The Novel Calcium Release-Activated Calcium (CRAC) Channel Inhibitor RP4010 Exerts Potent Antitumor Effects in NOD/SCID/IL2Rg-/- Mice with Diffuse Large B Cell Lymphoma (DLBCL) Cell Line Xenografts

Abstract

INTRODUCTION: Calcium ions (Ca2+) act as second messengers in cell signaling triggering various cellular processes, including gene transcription, secretion, cell proliferation, migration, and apoptosis. Store-operated Ca2+ entry (SOCE) is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and Orai1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways. As emerging evidence points to the involvement of aberrant CRAC channel activity in human diseases including Diffuse Large B Cell Lymphoma (DLBCL), we hypothesize that the CRAC channel inhibitorRP4010 might represent a unique therapeutic opportunity for DLBCL patients. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro . This study aimed at investigating the activity of RP4010 in preclinical DLBCL models.METHODS: The DLBCL cell lineswith GCB phenotype (SUDHL-4, SUDHL-6, SUDHL-16, and SUDHL-10) and ABC phenotype (SUDHL-8, RCK-8, RIVA, and SUDHL-2) were used to investigate in vitro the effects of RP4010 on cell growth and cell death and gene expression. The antitumor efficacy and of RP4010 was also analyzed in NOD/SCID/IL2Rg-/-mice bearing DLBCL cell line xenografts.RESULTS: Incubating GCB-DLBCL and ABC-DLBCL cell lines for up to 72 hours with increasing doses of RP4010 (1.25 - 10 µM)resulted in a significant dose- and time-dependent decrease in cell proliferation. For all cell lines, the peak of the cytostatic effect was detected upon incubation with 10 µM of RP4010 for 72 hours, when an average 80% decrease in cell proliferation over vehicle-treated controls (P ≤ 0.0001) was detected. RP4010 treatment resulted in a significant time- and dose-dependent increase in cell death, as assessed by AnnexinV/PI staining, in GCB-DLBCL cell lines and in two out of four ABC-DLBCL cell lines (SUDHL-8 and SUDHL-2), whereas modest or no effects were detected in the RCK-8 and RIVA ABC-DLBCL cell lines. Exposure to 5 µM RP4010 for 72 hours significantly increased cell death over vehicle-treated controls in SUDHL-4 (90 ± 4% vs. 11 ± 2%), SUDHL-6 (39 ± 4% vs. 16 ± 3%), SUDHL-16 (70 ± 5% vs. 18 ± 4%), SUDHL-10 (49 ± 5% vs. 15 ± 1%), SUDHL-8 (78 ± 4 vs. 8 ± 1%) and SUDHL-2 (57 ± 5 vs. 26 ± 4%) cell lines (P ≤ 0.0001). Interestingly, RP4010 (1.25 - 10 µM) failed to induce any cytotoxic effects on the in vitro growth of hematopoietic colony-forming cells from healthy volunteers. While no activation of caspase-8, -9, -3, or PARP were observed in ABC-DLBCL cell lines treated with RP4010, a potent caspase cleavage was observed upon RP4010 treatment in GCB-DLBCL cell lines. Pretreating cells with the pan-caspase inhibitor Z-VAD-fmk abrogated apoptosis only in GCB-DLBCL cell lines, but not in the ABC-DLBCL cell lines, supporting that RP4010 induces both caspase-dependent and caspase-independent cell death. Gene expression analysis revealed a strong correlation between Orai1 expression and sensitivity to RP4010-induced cell death. Thus, Orai1 has a pivotal role in the establishment of an apoptosis-resistant phenotype in ABC-DLBCL cells. Finally, in vivo experiments were conducted to investigate the antitumor activity of RP4010. Treatment of NOD/SCID/IL2Rg-/-mice bearing DLBCL tumor nodules with increasing dose of RP4010 (30, 60, 90 mg/Kg body weight, PO, 5 days/3 weeks) resulted in a dose-dependent inhibition of tumor growth (TGI) of SUDHL-8 [TGI = 35%, 74%, and 83% at 30, 60, and 90 mg/kg, respectively], RIVA and SUDHL-6[TGI = 66%, 52% at 90 mg/kg, respectively], compared to vehicle-treated controls. No mice experienced any apparent treatment-related toxicity. Despite the marked antitumor activity observed in DLBCL xenografts, RP4010 treatment did not induce any tumor necrosis in healthy tissues (including lungs, livers, spleens, and kidneys), suggesting that RP4010-induced growth inhibition was tumor restricted.CONCLUSIONS: RP4010 is associated with a potent anti-lymphoma activity, suggesting its therapeutic potential in DLBCL. A Phase 1/1b study in Relapsed/Refractory Non-Hodgkin Lymphoma (NHL) patients is currently ongoing in the US (ClinicalTrials.gov Identifier: NCT03119467). DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; ADC Therapeutics: Research Funding; Rhizen Pharmaceuticals: Research Funding.