Kurzfassung: Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses after frontline therapy. Although the first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has achieved a 68% overall response rate in relapsed/refractory MCL patients, most experience disease progression after ibrutinib treatment. Furthermore, the diverse heterogeneity of molecular alterations in each patient makes improving patient outcome with a uniform regimen extremely challenging. Therefore, identification of effective drug therapies, especially personalized therapeutic strategies are urgently needed. In this study, clinical patient samples were submitted for molecular profiling analysis to identify the dysregulated pathways for each patient. Isolated tumor cells underwent an in vitro drug screen with a panel of clinic drug candidates. Final in vivo efficacy evaluation on the patient derived xenograft (PDX) mouse models could be utilized to predict and validate the clinical response. Methods: We collected fresh peripheral blood specimens, surgical biopsies, bone marrow aspirates and apheresis samples under established IRB-approved protocols. The extracted tumor RNA was subjected to a CLIA-validated nanoString nCounter analysis to interrogate the dysregulated signaling pathways, and whole-exome sequencing (WES) was conducted to reveal the somatic mutations and DNA copy number alterations. High throughput cell viability assays of 23 clinical drug agents targeting multiple pathways associated with MCL pathogenesis were tested per patient sample using the CellTiter-Glo luminescent assay (Promega). Meanwhile, subcutaneous, intravenous and subrenal injections of the purified patient tumor cells were performed on NSG-mice to create corresponding PDX mouse models and these models were used for in vivo validation of rational therapeutic treatment options for precision medicine. Results: We collected and submitted 21 clinical patient samples for molecular profiling analysis and screened them through the designed drug panel. We identified correlations between the WES and nanoString nCounter analysis to interrogate the dysregulated pathways for each patient. The PI3K/mTOR signaling, cell cycle regulation, and apoptosis pathways were among the three most markedly enriched pathways in the relapsed MCL samples based on the hallmark gene set analysis. For example, aberrant apoptosis pathway was identified as the predominant cancer hallmark in one of the patients, the Bcl-2 inhibitor venetoclax and MCL-1 inhibitor AZD5991 were chosen as rational therapeutic treatment options. Indeed, both venetoclax and AZD5991 dramatically induced cell death in both primary patient cells and isolated PDX tumor cells ex vivo. Furthermore, administration of venetoclax at 50mg/kg eradicated the tumor growth in the PDX mouse model established from this patient (p & lt;0.001). In another patient, dysregulated cell cycle pathway (E2F targets and G2M checkpoint) was the predominant cancer hallmark and treatment with CDK4/6 inhibitor abemaciclib (75mg/kg) significantly decreased the tumor size in the PDX model derived from this patient (p & lt;0.0001). Conclusions: Interrogation of dysregulated pathways could be achieved by molecular profiling analysis, and suggest rational treatment options. Follow-up in vitro & in vivo drug efficacy determination facilitates the identification of potential therapeutic options for precision medicine in MCL patient to ultimately improve patient survival outcome. Disclosures Wang: AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Molecular Templates: Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Nobel Insights: Consultancy; InnoCare: Consultancy; Lu Daopei Medical Group: Honoraria; Beijing Medical Award Foundation: Honoraria; Guidepoint Global: Consultancy; Pulse Biosciences: Consultancy; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Acerta Pharma: Research Funding; Verastem: Research Funding; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; Dava Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses.

Kurzfassung: 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.

Kurzfassung: 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.

Kurzfassung: Background Mantle Cell Lymphoma (MCL) is a rare incurable non-Hodgkin's lymphoma despite remarkable recent therapeutic innovations such as CAR T cell therapies. Drug sensitivity and immunotherapy efficacy assays using preclinical tumor models are important in new therapy development. As for preclinical tumor models, in addition to the commonly used mouse model such as patient-derived xenograft (PDX) and genetically engineered mouse models, a 3 rd tumor model, the patient-derived organoid (PDO) in 3D culture model has been established in many types of solid tumors. However, no PDO models have been generated from MCL yet. We have established MCL PDO model by optimizing cell isolation, culture add-ons, spatial and temporal conditions. Our protocol presents a versatile MCL PDO platform, suitable for quick drug screens and applicable for rapid immunotherapy evaluation. For proof of concept demonstration, we tested the therapeutic efficacy of CD19-targeted CAR T-cell in MCL-specific PDOs. The established PDO procedures can be used for diverse biopsies including whole blood, apheresis, bone marrow, lymph node and previously established PDX tumors. Methods For human blood, bone marrow and apheresis biospecimens, we first eliminated red blood cells using RBC lysis buffer. The cells were then spun down and cell pellets were resuspended in culture medium and aliquoted and spun-down into V-shaped 96-well plates at 1-6´10 6 cells per well. After overnight incubation, the cell culture medium was replaced by 60 ml of precooled 60% Matrigel per well. The 3D formed aggregates were gently transferred into 24-well plate and feed the solidified domes with the medium containing an in-house cytokine cocktail. The developed organoids reach the drug screening optimal phase within 3-6 days or it can be further expanded for future use. For PDX tumor and human lymph node biospecimens, we first sliced tumor tissue using surgical scalpel blade, which resulted in small tissue pieces that were then resuspended in HBSS. Debris ( & gt;100 mm) were removed by brief gravity sedimentation. The resultant tissue pieces were then resuspended in 60% Matrigel in culture medium and dispensed onto pre-warmed 24-well plates. The primary tumor organoids were observed for 3-5 days before drug testing on processed for further expansion. For organoid passaging, primary organoids were extracted in pre-cooled medium by mechanically breaking the gel domes. The extracted organoids were digested by collagenase/dispase. The resultant MCL and stromal cells were enforced to aggregate in V-shape wells as described in the apheresis procedure. Organoid cell composition was examined using FACS. Drug sensitivity and T-cell activity against the MCL organoids were assessed using CellTiter-Glo 3D kit. Results We have successfully established and optimized the PDO procedure from diverse MCL biopsies (Fig. 1A). The success rate of MCL organoid from apheresis was & gt; 80% with even higher success rates when using PDX tumors, lymph node and bone marrow samples ( & gt; 90%). One critical step for processing the biopsies is preserving the original stromal cells. FACS analysis showed that although & lt;5% of total cell population is composed of macrophages, circulating fibroblast, epithelial and endothelial cells and they are indispensable for organoid formation and expansion. Addition of hematopoietic cytokines in culture medium significantly improved the organoid formation and expansion from diverse biopsies. FACS with lineage markers revealed that the cell populations of the primary and secondary organoids were not significantly different (Fig. 1B). Tumor-killing activity of the CD19-targeting CAR T cells was assessed by co-culturing the CAR T cells with MCL organoids. Conclusion We have established an MCL PDO platform which is time-efficient, labor-saving, cost-effective and highly reproducible. This platform provides a rapid approach for immune cell activity assays and drug screening. The organoids have been successfully used to generate PDX models. This platform can also be used for investigating the mechanism of drug resistance in the context of different TMEs. Figure 1 Figure 1. Disclosures Wang: Acerta Pharma: Consultancy, Honoraria, Research Funding; BioInvent: Research Funding; Pharmacyclics: Consultancy, Research Funding; Lilly: Research Funding; CStone: Consultancy; Oncternal: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Anticancer Association: Honoraria; VelosBio: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Juno: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Bayer Healthcare: Consultancy; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Imedex: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; Dava Oncology: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Clinical Care Options: Honoraria; Chinese Medical Association: Honoraria; BGICS: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria.

Kurzfassung: 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.

Kurzfassung: Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; 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; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

Kurzfassung: 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.