Dual targeting of PI3K and BCL‐2 overcomes ibrutinib resistance in aggressive mantle cell lymphoma

Abstract Despite significant efficacy of ibrutinib therapy in mantle cell lymphoma (MCL), about one‐third of MCL patients will display primary resistance. In time, secondary resistance occurs almost universally with an unlikely response to salvage chemotherapy afterwards. While intense efforts are being directed towards the characterization of resistance mechanisms, our focus is on identifying the signalling network rewiring that characterizes this ibrutinib resistant phenotype. Importantly, intrinsic genetic, epigenetic and tumour microenvironment‐initiated mechanisms have all been shown to influence the occurrence of the ibrutinib resistant phenotype. By using in vitro and in vivo models of primary and secondary ibrutinib resistance as well as post‐ibrutinib treatment clinical samples, we show that dual targeting of the BCL‐2 and PI3‐kinase signalling pathways results in synergistic anti‐tumour activity. Clinically relevant doses of venetoclax, a BCL‐2 inhibitor, in combination with duvelisib, a PI3Kδ/γ dual inhibitor, resulted in significant inhibition of these compensatory pathways and apoptosis induction. Our preclinical results suggest that the combination of venetoclax and duvelisib may be a therapeutic option for MCL patients who experienced ibrutinib failure and merits careful consideration for future clinical trial evaluation.

would result in a synergistic anti-MCL activity and overcome BTKi resistance. In this study, we investigated the in vitro and in vivo efficacy of duvelisib and venetoclax combination in IR MCL cells.

| Primary patient samples
Patient samples were obtained through a protocol approved by the Institutional Review Board at MD Anderson Cancer Center.

| Cell viability, cell apoptosis assay and Western blotting
These assays were performed as described previously. 8

| Reverse phase protein array analysis
JeKo BTK KD cells were treated with duvelisib and/or venetoclax for 24 h and subjected to reverse phase protein array (RPPA) analysis as described previously. 8 Proteins with >twofold change between the combination and vehicle were selected to generate the heatmap.

| Stroma-mediated cell migration and viability assay
The bottom chamber of the Transwell system was pre-seeded with JeKo-1 cell viability in response to ibrutinib was tested in the presence and absence of HS-5 cells using a similar procedure but without the Transwell system. 2.7 | In vivo drug efficacy in mouse xenograft models 5 × 10 6 JeKo-Luc cells were injected subcutaneously into each 6-8-week-old NOD SCID IL2Rγ null (NSG) mice. Treatment was started 3 days following inoculation: vehicle, ibrutinib (50 mg/kg, oral, daily), venetoclax (50 mg/kg, oral, daily) and duvelisib (50 mg/ kg, oral, daily) alone or in combination for 3 weeks. Tumours were measured weekly by IVIS Spectrum In Vivo Imaging System (Perkin Elmer).

| Animal study approval
All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center.

| Statistics
Student's t-test was used to calculate statistical significance.

| Duvelisib and venetoclax in combination synergistically inhibited the cell growth of ibrutinibresistant MCL models
All ibrutinib-resistant cell lines and patient samples (except Pt4) were only slightly responsive to duvelisib as a single agent, whereas two cell lines (Z138 and Maver-1) and two patient samples (Pt1 and Pt3) were sensitive to venetoclax as a single agent ( Figure 1A Table S1. To understand the underlying mechanism of action, we performed an unbiased proteomic analysis by reverse-phase protein array (RPPA) analysis with 425 antibodies encompassing multiple signalling pathways ( Figure 1D). PI3K/AKT/mTOR, PKC and PLK1 signalling pathways were predominantly downregulated in the combination-treated cells, validating the intended targets. PI3K/ AKT/mTOR plays a crucial role in IR, 6 and blocking this pathway is a rational strategy for overcoming IR. PKC activation correlates with ibrutinib relapse in CLL. 10 BTK activates and upregulates the expression of BCL-2 family. 11 PLK1, a central cell cycle regulator at G2/M phase, is associated with IR as previously shown. 12 Moreover, the most upregulated proteins in the combination group were apoptosisrelated proteins such as caspases and Histone-H3 ( Figure 1D). These results were further validated by Western blotting ( Figure 1E).
Together, these data demonstrate that the combination of duvelisib and venetoclax overcomes IR synergistically through inhibition of the PI3K-Akt-mTOR pathway and activation of pro-apoptotic signalling.

| Duvelisib and venetoclax in combination inhibit significantly TME-mediated cell migration
The tumour microenvironment (TME) plays a critical role in drug resistance in many cancer types. 13 PI3K-AKT signalling plays a crucial role in regulating the tumour-TME interplay, and targeting this pathway via duvelisib has shown to disrupt the malignant B-cell homing process. [14][15][16] To investigate whether duvelisib and venetoclax in combination synergistically block directional cell migration, we performed a Transwell migration assay using IR JeKo BTK KD and human stroma cell line HS-5 cell monolayer as the TME attractant ( Figure S2A). A 30-min pre-treatment with the venetoclax and duvelisib combination significantly reduced cell migration towards the HS-5 monolayer compared with controls (p = 0.027) and with venetoclax and duvelisib alone (p = 0.036 and p = 0.056, respectively; Figure 1F and Figure S2B). These data suggest that this combination has the potential to interfere with the chemoattractant release and, therefore, impacting directional migration.

| The combination of duvelisib and venetoclax overcomes TME-mediated ibrutinib-resistance
To investigate the role of TME in tumour phenotypic changes, we co- became IR when co-cultured with HS-5 cells in vitro (p < 0.0001; Figure 2A). The effect of TME shifting the JeKo-1 phenotype from sensitive to IR was also confirmed in vivo as mice bearing JeKo-Luc-derived xenografts showed no response to ibrutinib treatment ( Figure 2B,C). Next, we determined whether duvelisib and venetoclax in combination could overcome TME-associated IR. Similar to our primary IR models (JeKo BTK KD, Maver-1 and Z138 cells) and the acquired IR model (JeKo R), venetoclax and duvelisib in combination overcame IR in the stroma/JeKo-1 co-culture (p = 0.0007; Figure 2D-F). Furthermore, JeKo-Luc-derived xenograft models were resistant to single agent treatment but sensitive (p = 0.003) to the combination ( Figure 2G,H). Altogether, these data demonstrate that this combination may potentially subvert primary, acquired and TME-associated MCL IR in both, in vitro and in vivo.
Here, we demonstrated that duvelisib and venetoclax in combination overcomes IR-MCL synergistically in vitro and in vivo. We investigated different types of IR including intrinsic, acquired and TME-associated resistance mechanisms. The combination reduced tumour growth and migration in all IR cell types, indicating that this is a robust and sustained therapeutic strategy for overcoming Vehicle I bruƟnib IR. While the exact mechanism is still under investigation, we report here that PI3K-AKT/mTOR and Bcl-2 signalling are the main IR phenotype drivers. There are four PI3K inhibitors: idelalisib, 17 duvelisib, 18 alpelisib 19 and copanlisib 20 that have been approved by FDA. We tested three of the four PI3K inhibitors in MCL cells, excluding alpelisib (targeting PI3Kα) since PI3Kδ is highly expressed in haematologic malignancies, 21 including MCL ( Figure S3). Both duvelisib (targeting PI3Kδ/PI3Kγ) and copanlisib (pan-class I PI3K inhibitor), but not idelalisib (targeting PI3Kδ), showed potent anti-MCL activity (data not shown). MCL cells express all four isoforms of PI3K ( Figure S3), so it is likely that targeting PI3Kδ only by idelalisib is not sufficient for inducing cytotoxicity in MCL.

F I G U R E 2 Duvelisib and venetoclax in combination circumvents
Expression of other isoforms in addition to PI3Kδ may compensate for the PI3K-mediated tumour cell survival and growth. Our data on duvelisib and venetoclax in combination provides evidence to support the dual targeting of PI3K and BCL-2 as a promising therapeutic strategy to overcome ibrutinib resistance in R/R MCL. It is likely copanlisib and venetoclax in combination will also show anti-MCL synergy.

ACK N OWLED G EM ENTS
The authors acknowledge the patients and their families who contributed to this research study. We thank Dr. Liana Adam and Alexa Jordan for review and editing, and Dr. Lei Nie for discussion and manuscript revision.