Abemaciclib in Combination with Copanlisib to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Abstract

IntroductionAlthough novel therapeutic strategies including BTK and Bcl-2 inhibitors have dramatically improved the prognosis of MCL patients, resistance to these treatments is inevitable. We recently reported that the tumor suppressor gene CDKN2A were commonly deleted in ibrutinib-resistant tumors, leading to upregulation of CDK4/6 signaling. Among the other hallmarks are the mTOR/PI3K, Myc and OXPHOS pathways. Therefore, we attempt to exploit combinatory targeting of CDK4/6 and PI3K pathways to overcome therapy resistance using in vitro and PDX models.MethodsIbrutinib or venetoclax sensitive and resistant MCL cell lines were used in this preclinical study. 1x10 4 cells per well are seeded in 96-well plates and treated with abemaciclib monotherapy or in combination with copanlisib (PI3K inhibitor) in triplicate for 72h and then mixed with CellTiter-Glo Luminescent Cell viability Assay Reagent. For cell cycle assay, cells were seeded in 6 well plates and treated with vehicle or abemaciclib for 24h. Cells were fixed in 70% pre-cold ethanol and stained with propidium iodide. The cell cycle stages were quantified through the Novocyte Flow Cytometer. The molecular events at the protein level after treatment were determined by immunoblotting. For in vivo experiment, the combination of abemaciclib (25mg/kg, oral, daily) and copanlisib (5mg/kg, IP, three times a week) was assessed in Mino-venetoclax-resistant xenograft model. IC50 values were calculated using GraphPad Prism 8 for each cell line. Student's t-test was performed to compare the difference between vehicle and treated groups. Two-way analysis of variance (ANOVA) was conducted to analyze the tumor growth in vivo experiments. P values less than 0.05 were considered statistically significant.ResultsOur previous studies have identified a subset of MCL cells that were resistant to venetoclax (JeKo-1) or ibrutinib treatment (Maver-1 and Z-138). To overcome the resistance, we first treated MCL cell lines with abemaciclib and the result showed that abemaciclib as a single agent showed potent anti-MCL activity in a subset of MCL cell lines (IC 50 = 70-952 nM) including venetoclax sensitive- (Mino, Rec-1, Maver-1, and Z138) and primary resistant- MCL cells (JeKo-1). However, the cell lines Mino-ven-R and Rec-ven-R with acquired venetoclax resistance are highly resistant to abemaciclib treatment (IC 50 = 6.0 and 4.4 µM). PI3K/ATK pathway has been reported to be highly upregulated in Mino-ven-R and Rec-ven-R cells compared to their parental cells. To further increase the efficacy of the targeted therapy, we treated the resistant MCL cells with a combination of abemaciclib and copanlisib and the result showed synergistically enhanced cytotoxicity in ibrutinib or venetoclax-resistant MCL cell lines. Consistent with the role of CDK4/6 in cell cycle progression, inhibition of CDK4/6 with abemaciclib resulted in the cell cycle arrest at G1 phase in MCL cell lines.To validate whether abemaciclib in combination with copanlisib can overcome venetoclax resistance in vivo, we assessed the antitumor effect of abemaciclib in combination with copanlisib using a venetoclax-resistant xenograft models derived from Mino-ven-R cell line in immunodeficient NSG mice. As a result, abemaciclib (25 mg/kg, oral, daily), but not venetoclax (5 mg/kg, oral, daily) or copanlisib (5 mg/kg, IP, three times a week), significantly reduced tumor volume compared to the vehicle control (n = 5, p < 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p<0.0001). Of note, the combination did not cause major decreases in body weight. Taken together, these results suggest that the combinatory therapy is effective in overcoming venetoclax resistance in MCL.ConclusionsCombinatory treatment with abemaciclib and copanlisib may achieve clinical actionable efficacy through overcoming the venetoclax-resistance in MCL that may become an effective treatment regimen for refractory/relapsed MCL patients in the future. DisclosuresWang: Dava Oncology: Honoraria; Imedex: Honoraria; CStone: Consultancy; Hebei Cancer Prevention Federation: Honoraria; OMI: Honoraria; Chinese Medical Association: Honoraria; Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; Bayer Healthcare: Consultancy; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; Physicians Education Resources (PER): Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; Acerta Pharma: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; BGICS: Honoraria; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding.