Abstract: Introduction Although 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. Methods Ibrutinib 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. Results Our 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 & lt; 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p & lt;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. Conclusions Combinatory 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. Disclosures Wang: 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.

Abstract: Introduction Clinical relapse following CD19 CAR T therapy after failure to BTK inhibitors (BTKi) is a novel and fast growing medical challenge in treating patients with mantle cell lymphoma (MCL). Thus, developing a novel therapy to overcome this BTKi-CAR T dual resistance (Dual-R) is an urgent need. Our unpublished data revealed MYC targets and cyclin-dependent kinase 9 (CDK9) are highly upregulated in the Dual-R compared to BTKi-R samples. CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex. Its inhibition induces acute decline of short-lived mRNA and proteins, especially MYC and MCL-1, in acute myeloid leukemia and diffuse large B-cell lymphoma. Therefore, we hypothesize that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. BAY-1251152 is a novel selective CDK9 inhibitor with nanomolar potency. However, whether it has the potential to overcome BTKi-CAR T dual resistance has not been assessed. Methods To validate the correlation of MYC and CDK9 with drug resistance and clinical outcome, we performed whole transcriptomic profiling using primary patient samples. To assess the in vitro efficacy of BAY-1251152, we performed cell viability assay and cell apoptosis assay using MCL cell lines and primary patient samples and followed up with functional studies. furthermore, we assessed its in vivo efficacy using patient-derived xenograft (PDX) models derived from MCL patients including one with Dual-R. Results The expression of MYC oncogene associates with ibrutinib resistance and Dual-R, and poor clinical outcome. CDK9 expression does not correlates with ibrutinib resistance but it does associate with Dual-R and poor clinical outcome in CAR T-relapsed patients. CDK9 inhibition by BAY-1251152 is highly potent in anti-MCL activity in MCL cell lines with low nanomolar range of IC50 (59.6-172.3 nM) by inducing robust cell apoptosis. BAY-1251152 induces dose and time-dependent CDK9 inhibition. A rapid decline of phosphorylation of RNA polymerase II, MYC, MCL-1, and Cyclin D1 can be observed as early as 2 hours. Furthermore, BAY-1251152 (10mg/kg, QW) significantly inhibited tumor growth (p = 0.000003) in a PDX model derived from a Dual-R patient without causing apparent toxicity in NSG mice. In addition, BAY-1251152 also significantly suppressed tumor growth in the PDX models derived from a BTKi-R patient (p=0.00015) and a BTKi-venetoclax dual-resistant patient (p=0.009). Conclusion Our findings showed that targeting CDK9 by its specific inhibitor BAY-1251152 led to potent in vitro anti-MCL activity. BAY-1251152 induces fast CDK9 inhibition and rapid decline of MYC, MCL-1 and Cyclin D1 to induce robust cell death. BAY-1251152 is also potent in inhibiting tumor growth in PDX models. These data support that CDK9 is a promising target to overcome BTKi-CAR T dual resistance in MCL, which is in urgent need. Citation Format: Vivian Changying Jiang, Lingzhi Li, Alexa Jordan, Yu Xue, Fangfang Yan, Joseph McIntosh, Yang Liu, Yuxuan Che, Yijing Li, Qingsong Cai, Angela Leeming, Lukas Simon, Zhongming Zhao, Jia Zhou, Michael Wang. Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3955.

Abstract: Background Mantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model. Methods MCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination. Results Compared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p & lt;0.01). Treatment with pirtobrutinib (10μM) for 24 hours induced higher levels of apoptosis than that by ibrutinib in all the MCL cell lines tested (p & lt;0.05), which was also confirmed at the molecular level by stronger caspase-3 activation and PARP cleavage. To understand the mechanism of action, we performed whole transcriptomic profiling by RNAseq analysis using Z138 cells treated with/without pirtobrutinib or ibrutinib. Pirtobrutinib treatment resulted in upregulation of 137 genes and downregulation of 97 genes compared to the ibrutinib treatment (adjusted p & lt;0.05). In addition to the downregulated MYC targets and PI3K/Akt pathway, gene set enrichment analysis (GSEA) revealed a significant enrichment for G2/M checkpoints and E2F targets signatures (key genes: PLK1, CDKN1A and CCNB1) in pirtobrutinib treated cells. Consistently, follow-up studies showed that γH2AX level was highly increased upon pirtobrutinib treatment. Pirtobrutinib treatment but not ibrutinib treatment resulted in G2/M cell cycle arrest. The blockade of cell cycle progression is positively correlated with decreased protein levels of critical regulators of S and G2/M phase transition such as cyclin B and CDC25C. BTK inhibitor (ibrutinib) in combination with venetoclax has shown great efficacy in preclinical models and in MCL patients. Therefore, here we assessed the in vivo efficacy of pirtobrutinib in combination with venetoclax with side-by-side comparison to ibrutinib & venetoclax in the Mino-venetoclax-R mouse model. Pirtobrutinib & venetoclax combination enhanced the efficacy of pirtobrutinib in restraining the tumor size (p & lt;0.001) in the xenograft model. Notably, this novel combinatorial treatment exerted much higher potency than ibrutinib and venetoclax combination therapy (p & lt;0.001). In addition, the pirtobrutinib & venetoclax combination was well tolerated and did not reduce overall mouse body weights compared with the vehicle treated mice. Conclusions Pirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. Figure 1 Figure 1. Disclosures Wang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.