ALBERT

Description: Introduction: Multiple myeloma (MM) is characterized by deregulation of members of the Bcl-2 family of apoptotic regulatory proteins. This has led to the development of BH3-mimetics such as ABT-737 which inhibits Bcl-2/xL but not Mcl-1. Previously, we reported that simultaneous inhibition of Chk1 and MEK1/2 dramatically induced apoptosis in cultured and primary MM cells, including cells resistant to conventional agents, while sparing their normal counterparts (Pei et al., Blood 2007, 2011). Recently, we reported that this strategy circumvented MM cell resistance conferred by overexpression of Mcl-1, an important survival factor in this disease (Pei et al., PLoS One 2014). However, Bcl-2 overexpression confers significant resistance to the Chk1/MEK1/2 inhibition strategy. This raised the possibility that BH3-mimetics targeting Bcl-2 might circumvent this resistance mechanism. The purpose of the present studies was to determine whether BH3-mimetics could overcome such resistance while preserving anti-myeloma selectivity. An additional goal was to employ a new mathematical model to characterize interactions combining three novel agents that coordinately inhibit multiple survival signaling pathways. Methods: Various parental and Bcl-2 or Bcl-xL-over-expressing MM cell lines, as well as primary CD138+ MM cells were employed. ABT-737, the MEK1/2 inhibitor PD184352 (PD), and the Chk1/Wee1 inhibitor (Chk1i) were obtained from Abbott, Millipore and Calbiochem, respectively. Cells were exposed to agents alone or in various combinations for 4 -72 h, after which effects on apoptosis and signaling pathways were determined. Results: Co-administration of ABT-737 potentiated PD/Chk1i-mediated lethality in multiple parental MM cell lines, in association with Mcl-1 down-regulation, Bim up-regulation, and increased DNA damage (ΥH2A.X). Consistent with earlier findings, ectopic expression of Bcl-2 or Bcl-xL protected MM cells from the PD/Chk1i regimen. However, co-administration of ABT-737 significantly restored sensitivity towards PD/Chk1i lethality. Mathematical modeling indicated 3-agent synergistic interactions, particularly in Bcl-2 overexpressing cells. PD/Chk1i exposure inhibited phosphorylation (T705 and S727) of Stat3, another important survival factor for MM cells, while cells expressing constitutively active Stat3 (CA-STAT3) exhibited resistance to this regimen. However, the latter event was reversed by co-exposure to ABT-737. Moreover, combining ABT-737 with PD/Chk1i resulted in release of Bim from anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1, accompanied by Bak and Bax conformational change (activation). Knock-down of Bim by shRNA significantly protected cells from apoptosis induced by the 3-agent combination, indicating a functional role for Bim in anti-MM activity of this regimen. Furthermore, similar interactions, together with down-regulation of pStat3, were also observed in bortezomib-resistant MM cells, as well as in patient-derived CD138+ MM cells. In contrast, the regimen was minimally toxic to normal cord blood CD34+ cells or CD138- bone marrow cells. Finally, co-culture of parental or bortezomib-resistant MM cells with HS-5 stromal cells induced up-regulation of pStat3, while treatment with ABT-737 in combination with PD/Chk1i prevented Stat3 activation and robustly induced apoptosis despite the presence of stromal cells. Conclusion: ABT-737 co-administration synergistically potentiates the lethality of the PD/Chk1i regimen in MM cells, including bortezomib-resistant and primary MM cells, but not in normal hematopoietic progenitors. It also overcomes PD/Chk1i resistance conferred by overexpression of Bcl-2 or Bcl-xL, as well as by microenvironmental factors. Mechanisms responsible for these interactions are likely to be multi-factorial, including inactivation of Stat3, up-regulation of Bim, release of Bim from Bcl-2, Bcl-xL, and Mcl-1, and activation of Bak and Bax. Collectively, these findings demonstrate that combining BH3-mimetics with Chk1/MEK1/2 inhibition circumvents multiple forms of drug resistance in MM cells while exhibiting minimal toxicit toward normal hematopoietic cells. They also argue that a strategy targeting three coordinate survival signaling pathways may be highly effective in killing MM cells, particularly those resistant to current anti-MM therapies. Disclosures No relevant conflicts of interest to declare.

Description: M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, β5i, PSMB8) proteolytic subunit of the immunoproteasome; a crucial component of the cellular protein degradation machinery, which is highly expressed in malignant hematopoietic cells including multiple myeloma. M3258 was previously shown to deliver strong in vivo preclinical efficacy in multiple myeloma xenograft models, as well as a more benign non-clinical safety profile compared to approved pan-proteasome inhibitors, exemplified by a lack of effects on the central and peripheral nervous systems and cardiac and respiratory organs. Here we describe preclinical PK/PD and PK/efficacy modelling which led to a prediction of the PK profile, and the efficacious and safe dose ranges of M3258 in human which were used to guide the design of the phase I dose-escalation trial of M3258 in 〉3 line relapsed/refractory multiple myeloma (RRMM) patients. Mouse, rat, dog and monkey PK, plasma protein binding and intrinsic clearance data were used to estimate a half-life of approximately 6 hours for M3258 in human. The human total clearance and volume of distribution for M3258 were predicted to be 0.033 L/h/kg and 0.28 L/kg, respectively, whilst oral bioavailability was estimated to be above 80%. LMP7 proteolytic activity was assessed as a PD readout in human multiple myeloma tumor cells xenografted to mice as well as in dog peripheral blood mononuclear cells (PBMCs). A strong PK/PD relationship was observed for M3258 across both species. LMP7 inhibition by M3258 also correlated strongly with anti-tumor efficacy in multiple myeloma xenografts, with maximal efficacy observed at M3258 exposure delivering sustained inhibition of tumor LMP7 activity. Quantitative PK/PD/efficacy modeling predicted the biologically efficacious dose (BED) of M3258 upon oral application to be between 10 - 90 mg daily in human. By incorporating data from nonclinical safety studies, these data suggest an attractive human PK profile of M3258, enabling oral application, as well as an improved human therapeutic index compared to approved pan-proteasome inhibitors. M3258 is being investigated in a phase I, first-in-man, 2-part, open label clinical study designed to determine the safety, tolerability, PK, PD and early signs of efficacy of M3258 as a single agent (dose-escalation) and co-administered with dexamethasone (dose-expansion) in participants with RRMM whose disease has progressed following 〉 3 prior lines of therapy and for whom no effective standard therapy exists. Integration of these data will guide the selection of the BED for potential further clinical development of M3258. Disclosures Lignet: Merck Healthcare KGaA: Employment. Esdar:Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Becker:Merck Healthcare KGaA: Employment, Other: Holding shares with a value below 1000-USD. Drouin:EMD Serono Research and Development Institute: Employment. El Bawab:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Gimmi:Merck Healthcare KGaA: Employment. Haselmayer:Merck Healthcare KGaA: Employment. Jährling:Merck Healthcare KGaA: Employment. Sanderson:Merck Healthcare KGaA: Employment. Sloot:Merck Healthcare KGaA: Employment. Stinchi:Merck Healthcare KGaA: Employment. Victor:Merck Healthcare KGaA: Employment. Walter:Merck Healthcare KGaA: Employment. Rohdich:Merck Healthcare KGaA: Employment.

Description: Pharmacological inhibition of the canonical proteasome by inhibitors targeting its beta5 (PSMB5) subunit represent a cornerstone of the therapeutic management of plasma cell neoplasias. However, proteasome inhibitors have limited clinical applications in other hematologic malignancies. Notably, a broad spectrum of cell lines and patient-derived samples from lymphoma, leukemia and myeloma tend to express high levels of transcripts for and exhibit substantial activity of the immunoproteasome, an alternative proteasome particle which is enriched in hematopoietic/immune tissues. Established proteasome inhibitors used in clinical practice (e.g. bortezomib, carfilzomib, ixazomib) target both the canonical 20S proteasome and the immunoproteasome. We reasoned though that targeting of the immunoproteasome with more selective small molecule inhibitors that largely spare the canonical proteasome may provide insights into the specific functional role of the immunoproteasome in hematologic neoplasias, and importantly may also uncover underappreciated therapeutic opportunities related to targeting of hematologic neoplasias beyond myeloma, that might be more dependent on the immunoproteasome vs. the canonical proteasome; as well as potential benefits related to the therapeutic index of these agents. To address these questions, we characterized the activity of the previously disclosed selective immunoproteasome inhibitor M3258, which targets the LMP7 (beta5i subunit). The bortezomib-resistant cell line MM1VDR, which has a "gatekeeper" PSMB5 mutation preventing bortezomib binding to the canonical proteasome, remained sensitive to in vitro treatment with M3258, in further support of the functional specificity of this inhibitor. To obtain a comprehensive assessment of the activity of M3258 across hematologic malignancies, we studied a pool of 74 "DNA-barcoded" cell lines (PRISM system) from hematologic malignancies, including multiple myeloma (MM), leukemias and lymphomas, which were treated for 48hr with M3258. MM cell lines (9/16) were strongly represented among the sensitive lines which displayed 50% reduction in their cell viability in this analysis; and their responsiveness did not differ significantly from the M3258-responsive MM lines. Importantly, we observed no correlation between the responses of leukemia, lymphoma or MM lines to in vitro treatment with M3258 vs. clinically-relevant pulse of bortezomib. Furthermore, 7 of the 15 lymphoma/leukemia lines that were M3258-responsive had limited, if any, response to a clinically-relevant pulse of bortezomib. Taken together, these observations suggest that immunoproteasome inhibition with M3258 can be active against subsets of hematologic neoplasias with resistance/limited responsiveness to canonical proteasome inhibitors. To further examine the in vivo relevance of these observations, we studied the in vivo activity of M3258 against the MM.1S myeloma cell line, which has only moderate in vitro response to M3258 compared to other blood cancer cell lines tested in our "DNA-barcoded" pooled analysis. M3258 significantly increased the overall survival of mice with diffuse MM-bone lesions established after tail vein injection of MM.1S cells; and of mice harboring MM.1S lesions within a subcutaneously implanted ceramic scaffold-based system engineered to establish a bone marrow-like histological and functional unit with a "humanized" stromal compartment. The anti-tumor activity of M3258 in this scaffold model exhibited a trend for increased efficacy compared to twice weekly subcutaneous treatments with bortezomib. These results highlight that in vivo administration of M3258 exhibits anti-tumor activity against clinically-relevant models of MM lesions, even from cell lines that have modest in vitro responsiveness to this immunoproteasome inhibitor. Furthermore, our studies with a large panel of "DNA-barcoded" cell lines indicates that the anti-tumor effects of M3258 may extend beyond MM and into other classes of hematologic malignancies, including subsets of leukemias and lymphomas, identifying previously underappreciated therapeutic implications for the class of selective immunoproteasome inhibitors. Disclosures Walter: Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Sanderson:Merck Healthcare KGaA: Employment. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding.