Abstract: Inevitable relapses remain as the major therapeutic challenge in patients with mantle cell lymphoma (MCL) despite FDA approval of multiple targeted therapies and immunotherapies. Fc gamma receptors (FcγRs) play important roles in regulating antibody-mediated immunity. FcγRIIB, the unique immune-checkpoint inhibitory member of the FcγR family, has been implicated in immune cell desensitization and tumor cell resistance to the anti-CD20 antibody rituximab and other antibody-mediated immunotherapies; however, little is known about its expression and its immune-modulatory function in patients with aggressive MCL, especially those with multi-resistance. In this study, we found that FcγRIIB was ubiquitously expressed in both MCL cell lines and primary patient samples. FcγRIIB expression is significantly higher in CAR T-relapsed patient samples ( p   〈  0.0001) compared to ibrutinib/rituximab-naïve, sensitive or resistant samples. Rituximab-induced CD20 internalization in JeKo-1 cells was completely blocked by concurrent treatment with BI-1206, a recombinant human monoclonal antibody targeting FcγRIIB. Combinational therapies with rituximab-ibrutinib, rituximab-venetoclax and rituximab-CHOP also induced CD20 internalization which was again effectively blocked by BI-1206. BI-1206 significantly enhanced the in vivo anti-MCL efficacy of rituximab-ibrutinib ( p  = 0.05) and rituximab-venetoclax ( p  = 0.02), but not the rituximab-CHOP combination in JeKo-1 cell line-derived xenograft models. In patient-derived xenograft (PDX) models, BI-1206, as a single agent, showed high potency ( p   〈  0.0001, compared to vehicle control) in one aggressive PDX model that is resistant to both ibrutinib and venetoclax but sensitive to the combination of rituximab and lenalidomide (the preclinical mimetic of R 2 therapy). BI-1206 sensitized the efficacy of rituximab monotherapy in a PDX model with triple resistance to rituximab, ibrutinib and CAR T-therapies ( p  = 0.030). Moreover, BI-1206 significantly enhanced the efficacy of the rituximab-venetoclax combination ( p   〈  0.05), which led to long-term tumor remission in 25% of mice. Altogether, these data support that targeting this new immune-checkpoint blockade enhances the therapeutic activity of rituximab-based regimens in aggressive MCL models with multi-resistance. Graphical Abstract

Abstract: Introduction As a rare form of non-Hodgkin's lymphoma, mantle cell lymphoma (MCL) is an aggressive subtype. This is largely due to frequent relapses after therapies including paradigm shifting therapies BTK inhibitors (BTKi), such as ibrutinib and acalabrutinib, and Bcl-2 inhibitor (Bcl-2i) venetoclax after long-term treatment in the clinic. Dysregulation of Bcl-2 and Bcl-X L, contributes to therapeutic resistance in MCL. AZD0466 is a novel and highly potent Bcl-2/X L dual inhibitor with active moiety AZD4320. Our preliminary data showed AZD4320 is potent in inhibiting cell viability of MCL cells (IC 50 = 1.6-78 nM). In this study, we assessed the combination efficacy of AZD4320/AZD0466 and acalabrutinib on preclinical MCL models. Methods Cell viability assay was performed to assess the in vitro efficacy of AZD4320 and acalabrutinib alone or in combination in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines. Cell apoptosis assay was also performed to determine if AZD4320 and acalabrutinib enhanced cell death by cell apoptosis in MCL cell lines. Protein expression profiles of a panel of pro- and anti-apoptotic proteins and other relevant proteins were detected by immunoblotting. Since AZD4320 is limited in preclinical model due to physicochemical properties and dose limiting cardiovascular toxicity, AZD0466, the drug-dendrimer conjugate of AZD4320, was used for in vivo experiment. In vivo efficacy of AZD0466 (34 mg/kg, weekly, iv) and acalabrutinib (20 mg/kg, BID, oral) alone or in combination was evaluated using a Mino-venetoclax-R (Mino-R) cell xenograft model and a PDX model derived from an ibrutinib-CAR-T dual-resistant MCL patient. Results AZD4320 in combo with acalabrutinib inhibited cell proliferation synergistically in both ibrutinib/venetoclax-sensitive and -resistant cell lines (combination index = 0.17-0.93). Compared to vehicle or either single agent, the combination enhanced cell apoptosis by increasing pro-apoptotic markers cleaved caspase 3 and cleaved PARP. In the xenograft mouse model derived from venetoclax-resistant Mino-R cells, co-treatment of AZD0466 and acalabrutinib decreased tumor size significantly compared to vehicle (n = 5, p & lt; 0.0001) or either single agent (n = 5, p = 0.0118 and 0.0070, respectively). Furthermore, in the PDX mouse model derived from a patient relapsed subsequently from ibrutinib and CAR T therapy, the combination of AZD0466 and acalabrutinib inhibited tumor growth compared to vehicle or either single agent. Acalabrutinib or AZD0466 improved survival compared with vehicle by 14 days or 32 days, respectively. Compared to Acalabrutinib or AZD0466, the combination therapy extended survival by 25 days and 7 days, respectively. All mice tolerated the treatment dose without any weight loss compared to the vehicle or either single agent group. Conclusion Compared to AZD4320/AZD0466 and acalabrutinib, combination therapy demonstrated anti-MCL synergy both in vitro and in vivo. These findings suggest that targeting Bcl-2/X L and BTK is promising to overcome multiple acquired resistance phenotypes, including CD19 CAR T-cell therapy. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang: DTRM Biopharma (Cayman) Limited: Consultancy; BeiGene: Consultancy, Honoraria, Research Funding; Physicians Education Resources (PER): Honoraria; Anticancer Association: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; CAHON: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Epizyme: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; BGICS: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria; Celgene: Research Funding; Genentech: Consultancy; Loxo Oncology: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Molecular Templates: Research Funding; Lilly: Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Oncternal: Consultancy, Research Funding; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Scripps: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Chinese Medical Association: Honoraria; Pharmacyclics: Consultancy, Research Funding; Juno: Consultancy, Research Funding; CStone: Consultancy; Bayer Healthcare: Consultancy; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Moffit Cancer Center: Honoraria; Mumbai Hematology Group: Honoraria.

Abstract: Introduction Mantle cell lymphoma (MCL) patients often presents at later stages and progress through its disease course by frequent involvement of multiple dissemination sites including spleen, liver, bone marrow (BM), peripheral blood (PB), and gastrointestinal tract (GI). This devious behavior translates into high degree of clinicopathologic heterogeneity, which may compromise therapies and promote relapse. Therefore, dissecting the cellular and molecular profiling and trafficking is critical in understanding the role of tissue tropism and evolution patterns contributing to its biological behavior. Since it is almost unfeasible to perform spatiotemporal collection in patients, in this study we took advantage of PDX models with serial samples and single cell transcriptomic profiling to address this important biology issue for the first time on MCL. Method Orthotopic PDX models (n = 6) were established via intravenous (IV) inoculation of primary MCL patient samples collected from PB (n = 5) or from LN (n = 1). These mouse models displayed similar dissemination patterns as the parental tumors. Cells from the predominant site of generation 1 (G1) were used to pass onto next generations (up to G9). For heterotopic PDX models, subcutaneous (SC) models were generated in parallel from two independent lines (up to G6) and exhibited predominant tumor growth at primary injection site with tumor spread to secondary sites only at very late stage. PDX samples from IV models (spleen, liver, BM, PB) and SC models across generations (n = 36) were collected and subjected to scRNA-seq profiling together with parental patient samples (n = 6) and healthy donor PBMC samples (n = 2). Results All six PDX models at G1 faithfully mirrored parental samples by displaying similar cancer hallmarks. Interestingly, MYC and OXPHOS signaling were predominantly and progressively augmented with each IV passage, and to a lesser extent across SC passages, suggesting a higher degree of selection and evolution processes during orthotopic passage. With spatial collection at distinct dissemination sites (spleen, liver, BM and PB) within same generations, we revealed that heterogenous transcriptomic profiles were more evident across tissues than generations. Specifically, cancer hallmarks such as MYC (NES = 8.4, FDR & lt; 0.01), OXPHOS (NES = 8.9, FDR & lt; 0.01) and mTORC1 (NES = 6.6, FDR & lt; 0.01) signaling were highly enriched in cells from PB, and to a lesser extent in spleen and liver when compared to the cells in BM. More intriguingly, 55-60% of tumor cells in PB clustered together and showed enhanced cancer hallmarks for tumor migration and invasion (NES = 7.9, FDR & lt; 0.01), higher de-differentiation scores (cytoTRACE) and G0/G1 cell cycle stage. This suggests that these cells are quiescent, de-differentiated and disseminative. Importantly, a small fraction of cells from spleen (5-18%) and liver (12-18%), but not in BM, showed similar characteristics and clustered together with those from PB. Histopathologic analysis showed that tumor cells could be detected in blood only after cells settled and expanded in the spleen, liver or BM, whereas dissemination to LN, GI tract, lung and kidney were even later events. Therefore, it is likely that these disseminative MCL cells originate from tissues and represent the tumor seed cells for disease dissemination. More interestingly, the top differential expressed genes (DEGs) in these seed cells were also significantly upregulated in ibrutinib-resistant patients (p & lt; 0.01), compared to that in ibrutinib-sensitive patients based on bulk RNA sequencing (n = 69). This indicates that these seed cells are more resistant to ibrutinib and may drive therapeutic relapse. Targetable molecules are under active investigation to eradicate this ibrutinib-resistant seed cells. Conclusion MCL tissue tropism results in distinct transcriptomic profiles. A special cell population of tumor seed cells was identified to be quiescent, de-differentiated and disseminative, and may drive tumor spread, disease progression and therapeutic resistance (Figure 1). These observations provide biological insights into MCL disease progression in multiple MCL sites. Figure 1 Figure 1. Disclosures Wang: InnoCare: Consultancy, Research Funding; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Pharmacyclics: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; OMI: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; Oncternal: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Chinese Medical Association: Honoraria; Celgene: Research Funding; Imedex: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria; BioInvent: Research Funding; Physicians Education Resources (PER): Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Moffit Cancer Center: Honoraria; Newbridge Pharmaceuticals: Honoraria; Lilly: Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Genentech: Consultancy; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; Mumbai Hematology Group: Honoraria; CStone: Consultancy; Bayer Healthcare: Consultancy; Anticancer Association: Honoraria; Scripps: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Clinical Care Options: Honoraria; BGICS: Honoraria; 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: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by frequent relapses. The development of resistance to ibrutinib therapy remains a major challenge in MCL. We previously showed that glutaminolysis is associated with resistance to ibrutinib. In this study, we confirmed that glutaminase (GLS), the first enzyme in glutaminolysis, is overexpressed in ibrutinib-resistant MCL cells, and that its expression correlates well with elevated glutamine dependency and glutaminolysis. Furthermore, we discovered that GLS expression correlates with MYC expression and the functioning of the glutamine transporter ASCT2. Depletion of glutamine or GLS significantly reduced cell growth, while GLS overexpression enhanced glutamine dependency and ibrutinib resistance. Consistent with this, GLS inhibition by its specific inhibitor telaglenastat suppressed MCL cell growth both in vitro and in vivo. Moreover, telaglenastat showed anti-MCL synergy when combined with ibrutinib or venetoclax in vitro, which was confirmed using an MCL patient-derived xenograft model. Our study provides the first evidence that targeting GLS with telaglenastat, alone or in combination with ibrutinib or venetoclax, is a promising strategy to overcome ibrutinib resistance in MCL.

Abstract: Introduction Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma. Frequent relapse from prior therapies remains a major medical challenge. BTK inhibitors (BTKi), such as ibrutinib and acalabrutinib, have demonstrated clinical benefit in MCL patients, however, resistance to BTKi is acquired by most MCL patients following initial response in the clinic. Unbalanced pro- and anti-apoptotic proteins have been shown to contribute to therapeutic resistance. Bcl-2 inhibitor venetoclax was approved by FDA to treat chronic lymphocytic leukemia, small lymphocytic lymphoma, or acute myeloid leukemia, and is currently under investigation in MCL patients. Venetoclax is efficacious with ORR up to 75% in treating BTKi-naive MCL patients and 53% BTKi-R/R patients. Interestingly, increased expression of Mcl-1 and Bcl-x L highly correlates with venetoclax resistance in MCL. AZD5991 is a highly selective Mcl-1 inhibitor and our preliminary data showed it is potent in targeting MCL cells (IC 50 =76-600 nM). Therefore, we hypothesize that dual targeting Bcl-2 by venetoclax and Mcl-1 by AZD5991 will achieve synergistic effect and significantly improve treatment outcome for aggressive R/R MCL patients. In this study, we assessed the synergistic efficacy of AZD5991 in combination with venetoclax in MCL preclinical models with R/R phenotype to ibrutinib, venetoclax or CD19 CAR T therapies. Methods Cell viability assay was performed to assess the in vitro efficacy of AZD5991 and venetoclax alone or in combination in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines. Cell apoptosis assay was also performed to determine if AZD5991 and venetoclax induce cell death by cell apoptosis in MCL cell lines. Protein expression profiles of a panel of pro- and anti-apoptotic proteins and other relevant proteins were detected by western blots. In vivo efficacy of AZD5991 (30/30 mg/kg, intravenously, weekly) and venetoclax (5 mg/kg, oral, daily) alone or in combination was evaluated using PDX models derived from an ibrutinib-resistant patient or an ibrutinib-CAR-T dual-resistant MCL patient. Results AZD5991 and venetoclax combination potently and synergistically inhibited cell viability (combination index = 0.16-0.88) and enhanced cell apoptosis in vitro in both ibrutinib/venetoclax sensitive and resistant cell lines. Consistently, pro-apoptotic markers cleaved caspase 3 and cleaved PARP were increased. In an ibrutinib-resistant PDX mouse model, the combination of AZD5991 and venetoclax resulted in anti-MCL synergistic response. Consistently, the amount of β2M in the mouse plasma from the combo group was much lower than vehicle group (n =5, p = 0.010) and the two single agent groups (n =5, p = 0.005 and 0.013, respectively). In an ibrutinib-CAR-T dual-resistant PDX mouse model, co-treatment of AZD5991 and venetoclax inhibited tumor growth significantly and prolonged mouse survival for at least 100 days compared to vehicle or either single agent. All mice tolerated the treatment dose without any weight loss. Conclusion Significant anti-MCL synergy was observed for AZD5991 and venetoclax combo in vitro in both pairs of Jeko-1/JeKo-ibrutinib-R and Mino/Mino-venetoclax-R. This was further validated in aggressive R/R PDX models including an ibrutinib-resistant PDX model and an ibrutinib-CD19 CAR-T dual-resistant PDX model. Altogether, these findings suggest that dual targeting Bcl-2 and Mcl-1 is synergistic and has the potential in overcoming multiple acquired resistance phenotypes, including CD19 CAR T-cell therapy. These data provide insights on therapeutic development to improve patient outcome by overcoming therapeutic resistance. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang: Clinical Care Options: Honoraria; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Genentech: Consultancy; Celgene: Research Funding; Molecular Templates: Research Funding; Dava Oncology: Honoraria; BGICS: Honoraria; Imedex: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Juno: Consultancy, Research Funding; Newbridge Pharmaceuticals: Honoraria; CAHON: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Scripps: Honoraria; Mumbai Hematology Group: Honoraria; OMI: Honoraria; Epizyme: Consultancy, Honoraria; Chinese Medical Association: Honoraria; Oncternal: Consultancy, Research Funding; The First Afflicted Hospital of Zhejiang University: Honoraria; Anticancer Association: Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; CStone: Consultancy; Loxo Oncology: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Physicians Education Resources (PER): Honoraria; Lilly: Research Funding.