Combination of the Dual Pan-PI3K/PIM Inhibitor Ibl-202 with the BCL-2 Inhibitor Venetoclax Enhances Apoptotic Priming and Is an Effective Targeted Treatment Approach in Mantle Cell Lymphoma

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Introduction: Mantle cell lymphoma (MCL) is a rare subtype of B-cell Non-Hodgkin-lymphoma, characterized by its heterogeneous clinical course ranging from indolent to highly aggressive cases with dismal prognosis. Targeted treatment approaches in first-line and relapse (r/r) settings are representing more and more the therapeutic standards of MCL. Excessive activation of the PI3K-Akt-mTOR pathway and the expression of PIM kinases play a significant role in MCL pathogenesis. Additionally, PIM kinases mediate resistance to PI3K inhibitors via various feedback mechanisms. This rationale led to the development of the pan-PI3K/pan-PIM inhibitor IBL-202. Venetoclax, a small-molecule inhibitor of the anti-apoptotic protein BCL-2, achieved high response rates in r/r MCL, however, duration of response is limited. Here, we investigated the efficacy of IBL-202 alone and in combination with venetoclax in MCL. Methods: MCL cell lines (Granta-519, JeKo-1, Rec-1), primary MCL patient samples and bone marrow (BM)- and lymph node (LN)-like fibroblasts HS5 and YK6 (±CD40L) were used for in vitro assays. Antitumor efficacy of IBL-202 (I) and venetoclax (V) alone or in combinations was analyzed using trypan blue staining, luminescent cell viability assay and AnnexinV flow cytometry. Peripheral blood mononuclear cells (PBMSc) were isolated using MACSprep™. Synergy was defined using Chou-Talalay's algorithm. Protein expression was assessed by western blot. BH3-profiling was performed to assess the cells apoptotic vulnerabilities. Co-culture experiments with HK5- and YK6 (±CD40L) were conducted to assess the impact of the microenvironment on IBL-202 antitumor activity. A chick embryo chorioallantoic membrane (CAM) xenograft model of MCL was employed to assess the combination in an immunocompetent in vivo setting. Results: IBL-202 proved to be effective as a monotherapy in MCL, with varying sensitivity among cell lines, as assessed by luminescent cell viability assay (Viability with 650nM: Granta 519 76%, Maver-1 76%, Jeko-1 34%, Rec-1 34%). Importantly, viability was unaffected in healthy donor PBMCs treated with IBL-202. Co-culture with either BM- or LN-like fibroblasts did not hamper this effectivity. Notably, CD40L stimulation significantly enhanced cytotoxicity of IBL-202, contributing to contradictory reports on the role of CD40L in MCL (Viability with 500 nM: Granta 519: 80% CD40- vs. 40% CD40+, p<0.05; Jeko-1: 53% CD40- vs. 33% CD40+, p<0.005). The combination with venetoclax was highly synergistic in all cell lines. Furthermore, this combination was able to overcome the intrinsic resistance of JeKo-1 cells to venetoclax, presumably caused by a lack of BIM protein expression. Similar significant efficacy of the I + V combination was observed in primary patient samples (Median viability: 37% I+V vs 56% I, p<0,05; vs 71% V, p<0,005). Protein expression analysis revealed an increased NOXA-MCL1 ratio in MCL cell lines treated with the I+V combination, resulting in enhanced apoptosis. Functional quantification of apoptosis (dynamic BH3-profiling) showed increased overall apoptotic priming of I+V compared to I, but not V monotherapy (cytochrome c release: 39% I+C vs 1% I, p<0.0001; vs 5% C, p=0.0003; 46% I+V vs 1% I, p<0.0001; vs 5% C, p<0.0001; vs 38% V, p=0.79). Furthermore, the I + V combination restored the decreased BCL-2 dependence that was observed with V alone (44% vs 24% cytochrome c release, p=0.0004). Moreover, we observed an increased MCL-1 dependence with I+V compared to untreated cells (cytochrome c release 26% vs 9%, p=0.02) and an increased BCL-XL dependence in I + V compared to I or V alone (cytochrome c release: 51% I+C vs 37% V, p=0.03; vs 27% I, p<0.002). Hence, dynamic BH3 profiling reveals that I+V accumulates apoptotic vulnerabilities. To validate the anti-lymphoma activity of the I+V combination in vivo, a CAM assay showed effective, albeit not significant tumor growth inhibition upon treatment with I alone and in combination with V compared to V alone, without any detectable toxicity. Conclusion: We showed for the first time that the novel dual pan-PI3K/PIM inhibitor IBL-202 is an effective and selective targeted treatment approach in MCL in both, in vitro and in vivo settings, and cooperates with venetoclax, presumably by altering the cellular dependencies on anti-apoptotic proteins, suggesting it a valuable candidate for combination therapy with venetoclax.