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 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.

Abstract: Bruton’s tyrosine kinase (BTK) is a great target in mantle cell lymphoma (MCL). This is evident by multiple FDA approvals of covalent BTK inhibitors (BTKi, e.g. ibrutinib, acalabrutinib and zanubrutinib) and recent exciting clinical data on non-covalent BTKi pirtobrutinib. However, resistance to BTKi is a major clinical challenge and the resistance mechanism is not yet fully understood. To address this, we generated JeKo BTK KD cells via BTK knockdown (KD) through by CRISPR Cas9 in BTKi-sensitive JeKo-1 cells, which resulted in superior resistance to BTKi and cell growth defects in vitro. Interestingly, JeKo BTK KD cells demonstrated early tumor cell engraftment and growth in subcutaneous xenograft models, while parental JeKo-1 cells showed much later engraftment but with much faster growth kinetics. To understand this and the BTKi-resistance mechanism, we first performed bulk RNA sequencing analysis and identified MALT1, but not its well-known binding partners CARD11 and BCL10, as one of the top overexpressed genes in BTKi-resistant MCL cells, including JeKo BTK KD cells. Genetic knockout (KO) of MALT1 or CARD11 by CRISPR Cas9 in JeKo-1 resulted in defects in cell growth in vitro and delayed tumor engraftment and growth in vivo. In contrast, MALT1 KO, but not CARD11 KO, in JeKo BTK KD cells remarkably suppressed cell growth in vitro, and tumor engraftment and growth in vivo. These data demonstrate that MALT1 overexpression can drive ibrutinib resistance via bypassing BTK-CARD11 signaling. BTKi-resistant cells including JeKo BTK KD cells showed much higher potency in adhesion to extracellular matrix or stromal cells compared to BTKi-sensitive cells. BTK inhibition or MALT1 inhibition significantly suppressed cell adhesion and migration to extracellular matrix or stromal cells. Furthermore, BTK KD and MALT1 KO but not CARD11 KO in JeKo-1 cells remarkably suppressed tumor cell dissemination and growth in spleen, liver, bone marrow and peripheral blood. MALT1 KO in JeKo BTK KD cells further suppressed tumor cell dissemination. Consistent with this, MALT1 inhibition greatly suppressed tumor cell dissemination and growth in spleen, bone marrow and peripheral blood of an ibrutinib-resistant patient-derived xenograft model. Therefore, both BTK and MALT1 are critical for tumor cell adhesion and dissemination in vivo in a CARD11-independent manner. Furthermore, co-targeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in BTKi-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that (1) BTK and MALT1 are key molecules that control MCL cell growth and dissemination, (2) MALT1 overexpression drives resistance to BTKi in MCL, (3) targeting MALT1 is a promising therapeutic strategy to overcome BTKi resistance, and (4) co-targeting of BTK and MALT1 improves efficacy and durability beyond single agents. Citation Format: Vivian Changying Jiang, Yang Liu, Qingsong Cai, Joseph M. McIntosh, Yijing Li, Zhihong Chen, Heng-Huan Lee, Wei Wang, Yixin Yao, Lei Nie, Michael Wang. BTK and MALT1 are critical for cell adhesion and dissemination in mantle cell lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3914.