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 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 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: 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.