ARTICLE - Non-Hodgkin Lymphoma Resistance to PI3Kδ inhibitors in marginal zone lymphoma can be reverted by targeting the IL-6/PDGFRA axis Alberto J. Arribas,1,2* Sara Napoli,1* Luciano Cascione,1,2 Giulio Sartori,1 Laura Barnabei,1 Eugenio Gaudio,1 Chiara Tarantelli,1 Afua Adjeiwaa Mensah,1 Filippo Spriano,1 Antonella Correspondence: A. Arribas Zucchetto,3 Francesca M. Rossi,3 Andrea Rinaldi,1 Manuel Castro de Moura,4 Sandra Jovic,5 alberto.arribas@ior.usi.ch Roberta Bordone-Pittau,6 Alessandra Di Veroli,7 Anastasios Stathis,6,8 Gabriele Cruciani,7 F. Bertoni Georg Stussi,6 Valter Gattei,3 Jennifer R. Brown,9 Manel Esteller,4,10-12 Emanuele Zucca,1,6 francesco.bertoni@ior.usi.ch Davide Rossi1,6 and Francesco Bertoni1,6 Received: September 4, 2021. Accepted: April 12, 2022. 1 Institute of Oncology Research, Faculty of Biomedical Sciences, Università della Svizzera Prepublished: April 28, 2022. Italiana, Bellinzona, Switzerland; 2SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland; 3Centro di Riferimento Oncologico di Aviano – CRO, Aviano, Italy; 4Josep https://doi.org/10.3324/haematol.2021.279957 Carreras Leukemia Research Institute (IJC), Badalona, Spain; 5Institute for Research in ©2022 Ferrata Storti Foundation Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland; 6Oncology Institute Published under a CC BY-NC license of Southern Switzerland, Bellinzona, Switzerland; 7Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; 8Faculty of Biomedical Sciences, Università della Svizzera Italiana, Bellinzona, Switzerland; 9Chronic Lymphocytic Leukemia Center, Division of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 10Centro de Investigacion Biomedica en Red Cancer (CIBERONC), Madrid, Spain; 11Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain and 12Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Spain. *AJA and SN contributed equally as co-first authors. Abstract PI3Kδ inhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here we present a model of secondary resistance to PI3Kδ inhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed character- ization of the model, pharmacological screens, silencing experiments, and validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation data highlighted an enrichment of upregulated transcripts and low-methylated promoters in resistant cells, including IL-6/STAT3- and PDGFRA-related genes and surface CD19 expression, alongside the repression of the let-7 family of miRNA, and miR-125, miR-130, miR-193 and miR-20. The IL-6R blocking antibody to- cilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3Kδ inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the antitumor activity of PI3Kδ inhibitors in B-cell lymphoid tumors. naling.1,2 Indeed, its inhibition is being extensively explored Introduction as a therapeutic approach for patients with lymphoid neo- The delta isoform of phosphoinositide kinase (PI3Kδ) plasms.1-3 Idelalisib was the first-in-class specific PI3Kδ shows prominent expression across hematopoietic tis- inhibitor to show clinical activity as a single agent in pa- sues and has a central role in B-cell receptor (BCR) sig- tients with follicular lymphoma, marginal zone lymphoma Haematologica | 107 November 2022 2685 ARTICLE - PI3K resistance A. Arribas et al. (MZL), chronic lymphocytic leukemia and mantle cell further experiments. Cell line identity was periodically lymphoma, and in combination with rituximab in pa- authenticated by short tandem repeat DNA profiling, as tients with chronic lymphocytic leukemia,1,2 and it re- previously described.14 Cells were periodically tested to ceived the approval from the U.S. Food and Drug confirm Mycoplasma negativity using the MycoAlert My- Administration (FDA) for the treatment of patients with coplasma Detection Kit (Lonza, Visp, Switzerland). relapsed follicular lymphoma or small lymphocytic lym- phoma after at least two prior systemic therapies, and, Treatments in combination with rituximab, for patients with re- Response to single drugs or drug combinations was as- lapsed chronic lymphocytic leukemia.3 Idelalisib has sessed after 72 h of exposure to increasing doses of the been successfully followed by a series of second-gen- drug followed by an MTT assay. Copanlisib, duvelisib, eration PI3Kδ inhibitors, such as parsaclisib and zandeli- umbralisib, everolimus, bimiralisib, vincristine, 5-azaciti- sib, and by compounds that inhibit additional kinases, dine, masitinib, stattic and tocilizumab were purchased such as copanlisib (PI3Kα/PI3Kδ), duvelisib (PI3Kδ/PI3Kg) from Selleckchem, Lin28-1632 (LIN1632) from R&D Sys- and umbralisib (PI3Kδ and casein kinase-1ε), achieving tems (Minneapolis, MN, USA), and human recombinant clinical responses in lymphomas, including follicular interleukin 6 (IL-6; CYT-213) from Prospec (Rehovot, Is- lymphoma, mantle cell lymphoma, MZL and chronic rael). Loncastuximab tesirine was kindly provided by lymphocytic leukemia.1-3 In particular, data have so far ADC Therapeutics (Epalinges, Switzerland). Details are led to the FDA approval of copanlisib for patients with provided in the Online Supplementary Methods. relapsed/refractory follicular lymphoma, duvelisib for patients with relapsed/refractory chronic lymphocytic Genomics and data mining leukemia, small lymphocytic lymphoma or follicular lym- Details of the genomics studies and data mining are pro- phoma, and umbralisib for the treatment of patients vided in the Online Supplementary Methods. with relapsed/refractory MZL or follicular lymphoma.3 The identification of the mechanisms underlying either Enzyme-linked immunosorbent assays primary or secondary resistance to PI3Kδ inhibitors is For enzyme-linked immunosorbent assays (ELISA) the fundamental to optimize the use of these drugs, and a conditioned medium was cultured for 72 h and col- number of studies have described mechanisms of resis- lected. The medium was then filtered twice (22 µm) and tance to this class of agents in lymphoid neoplasms, centrifuged at 4000 rpm for 30 minutes in Amicon Ultra- driven by activation of alternative signaling cascades.4-11 4 tubes (Ultracel 3k, Merk Millipore) to remove cells and Here we present a model of secondary resistance to particles, and analyzed via cytokine array or ELISA. Cyto- PI3Kδ inhibitors, including duvelisib, copanlisib and um- kine arrays (Human Cytokine Array Panel A, R&D Sys- bralisib, obtained by prolonged exposure of a splenic tems) were performed according to the manufacturer’s MZL cell line to idelalisib. protocols. ELISA on frozen human serum samples were performed using a Luminex Assay (R&D Systems) ac- cording to the manufacturer’s protocols. The serum samples were collected from patients treated with ide- Methods lalisib and enrolled on tissue banking protocols at Dana Development of resistant cell lines Farber Cancer Institute; all patients signed written in- VL51 cells were cultured according to the recommended formed consent prior to a sample being drawn, and the conditions, as previously described.12 To develop resis- protocols were approved by the Dana-Farber Harvard tance cell lines were exposed to a 90% inhibitory con- Cancer Center Institutional Review Board. centration (IC90) of idelalisib (Selleckchem, Houston, TX, USA) for several months until they acquired specific Flow cytometry and protein analyses drug resistance (resistant cells). In parallel, cells were Flow cytometry was performed to determine the surface cultured under similar conditions in the absence of drug expression of platelet-derived growth factor receptor A (parental cells). Proliferation of stable resistance was (PDGFRA), IL-6 receptor (IL-6R), IL-6 cytokine family sig- tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl nal transducer (IL-6ST), CXCR4 and CD19 (Online Sup- tetrazolium bromide (MTT) assay after 2 weeks of drug- plementary Table S1), levels of p-AKT, p-BTK, p-PLCG2, free culture. The multidrug resistance phenotype was p-mTOR and p-ERK (Online Supplementary Table S2); assessed by real-time polymerase chain reaction for and cell cycle phases. Immunoblotting was performed MDR1 and MDR2/3 genes using published primers.13 We to determine the expression of AKT/p-AKT, ERK/p-ERK developed biological replicates by splitting the resistant JAK/p-JAK, STAT/p-STAT and GAPDH (Online Supplemen- clones 1 month after the development of resistance, tary Table S3). Details are provided in the Online Sup- keeping them separate for 6 months before performing plementary Methods. Haematologica | 107 November 2022 2686 ARTICLE - PI3K resistance A. Arribas et al. Pharmacological combination screening the Online Supplementary Methods. Pharmacological screening was performed by exposing idelalisib-resistant cells in parallel with parental clones to 348 compounds, as single agents (1 µM) or in combina- tion with idelalisib (1 µM), from a custom library containing Results agents belonging to the following classes: “Kinase In- A splenic marginal zone lymphoma model of secondary hibitory”, “Epigenetic Compound”, “PI3K/Akt Inhibitor”, resistance to PI3Kδ inhibitors “Apoptosis”, “Anti-cancer Compound”, and “MAPK In- To create a novel model of resistance to the PI3Kδ in- hibitor” (Selleckchem). Cell viability was measured by MTT hibitors, we selected the splenic MZL-derived VL51 cell assay after 72 h of exposure to the agents. line, with transcriptome, somatic mutational status, DNA profile and methylome comparable to that of primary Gene silencing splenic MZL samples.15,16 VL51 harbors a series of lesions Details of the gene silencing experiments are provided in that are typical of splenic MZL, including truncating mu- A B C D E F G Figure 1. Profiles of drug sensitivity differ between parental and resistant cell lines. (A-G) Acquired resistance was tested by an MTT assay (72 h) in parental (black) and resistant (red) cells of the VL51 cell line. Drug sensitivity was evaluated in resistant and parental cells for the PI3K inhibitors idelalisib (A), copanlisib (B), duvelisib (C), umbralisib (D), the dual PI3K-mTOR inhibitor bimi- ralisib (E), the mTOR inhibitor everolimus (F) and the chemotherapy agent and the multidrug resistance substrate vincristine (G). Error bars correspond to standard deviation of the mean. Data are derived from at least three independent experiments. P values from a Z-test, statistically significant for values <0.05. Haematologica | 107 November 2022 2687 ARTICLE - PI3K resistance A. Arribas et al. tations of NOTCH2 and BIRC3, which activate downstream miRNA) and methylation, as shown by unsupervised ana- NOTCH and non-canonical NF-κB, respectively, and a nu- lyses and at the pharmacological profiling (Figure 1, Online clear localization signal mutation of KLF2.15,17,18 Supplementary Figures S2 and S11), but no exonic nonsyn- After 6 months of continuous exposure to the FDA-ap- onymous variants appeared to be acquired in the resistant proved PI3Kδ inhibitor idelalisib, VL51 developed stable re- cells. Online Supplementary Table S4 shows the single and sistance with an IC50 25-fold higher than that of its copy number variants identified by whole-exome se- parental counterpart (Figure 1A). The resistance was con- quencing in resistant cells compared to parental cells. firmed to be stable by repeating the IC50 measurements after 2 weeks in medium containing no drug, and multi- Resistance to idelalisib is driven by IL-6 and PDGFRA drug resistance was ruled out by demonstrating no To investigate the mechanism of resistance, we integrated changes in the expression levels of MDR1/2 genes by semi- the gene expression (gene expression profiling, RNA-se- quantitative real-time polymerase chain reaction (Online quencing), miRNA (RNA-sequencing) and methylation Supplementary Figure S1). (800k Illumina array) profiles obtained in resistant and The idelalisib-resistant cell line also showed resistance parental cells (Online Supplementary Table S4). When to other FDA-approved PI3K inhibitors, including the compared to its parental counterpart, the resistant cell PI3Kα/PI3Kδ inhibitor copanlisib, the PI3Kδ/PI3Kg inhibitor line was enriched in BCR/TLR/NF-κB (TLR4, CD19, SYK), IL- duvelisib, and the PI3Kδ/CK1ε inhibitor umbralisib (Figure 6/STAT3 (IL-6, CD44), chemokines (CXCL10, CXCR4, CXCR3), 1B-D). Conversely, they maintained sensitivity to the mTOR PDGFR (PDGFRA, PRKCE), IGF1R and RAS-RAF signaling inhibitor everolimus, to the dual PI3K/mTOR inhibitor bimi- pathways, epigenetic signatures (PRC2-complex targets ralisib (Figure 1E, F) and to the chemotherapy agent and and methylated genes in cancer) and genes upregulated MDR substrate vincristine (Figure 1G). in MZL. On the other hand, it showed decreased ex- Resistant cells clearly differed from their parental pression of transcripts involved in amino acid deprivation, counterparts based on RNA-sequencing (total RNA, antigen processing, drug metabolism, translation, proteo- Figure 2. Multi-omics signature of the VL51, resistant cell line identifies activation of the IL-6-PDGFRA axis. Heatmap of RNA (gene expression profiling), methylation and miRNA profiles of resistant cells compared to parental cells. Heatmap values rep- resent the differences between resistant and parental cells: fold change (log2 for RNA and miRNA) or D β-value (methylation), red for enrichment in resistant cells and blue for parental cells. Columns correspond to gene expression (gene expression profiling, RNA-sequencing) and methylation (MethylationEPIC BeadChip, Illumina) profiles of the top ten upregulated and top ten down- regulated genes; rows represent the differently expressed miRNA (RNA-sequencing) with values in the columns corresponding to the targeted gene. *Statistically significant differences (moderated t-test). GEP: gene expression profile; FC: fold change. Haematologica | 107 November 2022 2688 ARTICLE - PI3K resistance A. Arribas et al. some and hypoxia (Online Supplementary Figures S2B and were enriched. Integration of methylation and miRNA data S3, Online Supplementary Table S5). The integrative analy- identified fully methylated and repressed miRNA known sis of transcriptome and methylation data highlighted an to target upregulated genes: members of the let-7 family enrichment of upregulated transcripts among the low- of miRNA (let-7d, let-7e, let-7g) that directly target IL-6,19 methylated promoters in resistant cells, including IL-6 miR-125a that regulates IL-6, IL-6R and STAT3,20 and the and RAS-related genes (Figure 2, Online Supplementary negative regulators of PDGFR signaling, miR-130a,21 miR- Figure S3). Analysis of the miRNA profiles identified re- 193b, miR-20b and miR-1722,23 (Figure 2, Online Supplemen- pression of members of the let-7 family (let-7e, let-7g, let- tary Table S5). These data indicated that an epigenetic 7d, let-7f, let-7a), along with other miRNA including reprogramming could sustain the observed resistance. miR-125, miR-130, miR-193 and miR-20. Conversely, only Having observed an upregulation of the secreted factor one member of the let-7 family (let-7c) was among the IL-6 at the RNA level in resistant cells, we evaluated upregulated miRNA in resistant cells, together with miR- whether the transfer of conditioned media to parental 3196, miR-4492, miR-4516 and others (Online Supplemen- cells was able to transfer the resistance to PI3K inhibitors. tary Table S5). Among the repressed miRNA in resistant The media from resistant cells (taken at 48 h), but not cells, miRNA potentially targeting the IL-6-PDGFRA axis media taken from other cells, including parental cells, A B C Figure 3. Proteome profiles of resistant cells differ from those of parental cells. (A) Cell viability, determined by the MTT assay, of parental cells (black line) exposed to idelalisib in the presence or not of conditioned medium from VL51 idelalisib-resistant cells (red line), cultured for 48 h, and parental + RES-cond RPMI (dotted black line). Data were derived from the average of three independent experiments. (B) Expression of surface PDGFRA (top), CXCR4 (center) and CD19 (bottom) by fluorescence activated cell sorting in parental (gray) and resistant (red) cell lines. The dotted black line represents the negative control (neg-cnt). Data were derived from two independent experiments. (C) Levels of protein phosphorylation by immunoblot in resistant cells. Values correspond to average fold-change of resistant compared to parental cells in two independent experiments. Data were normal- ized to GAPDH levels. Error bars represent standard error of the mean. *Statistically significant differences (t-test). MFI: mean fluorescence intensity; FACS: fluorescence activated cell sorting. Haematologica | 107 November 2022 2689 ARTICLE - PI3K resistance A. Arribas et al. conferred resistance (Figure 3A). The secretion of IL-6 was stattic decreased cell viability and enhanced sensitivity to confirmed by ELISA (Online Supplementary Figure S4), and idelalisib in resistant cells, with minimal effects in par- the overexpression of surface PDGFRA, CXCR4 and CD19 ental cells (Online Supplementary Figure S10). was shown by flow cytometry (Figure 3B, Online Supple- Since members of the let-7 family were downregulated in mentary Figure S5). Increased levels of p-ERK (immunob- the resistant cells, we evaluated LIN1632, an inhibitor of lot and phosphoFlow) and p-STAT (immunoblot) were LIN28, an RNA binding protein involved in the downregu- observed in resistant cells (Figure 3C, Online Supplemen- lation of such miRNA. While the compound had no activity tary Figures S6 and S7). as a single agent, addition of LIN1632 to idelalisib im- While silencing of individual genes had only partial effects, proved the activity of the PI3Kδ inhibitor (1 µM) (Figure 5A), the concomitant silencing of both IL-6 and PDGFRA by and increased the expression of let-7 in resistant cells but siRNA reverted the resistance (Figure 4A, Online Supple- not in parental ones (Online Supplementary Figure S11). mentary Figure S8). Consistent with the effect of IL-6 si- While a low dose of LIN1632 (1 µM) was effective only in lencing, exposure of parental cells to recombinant IL-6 resistant cells, higher doses of LIN1632 (50 µM) increased induced resistance (Figure 4B). expression of let-7 in parental cells as well, confirming the activity of the LIN28/let-7 axis in VL51 cells (Online Targeting IL-6/STAT3, LIN28, or PDGFRA reverts the Supplementary Figure S11B). resistance to idelalisib Screening with 348 anticancer agents and compounds We then explored pharmacological approaches to over- targeting important biological pathways identified ac- come the acquired resistance. Based on IL-6 involvement quired sensitivity of the resistant cells to the PDGFR in- we tested tocilizumab, an IL-6R blocking antibody ap- hibitor masitinib (Online Supplementary Figure S12). The proved by the FDA for the treatment of various auto- efficacy of idelalisib was enhanced by the presence of immune disorders and cytokine release syndrome.24 The masitinib (500 nM) only in resistant cells and not in par- addition of tocilizumab to idelalisib overcame the resis- ental ones (Figure 5B, Online Supplementary Figure S13). tance increasing the potency (i.e., the minimal active con- centration) of the small molecule in the resistant cells CD19 upregulation in resistant cells determines (Figure 4B). No advantage was given by adding the anti- increased sensitivity to CD19-targeting agents body in parental cells. Tocilizumab as a single agent Due to the increased CD19 (RNA and surface) expression showed limited cytotoxicity in both parental and resistant in the idelalisib-resistant VL51 cell line compared to its cells (Online Supplementary Figure S9). To investigate the parental counterpart (Figures 6A and 3B bottom, Online signaling activation in resistant cells downstream of IL-6, Supplementary Figure S5E), we evaluated a CD19-targeting we combined idelalisib with the STAT3 inhibitor stattic. treatment, namely the CD19-directed antibody-drug con- Consistent with the involvement of IL-6/STAT3 activation jugate loncastuximab tesirine (ADCT-402), recently ap- in resistant cells, and similarly to tocilizumab, addition of proved by the FDA for the treatment of patients with A B Figure 4. Interfering with IL-6 or PDFGRA overcomes resistance in the VL51 model. (A) Small interfering RNA were used for gene expression silencing of IL6 alone (brown dotted line), PDGFRA alone (orange dotted line) or concomitant silencing of IL6 and PDGFRA (yellow dotted line). Black and red lines for parental and resistant controls. *Statistically significant differences when compared to resistant control (red line, Z-test P<0.05). (B) Stimulation with recombinant IL-6 (30 ng/mL) conferred resistance to idelalisib in the parental cells (dotted black line) and blocking of IL-6 signaling with the monoclonal antibody tocilizumab (25 µg/mL) overcame resistance to idelalisib (red dotted line). Black and red continuous lines represent parental and resistant con- trols. *Statistically significant differences when compared to parental control (black continuous line, Z-test P<0.05). Sensitivity to all treatments was tested by an MTT assay at 72 h. Data were derived from three independent experiments, error bars represent standard deviation of the mean. Haematologica | 107 November 2022 2690 ARTICLE - PI3K resistance A. Arribas et al. A B Figure 5. Combination with LIN28 or PDFGRA inhibitors restores sensitivity to idelalisib. (A) Addition of 1 µM of the LIN28 inhibitor LIN1632 restored sensitivity to idelalisib in the resistant cell line with no effect in the parental cells and very limited sensitivity as a single agent for both parental and resistant cell lines (Online Supplementary Figure S11B). (B) The combination of idelalisib with 500 nM of the PDGFR inhibitor masitinib increased sensitivity to idelalisib in the resistant cells with limited benefit in the parental cells. Treatment with masitinib as a single agent was beneficial only in resistant cells but not in parental ones (Online Supplementary Figure S13B). Sensitivity to all treatments was tested by an MTT assay at 72 h. Data were derived from three in- dependent experiments, error bars represent standard deviation of the mean. *Statistically significant differences when compared to treatment with idelalisib as a single agent (black continuous line, Z-test P<0.05). relapsed/refractory large B-cell lymphoma.25 The idelal- the VL51 model, we first took advantage of a large series isib-resistant cells were much more sensitive than their of lymphoma cell lines we had previously characterized parental counterpart (Figure 6B). at transcriptome level and for their sensitivity to idelal- isib.26 IL6, PDGFRA and LIN28 expression levels were in- The detected mechanisms of resistance are not limited versely correlated with idelalisib sensitivity, while the to idelalisib or the VL51 model latter was positively correlated with let-7 and miR-125 We then investigated whether the mechanism of resis- levels also in these additional B-cell lymphoma models tance identified might affect sensitivity to other PI3K in- (P<0.05) (Figure 7). Second, we selected two B-cell lym- hibitors, such as duvelisib, umbralisib and copanlisib. phoma models, another splenic MZL SSK41 and the dif- Addition of the IL-6R blocking antibody tocilizumab, the fuse large B-cell lymphoma-derived RCK8, based on their STAT3 inhibitor stattic, the PDGFR inhibitor masitinib or low sensitivity to idelalisib and their expression of IL6R, the LIN28 inhibitor LIN1632 improved the anti-lymphoma PDGFRA and LIN28. We tested the response to idelalisib, activity not only of idelalisib but also of the other clini- duvelisib, umbralisib and copanlisib in combination with cally relevant PI3K inhibitors in resistant cells but not in the corresponding inhibitors tocilizumab (anti-IL-6R), the parental VL51 cell line (Online Supplementary Figure masitinib (PDGFR inhibitor) and LIN1632 (LIN28 inhibitor). S14A, B). Conversely, as observed for idelalisib in parental In line with what was observed in the VL51 model, both cells, stimulation with recombinant IL-6 decreased sen- SSK41 and RCK8 benefited from the addition of tocilizu- sitivity to duvelisib, umbralisib and copanlisib, which was mab, masitinib and LIN1632 (Online Supplementary Figure restored by the addition of tocilizumab (Online Supple- S15A, B). Third, the effect of IL-6 stimulation on the sen- mentary Figure S14C, D). sitivity to idelalisib and to the additional PI3K inhibitors To further extend the significance of our findings beyond duvelisib, umbralisib and copanlisib was evaluated in pri- Haematologica | 107 November 2022 2691 ARTICLE - PI3K resistance A. Arribas et al. A B Figure 6. Idelalisib-resistant cells exhibited increased sensitivity to an anti-CD19 treatment. (A) CD19 expression was increased in resistant (red) compared to parental (grey) cells, both at the mRNA level (shown by RNA-sequencing) and with regards to sur- face expression (shown by fluorescence activated cell sorting, Figure 3B bottom panel). (B) The response to loncastuximab tesirine (ADCT-402) was tested by an MTT assay at 72 h of exposure. Data were derived from three independent experiments, error bars represent standard deviation of the mean. *Statistically significant differences between parental (black) and resistant (red) cell lines (Z-test, P<0.05). RNA-seq: RNA sequencing mary idelalisib-sensitive B-cell lymphoma cell lines with peared expressed in two series of retrospective collec- expression of IL6R, including the mantle cell lymphoma tions of MZL clinical specimens and in a large series of models Granta519 and JVM2. Similarly to VL51 parental diffuse large B-cell lymphomas27-29 (Online Supplementary cells, stimulation with recombinant IL-6 decreased sen- Figure S17A-D). Taking advantage of the previously re- sitivity to all PI3K inhibitors, and addition of tocilizumab ported gene expression profile of splenic MZL clinical restored response to the drugs (Online Supplementary specimens,28 we determined the top 200 genes positively Figure S15C, D). correlated (Pearson correlation) with the expression of either IL6 or PDGFRA, defining IL6 and PDFGRA signatures. Exposure to epigenetic drugs improves sensitivity to When applied to our resistant model, the two signatures idelalisib were enriched among the transcripts more expressed in Based on the different epigenetic profiles observed in the resistant cells than in the parental VL51 cells (Online parental and resistant cells, we evaluated whether resis- Supplementary Figure S17E), highlighting the similarities tance may be reverted using epigenetic drugs. Due to the between our model and the clinical setting. enrichment in resistant cells for targets of the PRC2- Finally, secreted levels of IL-6 were evaluated in the complex and methylated genes in cancer (Online Supple- serum of patients treated with idelalisib, comparing mentary Figure S2B), we tested the combination of chronic lymphocytic leukemia patients with primary or idelalisib with the EZH2 inhibitor tazemetostat and the acquired resistance to the PI3Kδ inhibitor to patients re- demethylating agent 5-azacitidine. Resistant and parental sponding to the drug and paired for similar clinical fea- cells were exposed to 5-azacitidine or to tazemetostat tures (Online Supplementary Table S6). In agreement with given concomitantly with idelalisib or 5 days before the the in vitro data, all samples but one secreting IL-6 were PI3K inhibitor. The concomitant combination exhibited non-responders to idelalisib (Figure 8A). Longitudinal very limited effects in either parental or resistant cells analyses, comparing responders and non-responders to (Online Supplementary Figure S16A). Nevertheless, and idelalisib were carried out for those patients with IL-6 se- consistent with a mechanistic role of methylation in the cretion and available data along time. Non-responders resistance to idelalisib, pre-treatment with either 5-aza- showed increasing IL-6 levels upon treatment, while the citidine (200 nM) or tazemetostat (1 µM) was beneficial in paired responders remained with no IL-6 expression (Fig- resistant cells and not in parental ones (Online Supple- ure 8B). mentary Figure S16B). These data suggest that the secreted factors identified in vitro can be present in the tumor microenvironment The factors associated with resistance in cell lines are and that lymphoma cells might express the correspond- also relevant in clinical specimens ing receptors to take advantage of available chemokines. To extrapolate our findings to the clinical context, we Thus, although different mechanisms may drive resis- studied both available expression datasets and a series tance to PI3K inhibitors in clinical cases, the mechanism of serum samples. PDGFRA, IL-6 and IL-6 receptor ap- identified here might apply to some of these patients. Haematologica | 107 November 2022 2692 ARTICLE - PI3K resistance A. Arribas et al. Figure 7. Expression levels of crucial factors are correlated with resistance to idelalisib. IL6, PDGFRA, and LIN28 expression levels are inversely correlated with idelalisib sensitivity. Conversely, expression of miR-125a and members of the let-7 family of microRNA is associated with sensitivity to idelalisib. Expression and sensitivity data were analyzed from a previous publication by our group in a panel of 34 B-cell lymphoma cell lines.26 Cell lines were split into two groups based on higher (positive, black bars) or lower (negative, grey bars) values than the median expression of the corresponding gene or miRNA. Means of the idelalisib IC50 were calculated for these two groups and compared by a t-test. *P<0.05. IC50: half maximum inhibitory concentration. resistance to PI3K inhibitors by promoting the secretion Discussion of cytokines; and (ii) the resistance can be overcome We developed and characterized a model of secondary re- using various drugs, which could be tested in novel clinical sistance to PI3K inhibitors in splenic MZL. Our results in- trials. dicate that: (i) epigenetic reprogramming can drive Idelalisib was the first-in-class PI3Kδ inhibitor and several Haematologica | 107 November 2022 2693 ARTICLE - PI3K resistance A. Arribas et al. additional PI3K inhibitors with different selectivity, includ- elevated expression of EZH2, and enrichment of PRC2- ing duvelisib, copanlisib and umbralisib, have entered the complex targets are associated with a more aggressive clinical setting as single agents and in combinations.1-3 The clinical outcome.16 model of secondary resistance we developed via pro- The resistant cell line secreted interleukins and chemo- longed in vitro exposure to idelalisib presented decreased kines, such as IL-6, and showed upregulation of prosur- sensitivity to other PI3K inhibitors, such as copanlisib, du- vival networks, including PDGFRA, JAK-STAT and NF-κB velisib and umbralisib. The role of the identified factors in pathways. Our findings are in line with the notion that se- the resistance to PI3K inhibitors has been further vali- creted factors can give resistance to PI3K inhibitors,35,36 dated in additional B-cell lymphoma in vitro models and and with the background of the cell line we have used, across a large series of cell lines derived from different representative of the recently described NNK splenic MZL types of lymphoma, and their expression demonstrated in subgroup, driven by mutations in genes involved in NF- various clinical specimens, including the serum of idelal- κB/NOTCH/KLF2.37 IL-6 can protect cancer cells from isib-resistant patients. apoptosis and DNA damage induced by drugs and can de- Targeting epigenetic vulnerabilities has been recently pro- crease sensitivity to tyrosine kinase inhibitors by activat- posed as a valuable strategy to overcome resistance to ther- ing different signaling pathways, such as the JAK-STAT, apy in cancer, including resistance to PI3K inhibitors.30-33 AKT-mTOR and NF-κB signaling pathways.38 Release of IL- Epigenetic cell plasticity, which might be pharmacologi- 6 mediates resistance to the BTK inhibitor ibrutinib in cally reverted, allows the development of drug-tolerant Waldenström macroglobulinemia.39 to duvelisib and co- subpopulations even in the absence of genetic lesions or panlisib in diffuse large B-cell lymphoma and T-cell lym- can provide a first permissible environment for the phoma cells,35 and, in head and neck squamous cell emergence, later on, of cells carrying DNA changes.34 A carcinoma cell lines, blockade of IL-6 signaling overcomes relevant role for methylation has been suggested in resistance to the pan-PI3K inhibitor buparlisib.36 In our re- splenic MZL, in which a hypermethylation phenotype, sistant cell line, upregulation of IL-6 was associated with A B Figure 8. Levels of serum IL-6 are higher in idelalisib-resistant chronic lymphocytic leukemia patients than in idelalisib-sensitive ones. (A) IL-6 secretion was evaluated in serum samples from patients with chronic lymphocytic leukemia at the end of the treatment by enzyme-linked immunosorbent assay (Luminex, R&D Systems). Patients who responded to idelalisib (responders, blue) or did not (non-responders, red) were compared by a t-test. P represents adjusted P-value. (B) Longitudinal analyses were performed on clinically-paired patients with secretion of IL-6. Secretion of IL-6 was compared between patients not responding to idelalisib (red) and their corresponding matched controls (responding, blue). MFI: mean fluorescence intensity. ELISA: enzyme- linked immunosorbent assay. Haematologica | 107 November 2022 2694 ARTICLE - PI3K resistance A. Arribas et al. downregulation of let-7 and miR-125 microRNA, which di- activity to the CD19 targeting antibody drug conjugate lon- rectly target the interleukin,19 and with the loss of methyl- castuximab tesirine, recently approved by the FDA for the ation in the IL6 gene promoter. The latter was methylated treatment of patients with relapsed or refractory large B- in the parental cell line but not in resistant lines, and cell lymphoma.25 In the clinical routine, CD19 could be methylation-based repression of the IL-6-targeting easily investigated (by immunohistochemistry or flow miRNA, miR-125 and members of let-7 family, might con- cytometry) and its expression provides a rationale for tribute to the increased IL-6 expression. LIN28 is an RNA testing CD19-targeting antibody drug conjugates, naked binding protein that functions as an oncogene inhibiting antibodies, and cellular therapies in patients exposed to the expression of let-7 family microRNA, dysregulating the PI3K inhibitors. normal balance between differentiation and cell growth.40 In conclusion, a model of secondary resistance to PI3K in- Since IL-6 induces transcription of NF-κB targets, a posi- hibitors, derived from splenic MZL, has revealed mechan- tive feedback loop leads to B-cell activation under the isms of resistance to these drugs and allowed the control of LIN28 and let-7.19 The miRNA mir-125 is located identification of a first series of active therapeutic ap- at 19q13 and frequently downregulated or deleted in proaches that could be explored further. cancer, and it can also regulate cell proliferation by tar- geting p53.41 Loss of miR-125 leads to high expression of Disclosures STAT3, IL-6 itself and of a subunit of IL-6 receptor com- AJA has received a travel grant from Astra Zeneca. LC has plex (IL-6R).20 Hence, in the context of NF-κB and JAK- received a travel grant from HTG. LB is currently a part- STAT activation, miR-125 might work concomitantly with time employee of Bright Peak Therapeutics. AS has re- let-7. Alongside IL-6, PDGFRA too was upregulated in the ceived institutional research funds from Bayer, resistant cell line. Active PDGFRA signaling, as a con- ImmunoGen, Merck, Pfizer, Novartis, Roche, MEI Pharma, sequence of genomic aberrations affecting PDGFRA, is as- ADC-Therapeutics and travel grants from AbbVie and Phar- sociated with tumor development and progression in solid maMar. Valter Gattei has received research funding from cancer,42 it mediates the activation of PI3K-AKT, JAK-STAT Menarini SpA, laboratory activity fees from Janssen, and and RAS-ERK signaling,43 and it confers resistance to the scientific advisory board fees from AbbVie. JB is a consul- tyrosine kinase inhibitor imatinib.44 PDGFR signaling is tant for Abbvie, Acerta, Astra-Zeneca, Beigene, Catapult, known to increase IL-6/IL-6R axis expression,45 and, fur- Dynamo Therapeutics, Eli Lilly, Genentech/Roche, Gilead, thermore, PDGFR is also linked with CXCR4,46 upregulated Juno/Celgene/Bristol Myers Squibb, Kite, Loxo, MEI in the resistant cells. CXCR4 is upregulated after exposure Pharma, Nextcea, Novartis, Octapharma, Pfizer, Phar- to BCR inhibitors, including PI3K and BTK inhibitors26 and, macyclics, Rigel, Sunesis, TG Therapeutics and Verastem; in diffuse large B-cell lymphoma models, it has been sug- has received research funding from Gilead, Loxo, Sun, TG gested as a potential mechanism of resistance to the ty- Therapeutics and Verastem; and has served on data safety rosine kinase inhibitors themselves.5,6 monitoring committees for Invectys. EZ has received insti- Since a multitude of experiments, including genetic si- tutional research funds from Celgene, Roche and Janssen, lencing and use of IL-6 recombinant protein, indicated advisory board fees from Celgene, Roche, Mei Pharma, that the mechanism of resistance to PI3K inhibitors in the Astra Zeneca and Celltrion Healthcare and travel grants VL51 model was driven by activation of the IL-6/STAT3 and from Abbvie and Gilead; and has provided expert state- PDFGRA signaling cascades and was dependent on the ments to Gilead, Bristol-Myers Squibb and MSD. DR has re- action of LIN28, we explored possible therapeutic inter- ceived grant support from Gilead, AbbVie, and Janssen, ventions. Tocilizumab is an IL-6R blocking antibody in honoraria from Gilead, AbbVie Janssen, and Roche; and clinical use to treat different autoimmune disorders and scientific advisory board fees from Gilead, AbbVie, Janssen, more recently for the cytokine release syndrome that can AstraZeneca, and MSD. GS has received travel grants from be seen with chimeric antigen receptor T-cell therapy or Novartis, Celgene and Roche, consultancy fees from No- in patients with coronavirus disease 2019.24 Masitinib, a vartis, scientific advisory board fees from Bayer, Celgene, small molecule that inhibits PDGFR signaling and targets Janssen and Novartis, and speaker fees from Gilead. FB the innate immune system, is under clinical development has received institutional research funds from Acerta, ADC for various indications, including systemic mastocytosis, Therapeutics, Bayer AG, Cellestia, CTI Life Sciences, EMD solid tumors, and amyotrophic lateral sclerosis.47 The Serono, Helsinn, ImmunoGen, Menarini Ricerche, NEOMED combination with these two already clinically available Therapeutics 1, Nordic Nanovector ASA, Oncology Thera- drugs, and with the LIN28 inhibitor LIN1632 or with the peutic Development, and PIQUR Therapeutics AG, has re- STAT3 inhibitor stattic, two compounds in the preclinical ceived consultancy fees from Helsinn and Menarini, has phase of investigation, restored sensitivity to PI3K in- provided expert statements to HTG, and has received travel hibition. Moreover, the resistant cells had higher CD19 ex- grants from Amgen, Astra Zeneca, Jazz Pharmaceuticals pression on their cell surface, which gave much higher and PIQUR Therapeutics AG. The other authors have no- Haematologica | 107 November 2022 2695 ARTICLE - PI3K resistance A. Arribas et al. thing to disclose. research and edited the manuscript, FB designed research, interpreted data, and co-wrote the manuscript. All authors Contributions approved the final manuscript. AJA and SN contributed equally. AJA performed experi- ments, analyzed and interpreted data, performed data Funding mining, prepared the figures and co-wrote the manuscript; This study was supported in part by the Swiss National SN performed silencing experiments and interpreted data; Science Foundation (SNSF 31003A_163232/1) with funds to LC performed data mining; GS, EG, CT, AM, FS and LB per- EZ, DR and FB. JRB was supported by National Institutes formed experiments; AZ, FR, RBP, GS and VG performed of Health grant RO1 CA 213442 (PI: Jennifer Brown). flow-cytometry analyses; AR performed genomics experi- ments; MCM and ME performed methylation profiling ex- Data-sharing statement periments and data mining, SJ performed ELISA Luminex Genomics data will be available in the GEO database experiments; AS provided advice; JRB collected and char- (under submission). acterized tumor samples; EZ and DR jointly designed the References 1. Phillips TJ, Michot JM, Ribrag V. Can next-generation PI3K 13. Hoellein A, Decker T, Bogner C, et al. Expression of multidrug inhibitors unlock the full potential of the class in patients with resistance-associated ABC transporters in B-CLL is B-cell lymphoma? Clin Lymphoma Myeloma Leuk. independent of ZAP70 status. J Cancer Res Clin Oncol. 2021;21(1):8-20.e3. 2010;136(3):403-410. 2. Kienle DL, Stilgenbauer S. Approved and emerging PI3K 14. 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