ALBERT

Description: Introduction: Both inter- and intra-tumoral heterogeneity are obstacles to improving oncology clinical outcomes. Mantle cell lymphoma (MCL) is an extremely heterogeneous disease in clinical, pathological, genetic, and transcriptomic profiling. Furthermore, MCL patients frequently develop therapeutic resistance after frontline therapies. In this study, we performed longitudinal transcriptomic analysis on primary patient MCL specimens at single-cell resolution, aiming to understand the dynamic and complex cellular and molecular changes underlying therapeutic resistance and identify potential targets to overcome dual resistance to ibrutinib and venetoclax. Methods: Sequential single-cell transcriptome sequencing (scRNA-seq) was performed on patient specimens collected during the course of treatment(s) from 5 MCL patients (3 ibrutinib responders and 2 ibrutinib-venetoclax non-responders). Integrative computational approaches were employed to characterize the cellular and molecular basis of therapeutic resistance and clonal evolution. An orthotopic PDX model derived from one of the non-responders was established and used to validate the novel findings and to investigate the in vivo efficacies of multiple novel potential targets. Results: The 3 ibrutinib responders and 2 ibrutinib-venetoclax non-responders were highly heterogeneous in clinical and pathological profiling. To dissect the inter- and intra-tumor heterogeneity underlying the therapeutic resistance, we performed sequential scRNA-seq analysis of 21 specimens collected at baseline, during treatment, and/or at disease remission/progression. The scRNA-seq analysis revealed a high degree of inter- and intra-tumor heterogeneity with distinct cellular and transcriptomic profiling within and across ibrutinib-responders and ibrutinib-venetoclax non-responders. Unsupervised pathway enrichment analysis identified more than 15 cancer hallmarks significantly upregulated in ibrutinib-venetoclax non-responders. We tracked the clinical ibrutinib-induced lymphocytosis at a single-cell transcriptomic level in ibrutinib responders and disease-progression-associated clonal evolution in non-responders. Multiple actionable targets were identified, and targeting these showed effective anti-MCL activity in the orthotopic PDX model derived from one of the ibrutinib-venetoclax non-responders. Conclusions: This study demonstrates the potential of longitudinal single-cell transcriptomic analysis to reveal the molecular mechanisms underlying tumor heterogeneity, clonal evolution, disease progress, and therapeutic resistance, and to identify potential novel targets to circumvent therapeutic resistance in mantle cell lymphoma and other diseases. Disclosures Wang: Pharmacyclics: Honoraria, Research Funding; Juno Therapeutics: Research Funding; Celgene: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; Loxo Oncology: Research Funding; VelosBio: Research Funding; BioInvent: Consultancy, Research Funding; Dava Oncology: Honoraria; Aviara: Research Funding.

Description: Introduction: Mantle cell lymphoma (MCL) is a rare but aggressive non-Hodgkin lymphoma (NHL). CD20 antibodies (e.g. rituximab), BTK inhibitors (e.g. ibrutinib and acalabrutinib), and BCL-2 inhibitors (e.g. venetoclax) alone or in combination have shown great anti-MCL efficacy. However, primary and acquired resistance to one or multiple therapies commonly occurs, resulting in poor clinical outcome. Overcoming such mechanisms of resistance holds promise to significantly improve survival, meeting a significant medical need for patients with refractory/relapsed MCL. Recent studies showed Fc gamma receptors (FcγRs) play important roles in controlling therapeutic efficacy. FcgRIIB (CD32B), the inhibitory FcγR, negatively controls antibody efficacy through distinct inhibitory mechanisms in immune effector cells and lymphoma cells, respectively. When expressed on leukemic or lymphoma cells, FcgRIIB promotes rituximab internalization and removal from the tumor cell surface, resulting in reduced immune effector cell activation and ultimately decreased in vivo therapeutic efficacy. We recently developed antagonistic antibodies to FcgRIIB and demonstrated that these antibodies blocked rituximab internalization and helped prevent and overcome rituximab resistance in a PDX model of CLL. In this study, we investigated the expression of FcgRIIB in MCL cell lines and primary patient MCL samples, and we assessed the in vivo efficacy of BI-1206, a monoclonal antibody against FcgRIIB, in MCL PDX models. Methods: Flow cytometry analysis was performed to examine the cell surface expression of FcgRIIB in MCL cell lines (n=8) and primary patient MCL samples (n=27). An orthotopic patient-derived xenograft (PDX) model was established from a MCL patient with dual resistance to ibrutinib-venetoclax. In the first mouse cohort, single-agent ibrutinib, venetoclax, or vehicle control were administrated in mice carrying the orthotopic PDX model to assess their in vivo efficacies. In the second mouse cohort, mice were treated with vehicle, single agent BI1206, rituximab plus lenalidomide, or a combination of BI-1206, rituximab, and lenalidomide (triple combination) to assess their in vivo efficacies in the same PDX model. Results: Flow cytometry analysis showed that all 8 MCL cell lines and all 27 primary patient MCL samples expressed high levels of FcgRIIB, highlighting the potential importance of FcgRIIB on the control of therapeutic efficacy in MCL. In the first mouse cohort, we validated the ibrutinib and venetoclax resistance in the PDX model established from a MCL patient with resistance to both therapies. In the second mouse cohort, single agent BI-1206 (10 mg/kg, twice a week) potently diminished PDX growth in spleen, liver, bone marrow, and peripheral blood (Figure 1). Treatment with rituximab (10 mg/kg, twice per week) plus lenalidomide (50mg/kg, daily) or the triple combination showed similar potency (Figure 1). To investigate whether BI-1206 mediates boosting of rituximab-based targeted drug therapies, and/or overcoming of resistance to such therapies, a follow-up experiment with revised treatment setting using the same PDX model or an alternative CD20/FcγRIIb co-expressing PDX model is currently under investigation. Conclusions: All MCL cell lines and all primary MCL cells tested highly express FcγRIIb on the tumor cell surface. Single agent BI-1206 has potent anti-MCL activity in the FcγRIIb-expressing MCL PDX model to overcome ibrutinib-venetoclax dual resistance. Our data suggests FcγRIIb may be an important target for anti-MCL therapies. Disclosures Wang: Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; Juno Therapeutics: Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; Dava Oncology: Honoraria.

Description: BTK inhibitors such as ibrutinib and BCL-2 inhibitors such as venetoclax are two of the most effective therapies used to treat patients with refractory/relapsed mantle cell lymphoma (MCL). However, MCL patients treated with these therapies alone or in combination frequently develop therapeutic resistance, which results in poor clinical outcome. Therefore, targeting the signaling pathways responsible for therapeutic resistance and disease progression will potentially overcome primary and acquired resistance in ibrutinib-venetoclax-refractory/relapsed MCL. Our previous study on a MCL patient cohort comparing ibrutinib-sensitive and -resistant patients revealed that PI3K-ATK signaling is closely associated with ibrutinib resistance. Our recent single-cell transcriptomic profiling of another MCL patient cohort comparing ibrutinib responders and ibrutinib-venetoclax non-responders revealed that PI3K-AKT-mTOR signaling and the G2/M checkpoint were significantly upregulated in ibrutinib-venetoclax non-responders and were highly associated with disease progression. The constitutive PI3K-AKT-mTOR signaling in ibrutinib-resistant or ibrutinib-venetoclax dual-resistant MCL cells may be important in overcoming the G2/M checkpoint and promoting cell survival and cell proliferation. All PI3K isoforms - α, β, γ and δ - are expressed in all the MCL cell lines tested. Copanlisib is a pan-PI3K inhibitor, which targets all PI3K isoforms, and was FDA-approved to treat follicular lymphoma. Compared to idelalisib, a PI3Kδ inhibitor, copanlisib is more potent in targeting MCL cells in vitro with IC50 at a nanomolar range. Copanlisib at low dosage of 6 mg/kg/2d significantly inhibited in vivo tumor growth of an ibrutinib-venetoclax dual-resistant PDX model. PLK1, a central player in regulating G2/M transition, acts upstream of PI3K/AKT signaling via phosphorylating PTEN to cause a tumor-promoting metabolic state. Volasertib, a specific PLK1 inhibitor, is under a Phase III clinical trial for patients with acute myeloid leukemia. Volasertib dramatically arrested MCL tumor cells at the G2/M phase, which caused cell apoptosis at a low nanomolar range. Volasertib at low dosage of 10 mg/kg/week diminished in vivo tumor growth of the ibrutinib-venetoclax dual-resistant PDX model. More interestingly, the combination of copanlisib and volasertib induced synergistic effects in ibrutinib-resistant, venetoclax-resistant, and ibrutinib-venetoclax dual-resistant cell lines in vitro. Moreover, combined treatment with copanlisib (6mg/kg/2d) and volasertib (5mg/kg/week) significantly enhanced anti-MCL activity in the same PDX model, compared to that of either single-agent treatment. Our data demonstrated that pan-PI3K inhibitor copanlisib and PLK1 inhibitor volasertib are potent agents in targeting MCL cells in vitro and in vivo, and they have great potential to overcome ibrutinib and venetoclax resistance in MCL. When combined, copanlisib and volasertib have even greater potential to overcome ibrutinib and venetoclax resistance in MCL. Disclosures Wang: Dava Oncology: Honoraria; Juno Therapeutics: Research Funding; Aviara: Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; BioInvent: Consultancy, Research Funding; Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: Introduction: Mantle cell lymphoma is an aggressive subtype of non-Hodgkin lymphoma with poor prognosis. MCL cells display complex inter- and intra-patient heterogeneity and various dissemination patterns involving nodal and extranodal sites across patients. Due to its microenvironment distinct from patients' microenvironments, heterotopic or subcutaneous patient-derived xenograft (PDX) models are not ideal for studying clinical disease pathology, tumor heterogeneity, and clinical therapeutic responses. In contrast, patient-derived orthotopic xenografts (PDOXs) have been shown in many cancer types to faithfully recapitulate the primary patient cancers. Although a few MCL PDOX models have reportedly been established, these models have not been clearly defined or applied in mechanistic studies or preclinical drug studies. Methods: We established multiple MCL PDOX models from primary patient samples via intravenous or intraosseous routes and characterized these models for the first time in multiple dimensions. We performed longitudinal pathological and histological characterization of PDOX cells involved in the spleen, liver, stomach, lymph nodes, bone marrow, and/or peripheral blood across generations and disease stages, whole exon sequencing across dissemination sites and generations, and single-cell RNA sequencing across dissemination sites. We evaluated in vivo response to ibrutinib and venetoclax in one PDOX model derived from an ibrutinib-venetoclax dual-resistant MCL patient. An in vivo drug efficacy screen was performed to search for potential therapies to overcome ibrutinib-venetoclax dual resistance. Results: Longitudinal pathological and histological characterization of the PDOX models revealed faithful recapitulation of the clinical disease of MCL patients, and the PDOX models can be stably passed on to a series of generations. In vivo drug treatments including ibrutinib and venetoclax recapitulated the clinical response to these therapies. Whole exon sequencing and single-cell RNA sequencing of one ibrutinib-venetoclax dual-resistant PDOX model and its parental primary patient cells reflected reliable recapitulation of primary patient disease in genetic, cellular, and transcriptomic profiles, providing insights into potential targets to overcome therapeutic resistance. Single-cell transcriptomic profiling also revealed increased cancer hallmark signaling in the PDOX model compared to the primary patient samples used to established the PDOX model. Based on this, we performed an in vivo drug screen for the PDOX model and identified promising drugs that dramatically inhibited tumor burden in the spleen, liver, bone marrow, and peripheral blood. Intriguingly, a subcutaneous model (scPDOX) was derived from this PDOX model via subcutaneous implantation of its G1 PDOX cells and was found to be ibrutinib-resistant but venetoclax-sensitive in vivo. To understand the differential response to venetoclax and differential microenvironment of the PDOX model and its derived scPDOX model, whole exon sequencing and single-cell RNA sequencing of the cells from these models is currently being pursued. Conclusions: Our MCL PDOX models faithfully resembled the original MCL disease in histopathology, disease progression, tumor heterogeneity, genetic-transcriptomic profiling, and therapeutic responses. Therefore, these models provide invaluable platforms for mechanistic studies and preclinical drug studies. Disclosures Wang: VelosBio: Research Funding; Guidepoint Global: Consultancy; BioInvent: Consultancy, Research Funding; Juno Therapeutics: Research Funding; Aviara: Research Funding; Dava Oncology: Honoraria; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Celgene: Honoraria, Research Funding.

Description: Introduction: Mantle cell lymphoma (MCL) is a rare and aggressive subtype of B-cell non-Hodgkin's lymphoma with high risk of relapse after frontline therapies. Ibrutinib and venetoclax are two efficacious therapies for refractory/relapsed MCL patients. However, resistance to these therapies occurs frequently and is an urgent unmet clinical need. To understand the underlying mechanism of how intra- and inter-tumor heterogeneity (ITH) and its immune microenvironment contributes to therapeutic resistance, we performed a state-of-art single cell RNA sequencing on longitudinal samples from ibrutinib and venetoclax dual-resistant MCL patients with side-by-side comparison to ibrutinib-sensitive patients in our discovery cohort. To support our novel findings, patient samples from multiple validation cohorts were collected and analyzed via various approaches. Methods: Patient specimens from our discovery cohort that included ibrutinib-sensitive and ibrutinib-venetoclax dual-resistant MCL patients were collected longitudinally and subject to single cell RNA sequencing using 10x genomics. Integrative computational analysis was conducted to uncover the ITH and tumor immune microenvironment at single cell resolution and the underlying mechanism of therapeutic resistance and clonal evolution. To validate the novel findings, additional cohorts of patient samples were collected and subject to bulk RNA sequencing, whole exome sequencing, and multi-color flow cytometry analysis. An orthotopic PDX model was established from one of the ibrutinib-venetoclax dual-resistant MCL patients and was used to validate the novel findings as well as to test the potential therapies in vivo to overcome resistance. Results: To understand the underlying mechanism of heterogeneity and therapeutic relapse, we carried out sequential single cell RNA sequencing on 21 specimens (18,794 cells in total) collected from ibrutinib-sensitive and ibrutinib-venetoclax dual-resistant MCL patients along the course of ibrutinib and/or venetoclax treatments. Integrative computational analysis revealed a high degree of ITH with distinct profiles of cellular and molecular transcriptome. We revealed 15 top cancer hallmarks associated with disease progression and therapeutic resistance, albeit with remarkable clinical, pathological, and genetic-based inter-patient heterogeneity. We observed appearance and clearance of multiple subpopulations in patient blood samples, which likely interprets the clinical ibrutinib-induced lymphocytosis phenomenon at single-cell resolution and disease-progression-associated clonal evolution, which were further validated. Our analysis revealed reprogramming of the tumor microenvironment and tumor immune evasion. Moreover, we revealed multiple actionable targets to help overcome therapeutic resistance as tailored anti-MCL strategies. We found that the 17q gain strongly correlated with this dual resistance and thus targeting survivin located at 17q by YM155 significantly inhibited tumor growth and prolonged mouse survival in the ibrutinib-venetoclax dual-resistant PDX model. Conclusions: This study is the first to describe the mechanisms underlying dual resistance to ibrutinib and venetoclax at the single cell level. We not only identified various pathways underlying this resistance, but also characterized the evolutionary dynamics by using a longitudinal sampling strategy to uncover the underlying mechanisms. We found that the 17q gain highly correlates with ibrutinib-venetoclax dual resistance and showed that inhibition of survivin, located at 17q, overcame this dual resistance. These data provide evidence that 17q gain may be the driving force of disease progression and therapeutic resistance. Moreover, for the first time in MCL, we characterized changes in tumor immune microenvironment and identified a T-cell exhaustion signature correlated with the dual resistance. These changes to the tumor microenvironment strongly suggest the role of immune resistance in mediating dual resistance to ibrutinib and venetoclax in MCL. Disclosures Wang: Lu Daopei Medical Group: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Dava Oncology: Honoraria; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.