ C. These reagents were diluted in dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Saint Louis, MO, United States) and Immunoprecipitations added to the culture media at the indicated concentration and Lysates were obtained following the protein extraction protocol incubation time for every experiment. but using immunoprecipitation (IP) lysis buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton X-100, 1.5 mM Cell Death Assay MgCl2 , 10% glycerol and EDTA-free Protease Inhibitor Cocktail After treatment, cells were resuspended in staining buffer (4693159001, Roche, MannKind, Germany). Again, the protein (100 mM HEPES free acid, 40 mM KCl, 1.4 M NaCl, 7.5 mM extracted was quantified with PierceTM BCA Protein Assay MgCl2 and 25 mM CaCl2 at pH 7.4) with Alexa Fluor Kit and stored at −20◦ C. Equivalent amount of protein was 647 conjugated Annexin V (640912, BioLegend) and DAPI R incubated at 4◦ C overnight with magnetic beads (161-4021, (62248, Thermo Fisher). Cells were analyzed with a Gallios flow Bio-Rad, Madrid, Spain) previously conjugated to 5 µg of cytometer (Beckman Coulter, Nyon, Switzerland) and results rabbit anti-MCL-1 antibody (CST94296, Cell Signaling, Leiden, were analyzed with FlowJo software to quantify viable cells Netherlands) or 5 µg of rabbit-IgG antibody (CST2729, Cell (Annexin V and DAPI negative events). Results were represented Signaling). A 30 µL of protein for each condition was stored as the mean of %Cell death (100-%viable cells) of at least three at −20◦ C as the input fraction. After incubation, tubes were independent experiments. magnetized to obtain the binding fraction. The supernatant was extracted and stored at −20◦ C as the unbound fraction. The Dynamic BH3 Profiling binding fraction was cleaned with PBS-T (PBS with 0.1% Tween Dynamic BH3 profiling (DBP) was performed as previously 20) and resuspended in 40 µL of 4× Laemmli sample buffer described (Ryan et al., 2016; Montero et al., 2019; Alcon (161-0747, Bio-Rad), then heated at 70◦ C to allow separation et al., 2020). After treatment, cells were stained using the between the target protein and the magnetic beads-antibody viability marker Zombie Violet (423113, BioLegend, Koblenz, complex. The sample was magnetized again and the supernatant Germany) for 10 min at room temperature, washed with PBS and containing the pulled-down proteins was stored at −20◦ C resuspended in 330 µL of MEB buffer (150 nM mannitol, 10 mM as IP fractions. HEPES-KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA and 5 mM succinate). In parallel, peptide solutions Immunoblotting were prepared using MEB buffer with 0.002% of digitonin (D141, An equal amount of protein was prepared in 4× Laemmli buffer Sigma-Aldrich) and 12 different peptide solutions with final (161-0747, Bio-Rad) and heated at 96◦ C for 10 min. The sample concentrations of 25 µM of alamethicin (BML-A150-0005, Enzo was loaded in an SDS-PAGE gel (456-1025, Bio-Rad) and proteins Life Sciences, Lörrach, Germany), 10 µM, 3 µM, 1 µM, 0.3 µM, were separated for 2 h. Then, proteins were transferred to a 0.1 µM, 0.03 µM, and 0.01 µM of BIM BH3 peptide, 0.1 µM PVDF membrane (10600023, Amersham Hybond, Pittsburgh, of BAD BH3 peptide, 100 µM of HRK BH3 peptide, 10 µM of PA, United States) at 55V for 2 h at 4◦ C. The membrane was MS1 BH3 peptide and a DMSO only control. A 25 µL of cells was blocked with 5% dry milk in TBS-T (50 mM Tris–HCl pH = 7.5, added to 25 µL of each peptide solution in a 96-well plate (3795, 150 mM NaCl and 1% Tween20) for 1 h and washed with Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance TBS-T. After blocking, the membrane was probed overnight RESULTS at 4◦ C in TBS-T with 3%BSA and 1:1,000 dilution of the primary antibody against the protein of interest: rabbit anti-BCL- DBP Predicts Cytotoxicity in BCP-ALL 2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Cell Lines Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit Targeted therapies are currently explored in clinical trials to treat anti-BIM (CST2933, Cell Signaling), rabbit anti-BAX (CST2772, pediatric and young adult BCP-ALL. For example, imatinib and Cell signaling), rabbit anti-BAK (CST12105, Cell signaling), dasatinib for patients presenting the BCR-ABL fusion protein phospho-p44/42 MAPK (Erk1/2) (Thr202/Thr204) (CST4370, (Ph + cases); trametinib for RAS-mutant patients (Jerchel et al., Cell signaling), phospho-BIM (Ser69) (CST4585, Cell signaling) 2018); sunitinib for cases with overexpression or activating and rabbit anti-Actin (CST4970, Cell Signaling) followed by 1 h mutations of FLT3 (Brown et al., 2005); ruxolitinib for tumors at room temperature with 1:3,000 of anti-rabbit IgG HRP-linked with constitutive activation of the JAK/STAT signaling pathway secondary antibody (CST7074, Cell Signaling). When necessary, (Ding et al., 2018), and ibrutinib when pre-BCR is active (Kim membranes were stripped using mild stripping buffer (0.1 M et al., 2017). We sought to explore the effects of these targeted glycine pH 2.5 and 2% SDS) for two cycles of 20 min at 50◦ C therapies, particularly on the apoptotic pathway, in BCP-ALL. and extensively washed with TBS (50 mM Tris–HCl pH = 7.5 To explore the pro-apoptotic effect of these therapies, we and 150 mM NaCl). Membranes were developed using Clarity performed DBP in two pediatric and young adult cell lines, ECL Western substrate (1705060, Bio-Rad) in a LAS4000 imager SEM (presenting KMT2A-rearrangement and high expression of (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Bands were FLT3) and NALM-6 (NRAS-mutant), respectively (Figure 1A). quantified using ImageJ software to measure the integrated After a short incubation with treatments, we observed that optical density. sunitinib induced a high increment in apoptotic priming when exposed to the BIM peptide in the SEM cell line, while Precursor BCP-ALL PDX Model trametinib increased it mildly in both cell lines. In contrast, Generation imatinib, dasatinib, ruxolitinib and ibrutinib did not produce The primary leukemia specimen was obtained from peripheral any induction of apoptosis (Figure 1B). To validate DBP’s blood of an infant patient with newly diagnosed pro-B ALL predictions, we treated these cells for longer timepoints with after informed written consent with the legal guardians and in the same therapies and assessed cell death induction. When accordance with the institution’s ethical review board. Xenograft comparing 1%priming and cytotoxicity, similarly to DBP’s cells were established by intravenous injection of ALL cells into predictions, we observed that sunitinib in the SEM cell line and female NOD/SCID mice (NOD.CB17-Prkdcscid, Charles River) trametinib in both cell lines were more efficient than the rest as described earlier (Meyer et al., 2011; Seyfried et al., 2019). of the therapies inducing cell death (Figure 1C). The receiver Animal experiments were approved by the appropriate authority operating characteristic curve analysis was then used to assess the (Tierversuch Nr. 1260, Regierungspräsidium Tübingen). predictive capacity of DBP on identifying cytotoxic treatments. Our results showed an area under the curve of 1, confirming that DBP is an excellent predictor for this experimental subset DBP With PDX Cells (Supplementary Figure 1A). Furthermore, 1%priming strongly After treatment, cells were stained using the viability marker correlated with cell death after 3 days of treatment, suggesting Zombie Violet (423113, BioLegend, Koblenz, Germany) for that a higher increase in apoptotic priming is an early predictor 10 min on ice, followed by staining for 30 min on ice with for cytotoxicity in these cells (Supplementary Figure 1B). 1:200 dilution of Alexa Fluor 488 anti-human CD45 antibody R Altogether, these results demonstrate that DBP could be used as a (368536, BioLegend) and PE anti-human CD19 antibody predictive biomarker to find effective therapies for BCP-ALL. (392506, BioLegend) in TBS with 10%FBS. Then, cells were washed with PBS and resuspended in 330 µL of MEB buffer. After Trametinib Induces MCL-1 Dependence this, DBP was done as explained in point 2.3. in NALM-6 Cell Line As previously mentioned, trametinib was the only targeted Statistical Analysis therapy tested that showed some efficacy in NALM-6 cells. For the ROC curve analysis, data sets were separated as However, only 20% of the cells were dead after 72 h of treatment, responders (cell death >10%) and non-responders (cell death showing a modest efficacy. Multiple studies previously reported <10%) and their corresponding values of 1%priming were used cancer cells’ adaptation to therapy and the key role of the anti- to perform the analysis. Synergies were calculated using the apoptotic BCL-2 family proteins in their resistance to cell death Bliss Independent model (Foucquier and Guedj, 2015) were the (Reed et al., 1996; Mansoori et al., 2017; Maji et al., 2018; Combination Index (CI) is calculated as CI = (CDA + CDB – Wei et al., 2020). We next sought to study the role of these CDA ∗ CDB )/CDcombination (CD stands for the percentage of proteins after trametinib treatment on NALM-6. We repeated cell death after treatment A, B or the combination of them). the DBP analyses, but instead of using the BIM BH3 peptide, Combinations with CI < 1 were considered synergistic. we used peptides mimicking the sensitizer members of the GraphPad Prism 9 was used to perform statistical analyses and BCL-2 family to specifically identify the anti-apoptotic proteins’ generate graphs. contribution. In this case, we used a low concentration of the Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance FIGURE 1 | Cytotoxicity induced by targeted drugs in BCP-ALL cell lines. (A) Graphical scheme of the DBP technique where cancer cells are plated and treated for 16 h. After drug exposure, cells are plated in 96-well plates and exposed to the different BH3 peptides. After 1 h, cells were fixed and stained with an anti-Cytochrome C antibody overnight. Finally, analyses were performed using a flow cytometer for drug-response curves. Designed with BioRender.com. (B) DBP with BIM BH3 peptide after 16 h of incubation with 1,000 nM imatinib, 100 nM dasatinib, 1,000 nM sunitinib, 100 nM trametinib, 100 nM ruxolitinib, and 1,000 nM ibrutinib in NALM-6 and SEM cell lines. 1%priming stands for the difference in %priming between treatment and control conditions. (C) Cytotoxicity expressed as percentage of dead cells after 72 h of treatment with the same therapies assessed by Annexin V/DAPI staining. All results are expressed as the mean ± standard error of the mean (SEM) of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test comparing to control condition and considering *p < 0.05 and **p < 0.01. BAD BH3 peptide, due to the exquisite sensitivity of both BCP- block anti-apoptotic proteins. When we sequentially combined ALL cell lines to this peptide. In brief, an increase in apoptotic trametinib with the BCL-2 inhibitor ABT-199 (venetoclax), the priming after incubation with BAD BH3 peptide would mean BCL-xL inhibitor A-1331852 or the MCL-1 inhibitor S63845, we that BCL-2 and perhaps BCL-xL are involved in cell resistance observed enhanced cytotoxicity in all the combinations tested to trametinib. Similarly, a gain in apoptotic priming with HRK compared to single agent treatment. However, the dual inhibition BH3 would indicate an enhanced BCL-xL contribution, while of MEK and MCL-1 was significantly more effective and clearly an MS1 BH3 signal increase would point to MCL-1. When we synergistic (CI = 0.28), achieving almost a complete elimination analyzed NALM-6 treated with trametinib, we observed a positive of these cells (Figure 2B). signal from all sensitizer peptides (Figure 2A), suggesting that We next examined the molecular mechanism behind the multiple anti-apoptotic proteins may be involved in cell survival strong trametinib and S63845 synergy. The BCL-2 family of after therapy. We next studied how to overcome this acquired proteins is a complex interactome, thus explaining the observed resistance by preincubating the cells with trametinib and then effectiveness could be challenging. It is well reported that adding specific BH3 mimetics – small molecules that specifically trametinib treatment leads to an increase of the activator Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance FIGURE 2 | Trametinib synergizes with S63845 in NALM-6 cells, increasing BIM and MCL-1 protein expression. (A) DBP results using sensitizer peptides to study anti-apoptotic dependence of BCL-2 and BCL-xL with BAD 0.1 µM, BCL-xL with HRK 100 µM and MCL-1 with MS1 10 µM after 16 h of incubation with trametinib 100 nM in NALM-6 cell line. (B) Cytotoxicity was assessed by Annexin V/DAPI staining after 96 h incubation with trametinib 100 nM, ABT-199 100 nM, A-1331852 100 nM and S63845 1,000 nM in NALM-6 cell line. BH3 mimetics in combination with trametinib were added 16 h after treatment. (C) Western blot analysis for anti-apoptotic and BIM proteins in NALM-6 cells after 16 h of treatment with trametinib 100 nM. Quantification of optical density for each protein was normalized to actin, and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± standard error of the mean (SEM) of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01. Significance was also calculated comparing combination conditions with both single agents and considering #p < 0.05 and ##p < 0.01. Combination Index (CI) is indicated on top of every combination where CI < 1 indicates synergy. BIM, which was also detected in the NALM-6 cell line phosphorylation (Supplementary Figure 2A), and avoided its (Figure 2C). MEK inhibition using trametinib caused a proteasomal degradation (O’Reilly et al., 2009). This increase reduction of ERK1/2 phosphorylation, that also reduced BIM in BIM causes priming for apoptosis, yet the anti-apoptotic Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance members of the BCL-2 family could sequester this protein displaced when S63845 was sequentially added (Figure 3C). and block the initiation of apoptosis. When we analyzed the These findings confirm MCL-1 as the main pro-survival protein anti-apoptotic proteins expression, we found that this MEK and correlate with the observed synergy (Figure 2B). inhibitor selectively promoted MCL-1 increase, while BCL-2 MCL-1 preferentially blocks the increase of BIM protein and BCL-xL levels remained unchanged (Figure 2C). In the after trametinib exposure, and when sequentially inhibited with case of the effector proteins, BAX was not detected in this the BH3 mimetic S63845, over 80% of the cells succumbed to cell line and BAK slightly increased after trametinib treatment this synergistic combination (Figure 2B). Interestingly, when (Supplementary Figure 2B). To confirm that MCL-1 is the main we combined trametinib with ABT-199 or A-1331852 only a protein binding to BIM, we decided to immunoprecipitate it modest cytotoxic effect was observed (Figure 2B). Based on these and study their interaction. MCL-1 was detected in the normal findings, we conclude that MCL-1 is the main anti-apoptotic rabbit-IgG unbound fraction but not in the anti-MCL-1 antibody protein against trametinib-induced apoptosis in NALM-6, and condition (Figure 3A), confirming that we were able to effectively that BCL-2 and BCL-xL play a minor role. pull it down. As previously observed, trametinib treatment caused an increased binding of MCL-1 with BIM, and S63845 caused a marked increase in MCL-1 (Figure 3B) due to protein Combination of Low-Dose Sunitinib and stabilization, as described elsewhere (Kotschy et al., 2016; Li et al., BH3 Mimetics Synergize to Maintain 2019; Montero et al., 2019). As expected, the interaction between Treatment Efficacy MCL-1 and BIM in the IP fraction increased when treated with Secondary effects resulting from anti-cancer therapy trametinib compared to control, and was almost completely are particularly threating for the pediatric population FIGURE 3 | Synergy of trametinib and S63845 in NALM-6 is explained by an increased interaction between MCL-1 and BIM. (A) Western blot analysis of the unbound fraction after MCL-1 immunoprecipitation. (B) Immunodetection of MCL-1 and BIM initial expression in cell lysates after 16 h of incubation with trametinib 100 nM, and 2 h of incubation with S63845 1,000 nM in the specified conditions. Quantification of optical density for each protein was normalized to actin and fold-change was calculated comparing to protein expression in the control condition. (C) Western blot of the immunoprecipitated fraction was used to study the interaction between MCL-1 and BIM after treatment with trametinib 100 nM and 2 h with S63845 1,000 nM. To quantify this binding, BIM optical density was normalized to MCL-1 optical density and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01. Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance (Sarosiek et al., 2017). As a result, there is a trend to substitute a combination with BH3 mimetics that could synergize with high-dose single agent treatment for low-dose combinations sunitinib, currently explored in pre-clinical investigations for targeting different vulnerabilities to maximize efficacy and adult and pediatric BCP-ALL (Brown et al., 2005; Griffith reduce toxicity (Satti, 2009). In this regard, we sought to find et al., 2016). In the SEM cell line, sunitinib, administered as a FIGURE 4 | Sunitinib synergizes with ABT-199 and S63845 in SEM cells but anti-apoptotic proteins are downregulated. (A) DBP results using sensitizers peptides to study anti-apoptotic dependence of BCL-2 and BCL-xL with BAD 0.1 µM, BCL-xL with HRK 100 µM and MCL-1 with MS1 10 µM after 16 h of incubation with sunitinib 1,000 nM in SEM cell line. (B) Cytotoxicity assessed by Annexin V/DAPI staining after 96 h of sunitinib 100 nM, ABT-199 10 nM, A-1331852 100 nM and S63845 100 nM exposure in SEM cells. BH3 mimetics in combination with sunitinib were added 16 h after treatment. (C) Western blot analysis for anti-apoptotic and BIM proteins in SEM cells after 16 h of treatment with sunitinib 100 nM and sunitinib 1,000 nM. Quantification of optical density for each protein was normalized to actin, and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01. Significance was also calculated comparing combination conditions with both single agents and considering #p < 0.05 and ##p < 0.01. CI is indicated on top of every combination where CI < 1 indicates a synergistic combination. Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance single agent, killed around 60% of the cells (Figure 1C), but we followed the previous approach and immunoprecipitated BCL- aimed to improve its efficacy. By performing DBP analyses, we 2, that was effectively pulled down (Figure 6A), and BIM found that sunitinib incubation, enhanced BAD, HRK and MS1 increase was observed after treatment with sunitinib in the sensitizer BH3 peptides priming, also suggesting a diversified cell lysates (Figure 6B). BCL-2 was clearly detected in the anti-apoptotic adaptation to this targeted therapy (Figure 4A). immunoprecipitated fraction and we could observe an increased We then explored reducing 10-fold the concentration of binding between BCL-2 and BIM in the SEM cells when sunitinib, aiming to diminish the potential secondary effects treated; and this interaction was displaced when sequentially in the clinic, and combining with BH3 mimetics to boost administering ABT-199 (Figure 6C). These results suggest that its anti-cancer efficacy. Two BH3 mimetics, ABT-199 and the increase in BIM expression and the induction of apoptosis S63845, also induced high levels of cytotoxicity as single agents after low-dose sunitinib treatment is neutralized preferentially by (Supplementary Figure 3), but were innocuous when lowering BCL-2 and MCL-1. When sequentially inhibiting BCL-2 or MCL- 10-fold their concentration (Figure 4B). To improve the 1 with the corresponding low-dose BH3 mimetic, BIM is then performance of the low-dose sunitinib treatment, we combined displaced and the start of the apoptotic process is inevitable – the it with three BH3 mimetics targeting different anti-apoptotic other anti-apoptotic proteins cannot prevent it. proteins. As anticipated by DBP, when blocking any of the three anti-apoptotic proteins studied following sunitinib treatment, Pediatric BCP-ALL PDX Recapitulates we found a significant increase in cytotoxicity. This synergistic SEM Anti-apoptotic Adaptation effect was especially notable when combining it with low-dose We next aimed to confirm these anti-apoptotic adaptations ABT-199 (CI = 0.73) or S63845 (CI = 0.74) (Figure 4B). using a PDX ALL sample derived from a pediatric BCP-ALL We hypothesized that the molecular mechanism explaining patient with the same KMT2A-rearrangement (KMT2A/AFF1) these combinations could be similar to the one found in NALM-6 present in the SEM cell line (Benito et al., 2015). Following after trametinib administration. Similarly, we observed a marked the same experimental conditions, we shortly incubated these increase in BIM expression after sunitinib treatment (Figure 4C), cells with the same panel of targeted therapies and studied by as previously described for other types of tumors (Yang et al., DBP the apoptotic induction in the blast population. Similarly as 2010). Interestingly, sunitinib also reduced ERK1/2 and BIM previously described for the cell line, we observed a significant phosphorylation (Supplementary Figure 4A), suggesting that 1%priming increase with the BIM peptide after sunitinib this inhibitor also affects MAPK pathway, as previously described treatment but a modest increase with trametinib. The other for other cancer types (Chahal et al., 2010; Fenton et al., 2010). targeted therapies did not produce any perceptible apoptotic Sunitinib clearly increases apoptotic priming in the SEM cell line; changes (Figure 7). Interestingly, we also observed an increase yet, it also activates the anti-apoptotic machinery to neutralize in priming with all three sensitizer peptides (BAD, HRK and the increase of pro-apoptotic proteins. When we analyzed the MS1) after sunitinib exposure and analogous protein expression BCL-2 family components, in contrast to what we observed in changes (Supplementary Figure 5), suggesting similar anti- NALM-6, there was a minor decrease in BCL-2 and a significant apoptotic adaptations as the ones observed in SEM cells reduction of MCL-1 expression (Figure 4C). No significant (Figure 7), which correlates with the akin genetic background. changes were observed for BAX and BAK (Supplementary Figure 4B). Surprisingly, these results seemed to antagonize the observed synergies with ABT-199 or S63845. DISCUSSION As already mentioned, when exposed to a perturbation like a cytotoxic agent, cancer cells may adapt using anti-apoptotic Pediatric and young adult BCP-ALL patients present an overall proteins. However, in this case, a counterintuitive decrease in survival of 90% (Möricke et al., 2010; Pui et al., 2015). these proteins was detected. To further elucidate how SEM cells However, the minor subset of R/R cases do worse and display survive sunitinib, we immunoprecipitated MCL-1 (Figure 5A) to a poor outcome to therapy (Einsiedel et al., 2005; Ko et al., study its interaction with BIM. As expected, cell lysates showed a 2010), consolidating ALL as the first cause of death for significant lower expression of MCL-1 and an increase in BIM pediatric cancer. Reinduction therapy for the most advanced after sunitinib treatment, and the stabilization of MCL-1 after cases often include the same treatments previously used but S63845 (Figure 5B). Interestingly, we observed that sunitinib at a much higher concentrations (Raetz and Bhatla, 2012); treatment, even if the overall MCL-1 expression decreased, which is not only ineffective but also increases detrimental promoted and increase of its binding to BIM. Then, when MCL- secondary effects. There is a clear need for new and effective 1 was blocked with S63845, BIM was displaced and apoptosis treatments to improve the survival in those patients that do was then restored (Figure 5C). These findings explain why the not respond to actual chemotherapy regimens. Multiple targeted combination of sunitinib and S63845 synergize. Similarly as therapies have been proposed as potential candidates to treat before, BCL-2 and BCL-xL could not neutralize BIM after MCL-1 different subtypes of BCP-ALL that present genetic targetable inhibition to prevent cell death. vulnerabilities (Kuhlen et al., 2019). Chemotherapy relies on the We aimed to further investigate the other significant “one size fits all” concept in which all patients are treated in synergy detected between sunitinib and the BCL-2 inhibitor the same way, but the inclusion of targeted therapies requires ABT-199. In contrast to MCL-1, BCL-2 expression slightly a personalized medicine approach, where individual patient decreased after exposing them to sunitinib (Figure 4C). We tumors are first studied to find a driving oncogene to be Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance FIGURE 5 | Increased binding of BIM to MCL-1 causes sunitinib and S63845 synergism. (A) Western blot analysis of the unbound fraction after MCL-1 immunoprecipitation. (B) Immunodetection of MCL-1 and BIM initial expression in cell lysates after 16 h of incubation with sunitinib 100 nM, and 2 h incubation with S63845 100 nM in the specified conditions. Quantification of optical density for each protein was normalized to actin and fold-change was calculated comparing to protein expression in the control condition. (C) Western blot of the immunoprecipitated fraction was used to study the interaction between MCL-1 and BIM after treatment with sunitinib 100 nM and 2 h with S63845 100 nM. To quantify this binding, BIM optical density was normalized to MCL-1 optical density and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01. exploited pharmacologically. Despite recent advances in genetic identified the MEK inhibitor trametinib as the most effective screening and current efforts to implement precision medicine in the young adult NALM-6 cell line. NALM-6 presents a to treat pediatric patients (Ginsburg and Phillips, 2018), there mutation in NRAS, and downstream activation of the MAPK is a clear complication: childhood cancers present less genetic pathway, that may explain its sensitivity to trametinib (Irving alterations compared to adults. More precisely, they present a et al., 2014). In another pediatric BCP-ALL cell line, named very low frequency of somatic mutations, thus reducing the SEM, DBP predicted trametinib and sunitinib effectiveness number of predictive biomarkers and novel targeted therapies that was later confirmed by cell death measurements – the (Gröbner et al., 2018). latter was particularly active as single agent. Sunitinib has The lack of genetic biomarkers is boosting functional assays been proposed as a potential candidate to treat FLT3-driven that directly expose cancer cells to selected potential treatment hematological malignancies (Ikezoe et al., 2006), an alteration to measure their efficacy. We and others have demonstrated that is present in the SEM cell line (Brown et al., 2005; that DBP is a useful technology to screen and identify Gu et al., 2011). These results demonstrate DBP’s capacity to effective treatments for many types of cancer, including pediatric functionally identify effective targeted agents without requiring (Montero et al., 2015, 2017, 2019; Townsend et al., 2016; Pallis genetic information, which we believe would help personalize et al., 2017; Grundy et al., 2018; Seyfried et al., 2019; Alcon R/R BCP-ALL patients treatment. et al., 2020; Foley, 2020). We performed DBP in two BCP- Since the approval of venetoclax for chronic lymphocytic ALL cell lines by exposing them to potential targeted therapies leukemia, BH3 mimetics have bloomed as potential treatments for this type of leukemia. From all the treatments tested, we for multiple types of cancer, predominantly hematological Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance FIGURE 6 | BCL-2 binds to BIM after sunitinib treatment promoting synergy with ABT-199. (A) Western blot analysis of the unbound fraction after BCL-2 immunoprecipitation. (B) Immunodetection of BCL-2 and BIM initial expression in cell lysates after 16 h of incubation with sunitinib 100 nM, and 4 h incubation with ABT-199 10 nM. Quantification of optical density for each protein was normalized to actin and fold-change was calculated comparing to protein expression in the control condition. (C) Western blot of the immunoprecipitated fraction to study the interaction between BCL-2 and BIM after sunitinib 100 nM treatment, and 4 h with ABT-199 10 nM. To quantify this binding, BIM optical density was normalized to BCL-2 optical density and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01. (Valentin et al., 2018). Despite impressive experimental and of these cancer cells. Similarly, sunitinib synergized with ABT- clinical results as single agents, increasing evidence showed that 199 and S63845 in the SEM cell line, despite reducing 10- BH3 mimetics real potential is enhancing other anti-cancer fold its concentration. Importantly, all these combinations were agents, both conventional chemotherapy and targeted (Montero anticipated by DBP after only a short incubation with the targeted and Letai, 2018; Oudenaarden et al., 2018; Savona and Wei, therapy, demonstrating the utility of this functional assay as 2019; Lin et al., 2020). Numerous studies have demonstrated predictive biomarker for synergistic combinations of anti-cancer that cancer cells often rely on anti-apoptotic proteins to acquire agents with BH3 mimetics. resistance to therapy, and BH3 mimetics can effectively block Anti-apoptotic adaptations in response to therapy may appear these adaptations (Hata et al., 2015; Maji et al., 2018). However, by multiple cellular processes. When we analyzed the BCL-2 with these new therapeutic strategies we face the same problem family of proteins after trametinib and sunitinib treatment, in described for targeted therapies: when and how to correctly both cases we observed a significant increase of pro-apoptotic use these BH3 mimetics in the clinic. We previously described BIM expression, priming cells for apoptosis. Its stabilization that DBP can identify anti-apoptotic adaptations after treatment after kinase inhibitors use has been previously reported and and guide effective combinations with BH3 mimetics to boost related to the loss of ERK1/2 phosphorylation (Cragg et al., therapy’s potency (Montero et al., 2019; Alcon et al., 2020). 2008; Tan et al., 2013; Elgendy et al., 2017), as we confirmed When we applied it to BCP-ALL, we found that trametinib for both agents. Some reports also point out that sunitinib as single agent only produced a modest cytotoxic effect on may cause its accumulation by inhibiting STAT3 and AKT NALM-6 cells, but when sequentially combined with the MCL- (Xin et al., 2009; Yang et al., 2010). Interestingly, changes 1 inhibitor S63845, it reached an almost complete elimination in anti-apoptotic BCL-2 family members were very different Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance FIGURE 7 | PDX cells presenting KMT2A rearrangement show a similar DBP profile as the SEM cells. DBP with BIM BH3, BAD BH3, HRK BH3, and NOXA BH3 peptides after 16 h of incubation with 1,000 nM imatinib, 100 nM dasatinib, 1,000 nM sunitinib, 100 nM trametinib, 100 nM ruxolitinib and 1,000 nM ibrutinib in PDX cells. 1%priming stands for the difference in %priming between treatment and control condition. All results are expressed as the mean ± standard deviation of two technical replicates. when exposed to both kinase inhibitors. Trametinib increased consideration that venetoclax is already approved for clinical MCL-1 expression to neutralize BIM, as previously described use and that multiple MCL-1 inhibitors are currently explored (Korfi et al., 2016). Although MEK inhibition was reported to in clinical trials, we believe that these synergistic combinations enhance BCL-2/xL inhibitors cell death induction in BCP-ALL that we here describe could likely improve R/R BCP-ALL patient by Korfi and colleagues, as we also confirmed, as far as we know treatment and clinical outcomes. this is the first time that it is shown that trametinib strongly synergizes with MCL-1 inhibition in this disease. In contrast, sunitinib promoted BIM binding to BCL-2 and MCL-1, despite DATA AVAILABILITY STATEMENT the overall expression decrease of these pro-survival proteins. Even if the adaptation mechanism was different, we could also The raw data supporting the conclusions of this article will overcome it by sequentially adding a BH3 mimetics, venetoclax be made available by the authors upon request without or S63845, to this targeted agent, demonstrating the therapeutic undue reservation. potential of these molecules. We validated these results using a pediatric BCP-ALL PDX sample obtained from a patient presenting the same KMT2A rearrangement as the SEM cell line ETHICS STATEMENT (Bhimani et al., 2020), detecting similar therapeutic responses. The animal study was reviewed and approved by Tierversuch Nr. DBP identified sunitinib as the most effective agent inducing 1260, Regierungspräsidium Tübingen. apoptotic priming; correlating with the results observed in the SEM cell line, but contrasting to NALM-6 cells that present a different genetic background. Furthermore, we observed similar AUTHOR CONTRIBUTIONS anti-apoptotic adaptations when exposed to this kinase inhibitor, predicting that low-dose sunitinib combination with ABT-199 AM-M performed all experiments under supervision of CA and or S63845 could enhance the therapeutic effect for this patient. JM. PM provided the cell lines and important advice. FS, K-MD, Altogether, these results demonstrate that DBP could be used and LM provided the BCP-ALL PDX cells and related relevant as companion diagnostic tool to stratify R/R BCP-ALL cases information. MR provided his expertise in the field. JS and JM and identify the optimal combination of targeted therapies with supervised the work. AM-M and JM wrote the manuscript with BH3 mimetics to maximize anti-cancer efficacy while decreasing the contributions from all authors. All authors contributed to the undesired secondary effects. Although sunitinib was previously article and approved the submitted version. characterized as a potential treatment for BCP-ALL as a FLT3 inhibitor (Griffith et al., 2016), to our knowledge this is the first time that it is described in combination with BH3 mimetics to FUNDING treat this disease. In summary, these findings could represent three new JM acknowledges the Ramon y Cajal Program, Ministerio de potential therapeutic strategies for BCP-ALL. Taking into Economia y Competitividad (RYC-2015-18357) and the Spanish Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance National Plan “Retos Investigacion” I + D + i (RTI2018-094533- Supplementary Figure 1 | (A) ROC curve analysis using the values of A-I00) from Ministerio de Ciencia, Innovación y Universidades. 1%priming in NALM-6 and SEM cell lines establishing 10% as the cell death threshold for responders and non-responders. (B) Correlation between This work was supported by the CELLEX foundation and the 1%priming and %cell death analyses. Networking Biomedical Research Center (CIBER), Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Supplementary Figure 2 | (A) Western blot analysis of phospho-ERK1/2 and phospho-BIM in NALM-6 cell line after 16 h of treatment with trametinib 100 nM. Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, (B) Western blot analysis of BAK and BAX in NALM-6 cell line after 16 h of and the Instituto de Salud Carlos III (RD16/0006/0012), with the treatment with trametinib 100 nM. Quantification of optical density for each protein support of the European Regional Development Fund (ERDF). was normalized to actin, and fold-change was calculated comparing to protein This work was also partially funded by the CERCA Program expression in the control condition. All results are expressed as the mean ± SEM and by the Commission for Universities and Research of the of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering Department of Innovation, Universities, and Enterprise of the ∗ p < 0.05 and ∗∗ p < 0.01. Generalitat de Catalunya (2017 SGR 1079). FS: Medical Faculty of Ulm University (Clinician Scientist Programme). K-MD and Supplementary Figure 3 | ABT-199 and S63845 induce cytotoxicity in the SEM cell line. Cytotoxicity expressed as percentage of dead cells after 72 h of treatment LM: German Research Foundation (DFG, SFB 1074). with 100 nM ABT-199 and 1,000 nM S63845, as assessed by an Annexin V/DAPI staining. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering ∗ p < 0.05 and ∗∗ p < 0.01. ACKNOWLEDGMENTS Supplementary Figure 4 | (A) Western blot analysis of phospho-ERK1/2 and phospho-BIM in the SEM cell line after 16 h of treatment with sunitinib 100 nM We would like to thank to the Cytometry Facility from the and 1,000 nM. (B) Western blot analysis of BAK and BAX in SEM cell line after University of Barcelona for assistance with flow cytometry 16 h of treatment with sunitinib 100 nM and 1,000 nM. Quantification of optical experiments and Virginia Rodriguez for helping with the cell lines density for each protein was normalized to actin, and fold-change was calculated used in this manuscript. comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering ∗ p < 0.05 and ∗∗ p < 0.01. Supplementary Figure 5 | BCL-2 family of proteins expression in BCP-ALL PDX SUPPLEMENTARY MATERIAL cells after sunitinib treatment. Western blot analysis for anti-apoptotic and BIM proteins in BCP-ALL PDX cells after 16 h of treatment with sunitinib 100 nM and The Supplementary Material for this article can be found sunitinib 1,000 nM. Quantification of optical density for each protein was online at: https://www.frontiersin.org/articles/10.3389/fcell.2021. normalized to actin, and fold-change was calculated comparing to protein 695225/full#supplementary-material expression in the control condition. REFERENCES Cragg, M. S., Jansen, E. S., Cook, M., Harris, C., Strasser, A., and Scott, C. L. (2008). Treatment of B-RAF mutant human tumor cells with a MEK inhibitor Alcon, C., Manzano-Muñoz, A., Prada, E., Mora, J., Soriano, A., Guillén, G., requires Bim and is enhanced by a BH3 mimetic. J. Clin. Invest. 118, 3651–3659. et al. (2020). Sequential combinations of chemotherapeutic agents with BH3 doi: 10.1172/JCI35437 mimetics to treat rhabdomyosarcoma and avoid resistance. Cell Death Dis. Deininger, M. W. (2008). Milestones and monitoring in patients with CML treated 11:634. doi: 10.1038/s41419-020-02887-y with imatinib. Hematology Am. Soc. Hematol. Educ. Program. 2008, 419–426. Benito, J. M., Godfrey, L., Kojima, K., Hogdal, L., Wunderlich, M., Geng, H., et al. doi: 10.1182/asheducation-2008.1.419 (2015). MLL-rearranged acute lymphoblastic leukemias activate BCL-2 through Deng, J., Isik, E., Fernandes, S. M., Brown, J. R., Letai, A., and Davids, M. S. H3K79 methylation and are sensitive to the BCL-2-specific antagonist ABT-199. (2017). Bruton’s tyrosine kinase inhibition increases BCL-2 dependence and Cell Rep. 13, 2715–2727. doi: 10.1016/j.celrep.2015.12.003 enhances sensitivity to venetoclax in chronic lymphocytic leukemia. Leukemia Bhimani, J., Ball, K., and Stebbing, J. (2020). Patient-derived xenograft models— 31, 2075–2084. doi: 10.1038/leu.2017.32 the future of personalised cancer treatment. Br. J. Cancer 122, 601–602. doi: Ding, Y. Y., Stern, J. W., Jubelirer, T. F., Wertheim, G. B., Lin, F., Chang, F., 10.1038/s41416-019-0678-0 et al. (2018). Clinical efficacy of ruxolitinib and chemotherapy in a child with Biondi, A., Schrappe, M., De Lorenzo, P., Castor, A., Lucchini, G., Gandemer, philadelphia chromosome-like acute lymphoblastic leukemia with GOLGA5- V., et al. (2012). Imatinib after induction for treatment of children JAK2 fusion and induction failure. Haematologica 103, e427–e431. doi: 10.3324/ and adolescents with Philadelphia-chromosome-positive acute lymphoblastic haematol.2018.192088 leukaemia (EsPhALL): a randomised, open-label, intergroup study. Lancet Einsiedel, H. G., Von Stackelberg, A., Hartmann, R., Fengler, R., Schrappe, M., Oncol. 13, 936–945. doi: 10.1016/S1470-2045(12)70377-7 Janka-Schaub, G., et al. (2005). Long-term outcome in children with relapsed Brown, L. M., Hanna, D. T., Khaw, S. L., and Ekert, P. G. (2017). Dysregulation ALL by risk-stratified salvage therapy: results of trial acute lymphoblastic of BCL-2 family proteins by leukemia fusion genes. J. Biol. Chem. 292, 14325– leukemia-relapse study of the berlin-frankfurt-Münster Group 87. J. Clin. 14333. doi: 10.1074/jbc.R117.799056 Oncol. 23, 7942–7950. doi: 10.1200/JCO.2005.01.1031 Brown, P., Levis, M., Shurtleff, S., Campana, D., Downing, J., and Small, D. (2005). Elgendy, M., Abdel-Aziz, A. K., Renne, S. L., Bornaghi, V., Procopio, G., FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells Colecchia, M., et al. (2017). Dual modulation of MCL-1 and mTOR determines with high levels of FLT3 expression. Blood 105, 812–820. doi: 10.1182/blood- the response to sunitinib. J. Clin. Invest. 127, 153–168. doi: 10.1172/JCI8 2004-06-2498 4386 Chahal, M., Xu, Y., Lesniak, D., Graham, K., Famulski, K., Christensen, J. G., Fenton, M. S., Marion, K. M., Salem, A. K., Hogen, R., Naeim, F., and Hershman, et al. (2010). MGMT modulates glioblastoma angiogenesis and response to the J. M. (2010). Sunitinib inhibits MEK/ERK and SAPK/JNK pathways and tyrosine kinase inhibitor sunitinib. Neuro. Oncol. 12, 822–833. doi: 10.1093/ increases sodium/iodide symporter expression in papillary thyroid cancer. neuonc/noq017 Thyroid 20, 965–974. doi: 10.1089/thy.2010.0008 Frontiers in Cell and Developmental Biology | www.frontiersin.org 13 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance Foley, J. F. (2020). Revealing the plasma membrane in GPCR signaling. Sci. Signal. leukemia: a therapeutic advances in childhood leukemia consortium study. 13:eaay1451. doi: 10.1126/scisignal.aay1451 J. Clin. Oncol. 28, 648–654. doi: 10.1200/JCO.2009.22.2950 Foucquier, J., and Guedj, M. (2015). Analysis of drug combinations: current Korfi, K., Smith, M., Swan, J., Somervaille, T. C. P., Dhomen, N., and Marais, R. methodological landscape. Pharmacol. Res. Perspect. 3:e00149. doi: 10.1002/ (2016). BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia prp2.149 cells by BCL-2 and MEK inhibitors. Cell Death Dis. 7, e2177–e2177. doi: 10. Frenzel, A., Grespi, F., Chmelewskij, W., and Villunger, A. (2009). Bcl2 family 1038/cddis.2016.70 proteins in carcinogenesis and the treatment of cancer. Apoptosis 14, 584–596. Kotschy, A., Szlavik, Z., Murray, J., Davidson, J., Maragno, A. L., Le Toumelin- doi: 10.1007/s10495-008-0300-z Braizat, G., et al. (2016). The MCL1 inhibitor S63845 is tolerable and effective Ginsburg, G. S., and Phillips, K. A. (2018). Precision medicine: from science to in diverse cancer models. Nature 538, 477–482. doi: 10.1038/nature19830 value. Health Aff. 37, 694–701. doi: 10.1377/hlthaff.2017.1624 Kuhlen, M., Klusmann, J. H., and Hoell, J. I. (2019). Molecular Approaches to Gore, L., Kearns, P. R., de Martino Lee, M. L., De Souza, C. A., Bertrand, Y., Hijiya, treating pediatric leukemias. Front. Pediatr 7:368. doi: 10.3389/fped.2019.00368 N., et al. (2018). Dasatinib in pediatric patients with chronic myeloid leukemia Letai, A. (2011). The control of mitochondrial apoptosis by the BCL-2 family. in chronic phase: results from a phase II trial. J. Clin. Oncol. 36, 1330–1338. Apoptosis Physiol. Pathol. 122, 44–50. doi: 10.1017/CBO9780511976094.005 doi: 10.1200/JCO.2017.75.9597 Letai, A., Bassik, M. C., Walensky, L. D., Sorcinelli, M. D., Weiler, S., and Griffith, M., Griffith, O. L., Krysiak, K., Skidmore, Z. L., Christopher, M. J., Klco, Korsmeyer, S. J. (2002). Distinct BH3 domains either sensitize or activate J. M., et al. (2016). Comprehensive genomic analysis reveals FLT3 activation mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell and a therapeutic strategy for a patient with relapsed adult B-lymphoblastic 2, 183–192. doi: 10.1016/S1535-6108(02)00127-7 leukemia. Exp. Hematol. 44, 603–613. doi: 10.1016/j.exphem.2016.04.011 Li, Z., He, S., and Look, A. T. (2019). The MCL1-specific inhibitor S63845 acts Gröbner, S. N., Worst, B. C., Weischenfeldt, J., Buchhalter, I., Kleinheinz, K., synergistically with venetoclax/ABT-199 to induce apoptosis in T-cell acute Rudneva, V. A., et al. (2018). The landscape of genomic alterations across lymphoblastic leukemia cells. Leukemia 33, 262–266. doi: 10.1038/s41375-018- childhood cancers. Nature 555, 321–327. doi: 10.1038/nature25480 0201-2 Grundy, M., Seedhouse, C., Jones, T., Elmi, L., Hall, M., Graham, A., et al. Lin, V. S., Xu, Z. F., Huang, D. C. S., and Thijssen, R. (2020). Bh3 mimetics for the (2018). Predicting effective pro-apoptotic anti-leukaemic drug combinations treatment of b-cell malignancies—insights and lessons from the clinic. Cancers using cooperative dynamic BH3 profiling. PLoS One 13:e0190682. doi: 10.1371/ (Basel) 12:3353. doi: 10.3390/cancers12113353 journal.pone.0190682 Locatelli, F., Schrappe, M., Bernardo, M. E., and Rutella, S. (2012). How i treat Gu, T. L., Nardone, J., Wang, Y., Loriaux, M., Villén, J., Beausoleil, S., et al. relapsed childhood acute lymphoblastic leukemia. Blood 120, 2807–2816. doi: (2011). Survey of activated FLT3 signaling in leukemia. PLoS One 6:e19169. 10.1182/blood-2012-02-265884 doi: 10.1371/journal.pone.0019169 Maji, S., Panda, S., Samal, S. K., Shriwas, O., Rath, R., Pellecchia, M., et al. Hata, A. N., Engelman, J. A., and Faber, A. C. (2015). The BCL2 family: key (2018). Bcl-2 antiapoptotic family proteins and chemoresistance in Cancer. mediators of the apoptotic response to targeted anticancer therapeutics. Cancer Adv. Cancer Res. 137, 37–75. doi: 10.1016/bs.acr.2017.11.001 Discov. 5, 475–487. doi: 10.1158/2159-8290.CD-15-0011 Malone, E. R., Oliva, M., Sabatini, P. J. B., Stockley, T. L., and Siu, L. L. (2020). Howard, C. M., Valluri, J., and Claudio, P. P. (2017). Functional drug response Molecular profiling for precision cancer therapies. Genome Med. 12:8. doi: assay for cancer stem cells in the era of precision medicine. Transl. Med. Reports 10.1186/s13073-019-0703-1 1, 7–10. doi: 10.4081/tmr.6421 Mansoori, B., Mohammadi, A., Davudian, S., Shirjang, S., and Baradaran, B. (2017). Howlader, N., Noone, A., Krapcho, M., Miller, D., Bishop, K., Altekruse, S., et al. The different mechanisms of cancer drug resistance: a brief review. Adv. Pharm. (2015). SEER cancer statistics review. Natl. Cancer Inst. Available online at: Bull. 7, 339–348. doi: 10.15171/apb.2017.041 https://seer.cancer.gov/csr/1975_2017/ (accessed March 29, 2021). Mathur, S., and Sutton, J. (2017). Personalized medicine could transform healthcare Iacovelli, S., Ricciardi, M. R., Allegretti, M., Mirabilii, S., Licchetta, R., Bergamo, (Review). Biomed. Rep. 7, 3–5. doi: 10.3892/br.2017.922 P., et al. (2015). Co-targeting of Bcl-2 and mTOR pathway triggers synergistic Meijer, T. G., Naipal, K. A., Jager, A., and Van Gent, D. C. (2017). Ex vivo tumor apoptosis in BH3 mimetics resistant acute lymphoblastic leukemia. Oncotarget culture systems for functional drug testing and therapy response prediction. 6, 32089–32103. doi: 10.18632/oncotarget.5156 Future Sci. OA 3:FSO190. doi: 10.4155/fsoa-2017-0003 Ikezoe, T., Nishioka, C., Tasaka, T., Yang, Y., Komatsu, N., Togitani, K., et al. Meyer, L. H., Eckhoff, S. M., Queudeville, M., Kraus, J. M., Giordan, M., Stursberg, (2006). The antitumor effects of sunitinib (formerly SU11248) against a variety J., et al. (2011). Early relapse in all is identified by time to leukemia in of human hematologic malignancies: enhancement of growth inhibition via NOD/SCID mice and is characterized by a gene signature involving survival inhibition of mammalian target of rapamcycin signaling. Mol. Cancer Ther. 5, pathways. Cancer Cell 19, 206–217. doi: 10.1016/j.ccr.2010.11.014 2522–2530. doi: 10.1158/1535-7163.MCT-06-0071 Miller, D. R. (2006). A tribute to sidney farber – the father of modern Irving, J., Matheson, E., Minto, L., Blair, H., Case, M., Halsey, C., et al. (2014). Ras chemotherapy. Br. J. Haematol. 134, 20–26. doi: 10.1111/j.1365-2141.2006. pathway mutations are prevalent in relapsed childhood acute lymphoblastic 06119.x leukemia and confer sensitivity to MEK inhibition. Blood 124, 3420–3430. Mody, R., Li, S., Dover, D. C., Sallan, S., Leisenring, W., Oeffinger, K. C., doi: 10.1182/blood-2014-04-531871 et al. (2008). Twenty-five-year follow-up among survivors of childhood acute Jameson, J. L., and Longo, D. L. (2015). Precision medicine-personalized, lymphoblastic leukemia: a report from the childhood cancer survivor study. problematic, and promising. Obstet. Gynecol. Surv. 70, 612–614. doi: 10.1097/ Blood 111, 5515–5523. doi: 10.1182/blood-2007-10-117150 01.ogx.0000472121.21647.38 Montero, J., Gstalder, C., Kim, D. J., Sadowicz, D., Miles, W., Manos, M., et al. Jerchel, I. S., Hoogkamer, A. Q., Ariës, I. M., Steeghs, E. M. P., Boer, J. M., Besselink, (2019). Destabilization of NOXA mRNA as a common resistance mechanism to N. J. M., et al. (2018). RAS pathway mutations as a predictive biomarker targeted therapies. Nat. Commun. 10:5157. doi: 10.1038/s41467-019-12477-y for treatment adaptation in pediatric B-cell precursor acute lymphoblastic Montero, J., and Letai, A. (2018). Why do BCL-2 inhibitorswork and where should leukemia. Leukemia 32, 931–940. doi: 10.1038/leu.2017.303 we use them in the clinic? Cell Death Differ. 25, 56–64. doi: 10.1038/cdd.2017. Khaw, S. L., Suryani, S., Evans, K., Richmond, J., Robbins, A., Kurmasheva, R. T., 183 et al. (2016). Venetoclax responses of pediatric all xenografts reveal sensitivity Montero, J., Sarosiek, K. A., Deangelo, J. D., Maertens, O., Ryan, J., Ercan, D., et al. of MLL-rearranged leukemia. Blood 128, 1382–1395. doi: 10.1182/blood-2016- (2015). Drug-induced death signaling strategy rapidly predicts cancer response 03-707414 to chemotherapy. Cell 160, 977–989. doi: 10.1016/j.cell.2015.01.042 Kim, E., Hurtz, C., Koehrer, S., Wang, Z., Balasubramanian, S., Chang, B. Y., Montero, J., Stephansky, J., Cai, T., Griffin, G. K., Cabal-Hierro, L., Togami, K., et al. (2017). Ibrutinib inhibits pre-BCR+ B-cell acute lymphoblastic leukemia et al. (2017). Blastic plasmacytoid dendritic cell neoplasm is dependent on BCL2 progression by targeting BTK and BLK. Blood 129, 1155–1165. doi: 10.1182/ and sensitive to venetoclax. Cancer Discov. 7, 156–164. doi: 10.1158/2159-8290. blood-2016-06-722900 CD-16-0999 Ko, R. H., Ji, L., Barnette, P., Bostrom, B., Hutchinson, R., Raetz, E., et al. (2010). Möricke, A., Zimmermann, M., Reiter, A., Henze, G., Schrauder, A., Gadner, H., Outcome of patients treated for relapsed or refractory acute lymphoblastic et al. (2010). Long-term results of five consecutive trials in childhood acute Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 September 2021 | Volume 9 | Article 695225 Manzano-Muñoz et al. MCL-1 Inhibition Overcomes BCP-ALL Resistance lymphoblastic leukemia performed by the ALL-BFM study group from 1981 Seyfried, F., Demir, S., Hörl, R. L., Stirnweiß, F. U., Ryan, J., Scheffold, A., et al. to 2000. Leukemia 24, 265–284. doi: 10.1038/leu.2009.257 (2019). Prediction of venetoclax activity in precursor B-ALL by functional Ni Chonghaile, T., Roderick, J. E., Glenfield, C., Ryan, J., Sallan, S. E., Silverman, assessment of apoptosis signaling. Cell Death Dis. 10:571. doi: 10.1038/s41419- L. B., et al. (2014). Maturation stage of T-cell acute lymphoblastic leukemia 019-1801-0 determines BCL-2 versus BCL-XL dependence and sensitivity to ABT-199. Tan, N., Wong, M., Nannini, M. A., Hong, R., Lee, L. B., Price, S., et al. Cancer Discov. 4, 1074–1087. doi: 10.1158/2159-8290.CD-14-0353 (2013). Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition O’Brien, M. M., Seif, A. E., and Hunger, S. P. (2018). Acute lymphoblastic leukemia alone and in combination with PI3 kinase inhibition in lung and pancreatic in children. Wintrobes Clin. Hematol. Fourteenth Ed. 373, 4939–5015. doi: 10. tumor models. Mol. Cancer Ther. 12, 853–864. doi: 10.1158/1535-7163.MCT-1 1056/nejmra1400972 2-0949 O’Reilly, L. A., Kruse, E. A., Puthalakath, H., Kelly, P. N., Kaufmann, T., Huang, Townsend, E. C., Murakami, M. A., Christodoulou, A., Christie, A. L., Köster, D. C. S., et al. (2009). MEK/ERK-mediated phosphorylation of bim is required J., DeSouza, T. A., et al. (2016). The public repository of Xenografts enables to ensure survival of T and B lymphocytes during mitogenic stimulation. discovery and randomized phase II-like trials in mice. Cancer Cell 29, 574–586. J. Immunol. 183, 261–269. doi: 10.4049/jimmunol.0803853 doi: 10.1016/j.ccell.2016.03.008 Oskarsson, T., Söderhäll, S., Arvidson, J., Forestier, E., Frandsen, T. L., Hellebostad, Valentin, R., Grabow, S., and Davids, M. S. (2018). The rise of apoptosis: targeting M., et al. (2018). Treatment-related mortality in relapsed childhood acute apoptosis in hematologic malignancies. Blood 132, 1248–1264. doi: 10.1182/ lymphoblastic leukemia. Pediatr. Blood Cancer 65:e26909. doi: 10.1002/pbc. blood-2018-02-791350 26909 Wei, M. C., Zong, W. X., Cheng, E. H. Y., Lindsten, T., Panoutsakopoulou, V., Oudenaarden, C. R. L., van de Ven, R. A. H., and Derksen, P. W. B. (2018). Re- Ross, A. J., et al. (2001). Proapoptotic BAX and BAK: a requisite gateway inforcing the cell death army in the fight against breast cancer. J. Cell Sci. to mitochondrial dysfunction and death. Science 292, 727–730. doi: 10.1126/ 131:jcs212563. doi: 10.1242/jcs.212563 science.1059108 Pallis, M., Burrows, F., Ryan, J., Grundy, M., Seedhouse, C., Abdul-Aziz, A., et al. Wei, Y., Cao, Y., Sun, R., Cheng, L., Xiong, X., Jin, X., et al. (2020). Targeting Bcl-2 (2017). Complementary dynamic BH3 profiles predict co-operativity between proteins in acute myeloid leukemia. Front. Oncol. 10:584974. doi: 10.3389/fonc. the multi-kinase inhibitor TG02 and the BH3 mimetic ABT-199 in acute 2020.584974 myeloid leukaemia cells. Oncotarget 8, 16220–16232. doi: 10.18632/oncotarget. Wu, S. C., Li, L. S., Kopp, N., Montero, J., Chapuy, B., Yoda, A., et al. 8742 (2015). Activity of the type II JAK2 inhibitor CHZ868 in B cell acute PDQ Pediatric Treatment Editorial Board. (2002). Childhood Acute Lymphoblastic lymphoblastic leukemia. Cancer Cell 28, 29–41. doi: 10.1016/j.ccell.2015. Leukemia Treatment (PDQ R ): Health Professional Version. Available online at: 06.005 http://www.ncbi.nlm.nih.gov/pubmed/26389206 (accessed April 14, 2021). Xin, H., Zhang, C., Herrmann, A., Du, Y., Figlin, R., and Yu, H. (2009). Sunitinib Pui, C. H., Yang, J. J., Hunger, S. P., Pieters, R., Schrappe, M., Biondi, A., inhibition of Stat3 induces renal cell carcinoma tumor cell apoptosis and et al. (2015). Childhood acute lymphoblastic leukemia: progress through reduces immunosuppressive cells. Cancer Res. 69, 2506–2513. doi: 10.1158/ collaboration. J. Clin. Oncol. 33, 2938–2948. doi: 10.1200/JCO.2014.59.1636 0008-5472.CAN-08-4323 Raetz, E. A., and Bhatla, T. (2012). Where do we stand in the treatment of relapsed Yang, F., Jove, V., Xin, H., Hedvat, M., Van Meter, T. E., and Yu, H. (2010). acute lymphoblastic leukemia? Hematology Am. Soc. Hematol. Educ. Program. Sunitinib induces apoptosis and growth arrest of medulloblastoma tumor cells 2012, 129–136. doi: 10.1182/asheducation.v2012.1.129.3800156 by inhibiting STAT3 and AKT signaling pathways. Mol. Cancer Res. 8, 35–45. Reed, J. C., Miyashita, T., Takayama, S., Wang, H. G., Sato, T., Krajewski, S., doi: 10.1158/1541-7786.MCR-09-0220 et al. (1996). BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J. Cell. Biochem. 60, 23–32. Conflict of Interest: JM reports previous consulting for Vivid Biosciences and doi: 10.1002/(SICI)1097-4644(19960101)60:1<23::AID-JCB5<3.0.CO;2-5 Oncoheroes Biosciences, and his laboratory is currently collaborating with Ryan, J., Montero, J., Rocco, J., and Letai, A. (2016). IBH3: simple, fixable BH3 AstraZeneca. profiling to determine apoptotic priming in primary tissue by flow cytometry. Biol. Chem. 397, 671–678. doi: 10.1515/hsz-2016-0107 The remaining authors declare that the research was conducted in the absence of Sarosiek, K. A., Fraser, C., Muthalagu, N., Bhola, P. D., Chang, W., McBrayer, S. K., any commercial or financial relationships that could be construed as a potential et al. (2017). Developmental regulation of mitochondrial apoptosis by c-Myc conflict of interest. Governs age- and tissue-specific sensitivity to cancer therapeutics. Cancer Cell 31, 142–156. doi: 10.1016/j.ccell.2016.11.011 Publisher’s Note: All claims expressed in this article are solely those of the authors Satti, J. (2009). The emerging low-dose therapy for advanced cancers. Dose- and do not necessarily represent those of their affiliated organizations, or those of Response 7, 208–220. doi: 10.2203/dose-response.08-010.satti the publisher, the editors and the reviewers. 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