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The HER2/Neu protein is overexpressed in a large fraction of human breast cancers. NF-κB is one of several transcription factors that are aberrantly activated in HER2-positive breast cancers; however, the molecular mechanism by which HER2 activates NF-κB remains unclear. The CARMA3–BCL10–MALT1 (CBM) complex is required for GPCR- and EGFR-induced NF-κB activation. In the current study, the role of the CBM complex in HER2-mediated NF-κB activation and HER2-positive breast cancer was investigated. Interestingly, HER2-mediated NF-κB activation requires protein kinase C (PKC) activity rather than AKT activity. Using biochemical and genetic approaches, it was shown that the CBM complex is required for HER2-induced NF-κB activation and functionally contributes to multiple properties of malignancy, such as proliferation, avoidance of apoptosis, migration, and invasion, both in vitro and in vivo. In addition, CARMA3-mediated NF-κB activity was required for the upregulation of two matrix metalloproteinases (MMP), MMP1 and MMP13, both of which contribute to tumor metastasis. To further access the physiologic role of CBM complex-mediated NF-κB activation in HER2-positive breast cancer progression, Malt1 knockout mice (Malt1−/−) were crossed with MMTV-Neu mice, in which mammary tumors spontaneously developed with HER2 overexpression. We observed delayed onset and prolonged progression time in mammary tumors in Malt1 knockout mice compared with control mice. In summary, these data demonstrate that the CBM complex is a crucial component mediating HER2-induced NF-κB signaling and tumor malignancy in HER2-positive breast cancer.Implications: The CBM complex bridges key signaling pathways to confer malignant phenotypes and metastatic potential in HER2-associated breast cancer. Mol Cancer Res; 14(1); 93–102. ©2015 AACR.

Supplementary figure 1. Heregulin specifically activates HER2 receptor. Supplementary figure 2. Knockdown efficiency of CARMA3, BCL10 and MALT1. Supplementary figure 3. MALT1 is required for cell migration.

EGF activates NF-κB, and constitutively activated NF-κB contributes to EGFR mutation-associated tumorigenesis, but it remains unclear precisely how EGFR signaling leads to NF-κB activation. Here we report that CARMA3, a caspase recruitment domain (CARD)-containing scaffold molecule, is required for EGF-induced NF-κB activation. CARMA3 deficiency impaired the activation of the IKK complex following EGF stimulation, resulting in a defect of EGF-induced IκBα phosphorylation and NF-κB activation. We found that CARMA3 and Bcl10 contributed to several characteristics of EGFR-associated malignancy, including proliferation, survival, migration, and invasion. Most importantly, CARMA3 contributed to tumor growth in vivo. Our findings elucidate a crucial link between EGFR-proximal signaling components and the downstream IKK complex, and they suggest a new therapeutic target for treatment of EGFR-driven cancers. Cancer Res; 71(6); 2183–92. ©2011 AACR.

Supplementary Figures 1-8 from CARMA3 is Crucial for EGFR-Induced Activation of NF-κB and Tumor Progression

Background Chimeric antigen receptor (CAR) T-cell therapy using brexucabtagene autoleucel (BA) induces remission in many patients with mantle cell lymphoma (MCL), and BA is the only CAR T-cell therapy approved by the FDA for MCL. However, development of relapses to BA is recognized with poor patient outcomes. Multiple CAR T-cell therapies have been approved for other lymphomas and the resistance mechanisms have been investigated. However, the mechanisms underlying BA relapse in MCL have not been investigated and whether any previously reported resistance mechanisms apply to BA-relapsed patients with MCL is unknown. Methods To interrogate BA resistance mechanisms in MCL, we performed single-cell RNA sequencing on 39 longitudinally collected samples from 15 BA-treated patients, and multiplex cytokine profiling on 80 serial samples from 20 patients. Results We demonstrate that after BA relapse, the proportion of T cells, especially cytotoxic T cells (CTLs), decreased among non-tumor cells, while the proportion of myeloid cells correspondingly increased. TIGIT, LAG3, and CD96 were the predominant checkpoint molecules expressed on exhausted T cells and CTLs; only TIGIT was significantly increased after relapse. CTLs expanded during remission, and then contracted during relapse with upregulated TIGIT expression. Tumor cells also acquired TIGIT expression after relapse, leading to the enhanced interaction of tumor cell TIGIT with monocyte CD155/PVR. In myeloid cells, post-relapse HLA-II expression was reduced relative to pretreatment and during remission. Myeloid-derived suppressor cells (MDSCs) were enriched after relapse with elevated expression of activation markers, including CLU (clusterin) and VCAN (versican). Extracellular chemokines (CCL4, CXCL9, CXCL13), soluble checkpoint inhibitors (sPD-L1, sTIM3, s4-1BB), and soluble receptors (sIL-2R, sTNFRII) were decreased during remission but elevated after relapse. Conclusions Our data demonstrate that multiple tumor-intrinsic and -extrinsic factors are associated with T-cell suppression and BA relapse. Among these, TIGIT appears to be the central player given its elevated expression after BA relapse in not only CTLs but also MCL cells. The acquisition of TIGIT expression on tumor cells is MCL-specific and has not been reported in other CAR T-treated diseases. Together, our data suggest that co-targeting TIGIT may prevent CAR T relapses and thus promote long-term progression-free survival in MCL patients.

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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.

Despite significant efficacy of ibrutinib therapy in mantle cell lymphoma (MCL), about one-third of MCL patients will display primary resistance. In time, secondary resistance occurs almost universally with an unlikely response to salvage chemotherapy afterwards. While intense efforts are being directed towards the characterization of resistance mechanisms, our focus is on identifying the signalling network rewiring that characterizes this ibrutinib resistant phenotype. Importantly, intrinsic genetic, epigenetic and tumour microenvironment-initiated mechanisms have all been shown to influence the occurrence of the ibrutinib resistant phenotype. By using in vitro and in vivo models of primary and secondary ibrutinib resistance as well as post-ibrutinib treatment clinical samples, we show that dual targeting of the BCL-2 and PI3-kinase signalling pathways results in synergistic anti-tumour activity. Clinically relevant doses of venetoclax, a BCL-2 inhibitor, in combination with duvelisib, a PI3Kδ/γ dual inhibitor, resulted in significant inhibition of these compensatory pathways and apoptosis induction. Our preclinical results suggest that the combination of venetoclax and duvelisib may be a therapeutic option for MCL patients who experienced ibrutinib failure and merits careful consideration for future clinical trial evaluation.

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 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 R2 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 Graphical Abstract

Mantle cell lymphoma (MCL) is a rare, aggressive and incurable subtype of non-Hodgkin’s B-cell lymphoma. The principal barrier is frequent clinical relapse to multiple lines of therapies, including new FDA-approved biologics and cell therapy. Brexucabtagene autoleucel, the first and only FDA approved chimeric antigen receptor (CAR) T product in MCL, demonstrated unprecedented efficacy in overcoming resistance to Bruton’s tyrosine kinase inhibitors. However, relapses have inevitably occurred and once relapsed these patients display a very poor clinical outcome. Currently, there is no optional therapy specifically designed for these patients. The development of tailored and more efficacious therapies is therefore critical and represents a new medical need. We found that while the receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed across most of the MCL cells, it is significantly elevated in CAR T-relapsed MCL tumors. To see whether this aberrant ROR1 expression contributed to CAR T resistance, we targeted ROR1 using VLS-101, a monomethyl auristatin E conjugated anti-ROR1 antibody. VLS-101 showed potent anti-MCL activity in vitro in ROR1-expressing MCL cell lines and ex vivo in primary patient samples. Importantly, VLS-101 safely induced tumor regression in PDX models resistant to CAR T-cell therapy, ibrutinib and/or venetoclax. These data advocate for targeting ROR1 as a viable approach in the treatment of ROR1-positive MCL tumors, especially those with failure to prior therapies. These data also provide strong evidence for future enrollment of post-CD19 CAR T-cell relapsed MCL patients in a first in-human phase 1b VLS-101 trial. The upcoming testing in a clinical setting will provide important insights on this new therapeutic development aiming to overcome the CAR T resistance via targeting ROR1, which is a rising unmet clinical need in MCL. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01143-w.

Bruton's tyrosine kinase (BTK) is a key mediator of BCR-dependent cell growth signaling and a clinically effective therapeutic target in mantle cell lymphoma (MCL). The molecular impact of BTK inhibition remains unclear particularly in hematopoietic malignancies. We analyzed the molecular mechanisms of BTK inhibition with the novel inhibitor BGB-3111 (zanubrutinib) in MCL models. The efficacy of BGB-3111 was investigated using growth proliferation/cell viability and apoptosis assays in MCL cell lines and patient-derived xenograft (PDX) MCL cells. The activity and mechanisms of BGB-3111 were further confirmed using a cell line xenograft model, an MCL PDX mouse model, and a human phosphokinase profiler array and reverse phase protein array. Finally, the mechanisms related to resistance to BTK inhibition were analyzed by creating cell lines with low levels of BTK using CRISPR/Cas 9 genome editing. We found that inhibition of BTK leads to suppression of tumor growth, which was mediated via potent suppression of AKT/mTOR, apoptosis, and metabolic stress. Moreover, targeted disruption of the BTK gene in MCL cells resulted in resistance to BTK inhibition and the emergence of novel survival mechanisms. Our studies suggest a general efficacy of BTK inhibition in MCL and potential drug resistance mechanism via alternative signaling pathways.

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.

Background Due to excellent clinical response, brexucabtagene autoleucel (BA), an anti-CD19 chimeric antigen receptor T (CAR T) cell therapy, was approved by FDA to treat patients with MCL. However, outcomes of BA-relapsed patients are very poor. Therefore, in this study, we performed multi-omics profiling for BA-treated patients and explore the potential mechanisms of response/resistance to BA therapy in MCL. Methods We performed single cell transcriptomic profiling for longitudinally collected primary patient samples (n = 39) from 15 patients (1 refractory, 14 CR and 5 relapsed). Cytokine multiplex analysis of 80 analytes were performed for longitudinal patient plasma samples (n = 80) for 18 patients (17 CR, 5 relapsed and 1 refractory). 15 patients had data available for both analyses. Functional studies were performed to further validate the findings from multi-omic analysis. Results Using single cell RNA sequencing, we demonstrated that the fraction of CD4/CD8 cytotoxic T cells among total immune cells were significantly reduced at relapse compared to those at pretreatment or at BA remission. Furthermore, T cell exhaustion at relapse was demonstrated by upregulated immune checkpoint molecules TIGIT, CD96, and LAG3 in CD4/CD8 cytotoxic T cells. Ex vivo stimulation induced T cell activation to produce IFNγ and IL-2 in the samples collected at remission, but not at relapse. These data showed an acquisition of exhausted T cell state at relapse. In contrast, a significant elevation in myeloid cells was detected in samples at relapse compared to those at pretreatment or at BA remission. Myeloid-derived suppressive cells were enriched at relapse. Together, these data may indicate tumor immune escape at relapse by inducing a state of T cell exhaustion and expanded MDSCs. To further understand the tumor microenvironment, we performed longitudinal profiling of cytokines in patient plasma. A panel of soluble checkpoint molecules including PD-L1, PD-L2, TIM3, and LAG3 were significantly decreased at remission, compared to baseline and resurrected back to baseline at relapse. Moreover, soluble IL2 receptor (sIL2R) was significantly decreased at remission but was elevated at relapse at levels even higher than baseline. Ex vivo stimulation with IL-2 plus sIL2R enhanced cell growth compared to IL-2, or sIL2R alone in the sample collected at pretreatment, but diminished cell growth in samples collected at relapse. This suggests that sIL2R may play a suppressive function for T cell expansion and activation. Conclusion This study is the first study reporting on exploring potential mechanisms of resistance to BA therapy in patients with MCL. We demonstrate a reprogrammed tumor-immune landscape inducing T cell exhaustion, enrichment of suppressive MDSC, and elevated sIL-2R and check point molecules leading to resistance and relapses after BA therapy. Citation Format: Vivian Changying Jiang, Dapeng Hao, Preetesh Jain, Yijing Li, Qingsong Cai, Yang Liu, Yuxuan Che, Jovanny Vargas, Yixin Yao, Lei Nie, Enyu Dai, Guangchun Han, Ruiping Wang, Kunal Rai, Andrew Futreal, Christopher Flowers, Linghua Wang, Michael Wang. Multi-omics profiling can predict for relapse and response to brexucabtagene autoleucel CAR T-cell therapy in patients with mantle cell lymphoma [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 771.

Blastoid and pleomorphic mantle cell lymphomas (MCLs) are variants of aggressive histology MCL (AH-MCL). AH-MCL can arise de novo (AH-DN) or transform from prior classic variant MCL (AH-t). This study is the first integrated analysis of clinical and genomic characteristics of AH-MCL. Patient characteristics were collected from diagnosis (AH-DN) and at transformation (AH-t). Survival after initial diagnosis (AH-DN) and after transformation (AH-t) was calculated. Regression tree analysis was performed to evaluate prognostic variables and in univariate and multivariate analyses for survival. Whole-exome sequencing was performed in evaluable biopsy specimens. We identified 183 patients with AH-MCL (108 were AH-DN, and 75 were AH-t; 152 were blastoid, and 31 were pleomorphic). Median survival was 33 months (48 and 14 months for AH-DN and AH-t, respectively; P = .001). Factors associated with inferior survival were age (≥72 years), AH-t category, Ki-67 ≥50% and poor performance status. AH-t had a significantly higher degree of aneuploidy compared with AH-DN. Transformed MCL patients exhibited KMT2B mutations. AH-MCL patients with Ki-67 ≥50% had exclusive mutations in CCND1, NOTCH1, TP53, SPEN, SMARCA4, RANBP2, KMT2C, NOTCH2, NOTCH3, and NSD2 compared with low Ki-67 (

Polygalacturonase (PG) is an essential hydrolytic enzyme responsible for pectin degradation and thus plays an important role in fruit softening and other cell separation processes. PG protein is encoded by a multigene family, however, the members of PG gene family in kiwifruit (Actinidia chinensis) have not been extensively identified. In this study, a total of 51 AcPG genes in kiwifruit genome were identified. They are phylogenetically clustered into seven clades, and of them AcPG4 and AcPG18 with other known PG genes involved in fruit softening from peach, pear, papaya and melon form a small cluster together. The members of kiwifruit PG gene family consist of three to nine exons and two to eight introns, and their exon/intron structures are generally conserved in all clades except the clade D and E. During fruit softening of kiwifruit &lsquo, Donghong&rsquo, under ambient temperature, cell wall modifying enzymes, including PG, PL (pectate and pectin lyases), and PE (pectinesterase, also known as pectin methylesterase, PME) showed a different activity profile, and of them, PG and PE activities largely correlated with the change of pectin content and firmness. Moreover, only 11 AcPG genes were highly or moderately expressed in softening fruit, and of which three AcPG genes (AcPG4, AcPG18, and AcPG8, especially the former two) has been found to strongly correlate with the profile of PG activity and pectin content, as well as fruit firmness, suggesting that they maybe play an important role in fruit softening. Thus, our findings not only benefit the functional characterization of kiwifruit PG genes, but also provide a subset of potential PG candidate genes for further genetic manipulation.

Inhibition of the B-cell receptor (BCR) signaling pathway is highly effective in B-cell neoplasia through Bruton tyrosine kinase inhibition by ibrutinib. Ibrutinib also disrupts cell adhesion between a tumor and its microenvironment. However, it is largely unknown how BCR signaling is linked to cell adhesion. We observed that intrinsic sensitivities of mantle cell lymphoma (MCL) cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion signatures were simultaneously downregulated by ibrutinib in the ibrutinib-sensitive, but not ibrutinib-resistant, cells. Among the differentially expressed genes, RAC2, part of the BCR signature and a known regulator of cell adhesion, was downregulated at both the RNA and protein levels by ibrutinib only in sensitive cells. RAC2 physically associated with B-cell linker protein (BLNK), a BCR adaptor molecule, uniquely in sensitive cells. RAC2 reduction using RNA interference and CRISPR impaired cell adhesion, whereas RAC2 overexpression reversed ibrutinib-induced cell adhesion impairment. In a xenograft mouse model, mice treated with ibrutinib exhibited slower tumor growth, with reduced RAC2 expression in tissue. Finally, RAC2 was expressed in ∼65% of human primary MCL tumors, and RAC2 suppression by ibrutinib resulted in cell adhesion impairment. These findings, made with cell lines, a xenograft model, and human primary lymphoma tumors, uncover a novel link between BCR signaling and cell adhesion. This study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and drug development.

The mechanisms driving therapeutic resistance and poor outcomes of mantle cell lymphoma (MCL) are incompletely understood. We characterize the cellular and molecular heterogeneity within and across patients and delineate the dynamic evolution of tumor and immune cell compartments at single cell resolution in longitudinal specimens from ibrutinib-sensitive patients and non-responders. Temporal activation of multiple cancer hallmark pathways and acquisition of 17q are observed in a refractory MCL. Multi-platform validation is performed at genomic and cellular levels in PDX models and larger patient cohorts. We demonstrate that due to 17q gain, BIRC5/survivin expression is upregulated in resistant MCL tumor cells and targeting BIRC5 results in marked tumor inhibition in preclinical models. In addition, we discover notable differences in the tumor microenvironment including progressive dampening of CD8+ T cells and aberrant cell-to-cell communication networks in refractory MCLs. This study reveals diverse and dynamic tumor and immune programs underlying therapy resistance in MCL., Mantle cell lymphoma can be refractory to treatment. Here, the authors used single cell sequencing to study the tumours of patients that were responsive and resistant to treatment and find gains of 17q in resistant tumours, which they attribute to increased expression of Birc5 and validate these findings in mouse models of the disease.

Venetoclax is effective in relapsed patients with mantle cell lymphoma (MCL). Mechanisms of resistance to venetoclax in MCL are poorly understood. We describe the clinical outcomes and genomic characteristics of 24 multiply relapsed patients (median of five prior lines of therapy) who received venetoclax-based therapies; 67% had progressed on BTK inhibitors (BTKi) and 54% had blastoid or pleomorphic histology. Median follow up after venetoclax treatment was 17 months. The overall response rate was 50% and complete response (CR) rate was 21%, 16 patients had progressed and 15 died. The median progression free, overall and post venetoclax survival were 8, 13.5 and 7.3 months respectively. Whole-exome sequencing (WES) was performed on samples collected from seven patients (including five pairs; before starting venetoclax and after progression on venetoclax). The SMARCA4 and BCL2 alterations were noted only after progression, while TP53, CDKN2A, KMT2D, CELSR3, CCND1, NOTCH2 and ATM were altered 2-4-fold more frequently after progression. In two patients with serial samples, we demonstrated clonal evolution of novel SMARCA4 and KMT2C/D mutations at progression. Mutation dynamics in venetoclax resistant MCL is demonstrated. Our data indicates that venetoclax resistance in MCL is predominantly associated with non-BCL2 gene mutations. Further studies are ongoing in MCL patients to evaluate the efficacy of venetoclax in combination with other agents and understand the biology of venetoclax resistance in MCL.

To evaluate the feasibility and effectiveness of conducting a train-the-trainer (TTT) program for stable coronary artery disease (SCAD) management in community settings.The study involved two steps: (1) tutors trained community nurses as trainers and (2) the community nurses trained patients. 51 community nurses attended a 2-day TTT program and completed questionnaires assessing knowledge, self-efficacy, and satisfaction. By a feasibility and non-randomized control study, 120 SCAD patients were assigned either to intervention group (which received interventions from trained nurses) or control group (which received routine management). Pre- and post-intervention, patients' self-management behaviors and satisfaction were assessed to determine the program's overall impact.Community nurses' knowledge and self-efficacy improved (P0.001), as did intervention group patients' self-management behaviors (P0.001). The satisfaction of community nurses and patients was all very positive after training.The TTT program for SCAD management in community settings in China was generally feasible and effective, but many obstacles remain including patients' noncompliance, nurses' busy work schedules, and lack of policy supports.Finding ways to enhance the motivation of community nurses and patients with SCAD are important in implementing community-based TTT programs for SCAD management; further multicenter and randomized control trials are needed.

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 < 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.

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 (>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 > 80% with even higher success rates when using PDX tumors, lymph node and bone marrow samples (> 90%). One critical step for processing the biopsies is preserving the original stromal cells. FACS analysis showed that although 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.

Background Mantle cell lymphoma (MCL) is a rare and aggressive B-cell lymphoma characterized by poor prognosis. Although remarkable therapeutic advances have been made by covalent Bruton's tyrosine kinase (BTK) inhibition and CAR T cell therapy, therapeutic resistance inevitably occurs and leads to dismal clinical outcome. Pirtobrutinib (LOXO-305) is a next-generation, highly selective and non-covalent BTK inhibitor. A phase 1/2 BRUIN study showed that pirtobrutinib demonstrated promising efficacy in heavily pretreated MCL patients with or without prior covalent BTK inhibition. Here, we investigated the mechanism of action of pirtobrutinib in MCL cells in vitro and proposed the potential combination therapy in a venetoclax-resistant xenograft model. Methods MCL cell proliferation was monitored by trypan blue exclusion assay after 24-, 48- and 72-hour treatment with pirtobrutinib and ibrutinib. We performed Annexin V/PI staining to measure the apoptosis inductive effects. Cell cycle analysis using propidium iodide (PI) DNA staining was conducted to compare cell cycle progression kinetics between pirtobrutinib and ibrutinib. We performed RNAseq analysis in Z138 cells to compare differentially expressed genes (DEGs) between pirtobrutinib and ibrutinib treatment. Western blotting was utilized to detect specific signaling proteins. Mino-venetoclax-R cells were inoculated subcutaneously into NSG mice and used for in vivo drug efficacy determination. Results Compared to covalent BTK inhibitor ibrutinib, the novel non-covalent BTK inhibitor pirtobrutinib was more potent in inhibiting MCL cell proliferation in a panel of MCL cell lines, especially in ibrutinib/venetoclax resistant cell lines (pirtobrutinib vs. ibrutinib, p Conclusions Pirtobrutinib overcame both ibrutinib and venetoclax resistance in MCL cells in vitro and in vivo. G2/M checkpoints and E2F targets pathways were significantly enriched in both cases. Pirtobrutinib & venetoclax showed better in vivo efficacy in MCL models than combination of ibrutinib & venetoclax. Figure 1 Figure 1. Disclosures Wang: Genentech: Consultancy; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; CAHON: Honoraria; InnoCare: Consultancy, Research Funding; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Lilly: Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; OMI: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy; Chinese Medical Association: Honoraria; Dava Oncology: Honoraria; Celgene: Research Funding; Mumbai Hematology Group: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; Newbridge Pharmaceuticals: Honoraria; CStone: Consultancy; BGICS: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Scripps: Honoraria; Epizyme: Consultancy, Honoraria; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; VelosBio: Consultancy, Research Funding; Anticancer Association: Honoraria.

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 < 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p 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.

Introduction FDA approval for CD19 chimeric antigen receptor (CAR) T therapy with Brexucabtagene Autoleucel (BA) for relapsed mantle cell lymphoma (MCL) is a milestone advance. However, most patients relapsed after Bruton's tyrosine kinase (BTK) inhibitor ibrutinib therapy and CAR T therapy, making it urgent and essential to uncover the underlying mechanisms that govern resistance to ibrutinib and BA, as well as potential therapeutic targets. Method Two cohorts of primary patient samples were longitudinally collected at pre- and post-ibrutinib therapy (cohort 1, n = 12, samples = 28) or at pre- and post-BA therapy (cohort 2, n = 15, samples = 39) and subject to single-cell RNA sequencing (Figure 1A). Two healthy PBMC samples were included as normal controls. All 67 patient samples were divided into four major groups based on clinical outcomes: samples with fast response to ibrutinib (IBN-S), samples with slow response (IBN-Slow), samples with resistance (IBN-R), and samples with dual resistance to ibrutinib and BA (Dual). Data was integrated across cohorts and experimental batches. In-silico isolation of B cells was followed by differential gene expression analysis using a mixed model with random effect accounting for inter-patient variation. Gene Set Enrichment Analysis (GSEA) was performed to link clinical outcomes to dysregulated cancer hallmarks. Trajectory analysis further modeled the sequential changes resulting in different clinical outcomes. Copy number variation (CNV) analysis was performed to infer chromosomal aberrations. Results A high degree of transcriptomic heterogeneity was observed in tumor cells among patients within the same or across different clinical outcomes. Differential gene expression analysis revealed a set of outcome-specific genes across all patients. A list of 37 genes was consistently altered between Dual and IBN-R samples across both cohorts (FDR To capture the transitions of biological processes between different clinical outcomes, we performed trajectory analysis. The major trajectory stemmed from normal to IBN-S samples, then branched into IBN-Slow and IBN-R/Dual, which further branched into IBN-R and Dual (Figure 1C). A list of differentially expressed genes near the bifurcation point of trajectories (IBN-R/Dual) was identified (adjusted p-value < 2e-16) . To further understand the evolution associated with therapeutic resistance at the genomic level, we conducted the CNV analysis. We then quantified the genome instability score using chromosomal aberrations and observed a significant positive association with tumor aggressiveness (ANOVA test, p-value < 2e-16). Dual samples had the highest score, followed by IBN-R, IBN-Slow, and IBN-S (Figure 1D). Of note, chr22p gain was exclusive to the CAR T-resistant samples (4 out of 6), with specific aberrations in immunoglobulin lambda variable (IGLV) and constant (IGLC) genes, suggesting its association with BA resistance. Together with previously identified DEGs, targetable molecules are under active investigation for therapeutic development to overcome BA resistance. Conclusion In this study, we found co-enrichment of the OXPHOS and MYC pathways in each resistant cohort, suggesting their predominant role in ibrutinib resistance and BA resistance. Trajectory analysis detected genes differentially expressed at the branch point, which may represent novel early drivers of therapeutic resistance and disease progression. Acquired genome instability is positively correlated with tumor aggressiveness. The exclusive chr22p gain and immunoglobulin lambda alterations in Dual samples may suggest novel therapeutic targets to overcome the resistance for relapsed MCL patients. Collectively, our findings gained novel insight into the underlying mechanisms of resistance to ibrutinib and BA, as well as therapeutic vulnerabilities that can be targeted to overcome resistance. Figure 1 Figure 1. Disclosures Jain: Kite: Consultancy; Lilly: Membership on an entity's Board of Directors or advisory committees. Wang: Hebei Cancer Prevention Federation: Honoraria; Dava Oncology: Honoraria; Genentech: Consultancy; Bayer Healthcare: Consultancy; Clinical Care Options: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Imedex: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; InnoCare: Consultancy, Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Mumbai Hematology Group: Honoraria; DTRM Biopharma (Cayman) Limited: Consultancy; Epizyme: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; VelosBio: Consultancy, Research Funding; CStone: Consultancy; BGICS: Honoraria; OMI: Honoraria; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding; CAHON: Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Chinese Medical Association: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Moffit Cancer Center: Honoraria; Newbridge Pharmaceuticals: Honoraria; Scripps: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria.

Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment, and the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of ibrutinib resistant (IBN-R) cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their growth and survival. Recently, glutamine has been proposed to be the essential amino acid in the malignant metabolism, confers those specific tumors with glutamine addiction. Accordingly, such addiction is interplayed with several oncogenic pathways including Myc and mTOR. Our previous studies indicate the increased Myc copies in IBN-R MCL. Therefore, our aim of this work was to uncover the relationship between glutamine addiction with MCL resistance. In the work presented, we focused on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. Targeting glutamine addiction and glutamine-fueled OXPHOS would act as a potential therapeutic strategy in conquering drug resistance in glutamine-addicted MCL. We presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples by immunoblotting. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigations on solid tumors. Inhibiting GLS1 with CB-839 decreases the cell viability in MCL (IC50=0.56nM-308.4nM). Of note, the combinatory treatment of GLS and OXPHOS inhibitor, IACS-010759, induces apoptosis (53% compared with control, and 33%-50% with single agent). Furthermore, the combinatory treatment also shows synergistic effects in decreasing cell viability in IBN-R MCL cell line, Z-138 (CI=0.44), as well as in decreasing mitochondrial membrane potential (≈16% compared with single agent, and 32% with control) and increasing ROS generation (6 times to control, and 3-4 times to single agent). Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the GLS inhibitor, CB-839, may present a clinical potential for a new indication and the combinatory treatment with our in-house inhibitor, IACS-010759, warrants more in-depth investigation as a novel therapeutic regimen. Citation Format: Lingzhi Li, Changying Jiang, Lucy J. Navsaria, Yang Liu, Joseph M. McIntosh, Michael Wang, Yinxin Yao. Exploiting oncogene-conferred glutamine addiction as a therapeutic vulnerability in resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1112.

Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that accounts for 5-8% of all non-Hodgkin lymphomas. Despite the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax (ABT-199) have proven to be effective therapeutic strategies for MCL, most patients often experience disease progression after treatment. Thus, developing a novel drug to overcome this aggressive relapsed/refractory malignancy is an urgent need. Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase belonging to the CDK family which regulates multiple cellular processes, particularly in driving and maintaining cancer cell growth. Unlike classical CDKs, CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex that mediates transcription elongation and mRNA maturation via phosphorylating RNA polymerase II (RNAP2). Previous studies demonstrated that CDK9 inhibition downregulates transcription levels of MCL-1 and MYC, which are crucial in both survival and proliferation of acute myeloid leukemia and diffuse large B-cell lymphoma. We and others found that the MYC signaling pathway was enhanced in MCL, especially in ibrutinib-resistant MCL patients. MYC is a core transcription factor driving lymphomagenesis. It does not possess enzymatic activity and has long been considered to be undruggable. MCL-1 is a key anti-apoptotic protein and is overexpressed in several hematologic malignancies. It was also found to be overexpressed in ibrutinib or venetoclax-resistant MCL cells. Thus, CDK9 is considered as a potential target that may inhibit MYC and MCL-1 pathways. Although recently it was shown that MC180295, a novel selective inhibitor of CDK9, has nanomolar levels anti-cancer potency, whether its beneficial effects extend to relapsed/refractory MCL has not yet been assessed. Methods We use three paired MCL cells sensitive/resistant to ibrutinib or venetoclax to test the efficacy of CDK9 inhibitor MC180295. Cell viability was measured by using Cell Titer Glo (Promega). Cell apoptosis assay and western blot analyses were used to identify affected pathways after MC180295 treatment. Finally, we used patient-derived xenograft (PDX) mouse models to test the therapeutic potential of MC180295 in MCL. Results First, we examined the potential efficacy of a CDK9 inhibitor MC180295 in MCL cells. MC180295 treatment results in growth inhibition of ibrutinib-resistant or venetoclax-resistant MCL cells. By assessing the caspase 3 and PARP activity, we found that MC180295 treatment induces cell death via cell apoptosis in MCL cell lines. Meanwhile, we found that RNAP2 phosphorylation at Ser2, the active form of RNAP2, is downregulated in MC180295 treated MCL cell lines. Consistent to previous studies, MC180295 treatment significantly reduces the protein level of MYC and MCL-1. In addition, we identified several other important proteins, such as cyclin D1 and BCL-XL, were also downregulated upon MCL180295 treatment. MC180295 was able to overcome ibrutinib-venetoclax dual resistance in PDX mouse models without severe side effects. To improve the efficacy of MC180295 as a single agent, we performed in vitro combinational drug screen with a number of FDA-approved or investigational clinical agents and found that MC180295 had a synergistic effect with venetoclax. We are currently investigating the underlying mechanism of action. Conclusion Taken together, our findings showed that targeting CDK9 by its specific inhibitor MC180295 is effective in targeting MCL cells, especially those with ibrutinib or venetoclax resistance and therefore supports the concept that CDK9 is a new target to overcome ibrutinib/venetoclax resistance in MCL. Disclosures Wang: MoreHealth: Consultancy; Dava Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: 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; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Lu Daopei Medical Group: Honoraria; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding.

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.

Background: Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment. Despite the encouraging initial MCL tumor response to ibrutinib (IBN), relapse occurs only after few months of treatment due to multiple resistance mechanisms. Thus, the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of IBN-R cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their grow and survival. The aim of this work was to uncover potential targets responsible for the upregulation of OXPHOS pathway in the refractory/relapsed (R/R) MCL by using multiple biochemical and biological strategies. We focused the present study on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate (α-KG) that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. To further demonstrate the reliance of OXPHOS on glutamine anaplerosis, we have further tested the combinatory effects of targeting GLS and OXPHOS using their respective inhibitors, CB-839 and IACS-010759, on tumor killing activity in R/R MCL. Methods:Primary MCL cells from patient leukapheresis or whole blood specimens, as well as established MCL cell lines were used as experimental models of MCL. Metabolomic profiling was used to determine intracellular metabolite fluxes and levels. Cell Titer Glo assay was used to measure cell proliferation/viability after treatment with inhibitors. Annexin V and propidium iodide were used to measure cell apoptosis and cell cycle arrestviaflow cytometry analysis. Magnetic microbeads-based B-cell isolation method were used for the purification of malignant B cells from patient samples. Western blot analysis was used to evaluate protein level expression. Patient-derived Xenograft (PDX) mouse model created from patients with MCL was used to evaluate the in vivo anti-tumor activity and potential clinical value of GLS and OXPHOS inhibitors. Results:Our recent metabolomic profiling studies have demonstrated that glutaminolysis and OXPHOS are upregulated in IBN-R MCL, manifested by increased glutamine uptake in the ibrutinib-resistant MCL cell lines (p=0.03).Inhibition of glutamine metabolism with the allosteric GLS1-selective inhibitor BPTES resulted in inhibition of cell viability (0.2381uM-9.98uM), indicating that MCL cells are dependent on glutamine metabolism for their proliferation. To corroborate with the above finding, we also presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples and cell lines confirmed by immunoblotting. Inhibiting of GLS would lead to significant reduction in OXPHOS, mitochondria membrane potential and ATP production, as either single drug or in combination with other targeting agents. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigation on solid tumors. Inhibiting GLS1 with CB-839 leads to the decreased cell viability in MCL (0.5626nM-308.4nM). Of note, the treatment with CB-839 to MCL cell lines induces S phase reduction in both Jeko-1 (17.23%) and Z-138 (14.01%), as well as induces significant apoptosis (p=0.013 and p=0.002 in Jeko-1 and Z-138 cells). GLS inhibition will be further explored in the context of mitochondria defect or hypoxia, where OXPHOS maybe deficient. Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. Conclusion:In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the new GLS inhibitor, GCB-839, may present a clinical potential for a new indication and warrants more in-depth investigation. Deciphering the mechanisms involved in MCL metabolic heterogeneity and adaptability during drug resistance development would be crucial to identify key actors enabling MCL cells to escape from therapy. Disclosures Wang: Acerta Pharma:Research Funding;Molecular Templates:Research Funding;InnoCare:Consultancy;Oncternal:Consultancy, Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Targeted Oncology:Honoraria;MoreHealth:Consultancy;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Lu Daopei Medical Group:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Verastem:Research Funding;Nobel Insights:Consultancy;BioInvent:Research Funding;Guidepoint Global:Consultancy;AstraZeneca:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Pharmacyclics:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Janssen:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Loxo Oncology:Consultancy, Research Funding;Pulse Biosciences:Consultancy;OncLive:Honoraria;Beijing Medical Award Foundation:Honoraria;VelosBio:Research Funding.

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.

Opportunistic fungal infections are a leading cause of death among immune-compromised patients, and there is a pressing need to develop new antifungal therapeutic agents because of toxicity and resistance to the antifungal drugs currently in use. Although C-type lectin receptor- and Toll-like receptor-induced signaling pathways are key activators of host antifungal immunity, little is known about the mechanisms that negatively regulate host immune responses to a fungal infection. Here we found that JNK1 activation suppresses antifungal immunity in mice. We showed that JNK1-deficient mice had a significantly higher survival rate than wild-type control mice in response to Candida albicans infection, and the expression of JNK1 in hematopoietic innate immune cells was critical for this effect. JNK1 deficiency leads to significantly higher induction of CD23, a novel C-type lectin receptor, through NFATc1-mediated regulation of the CD23 gene promoter. Blocking either CD23 upregulation or CD23-dependent nitric oxide production eliminated the enhanced antifungal response found in JNK1-deficient mice. Notably, JNK inhibitors exerted potent antifungal therapeutic effects in both mouse and human cells infected with C. albicans, indicating that JNK1 may be a therapeutic target for treating fungal infection.

Crosstalk between tumor cells and other cells within the tumor microenvironment (TME) plays a crucial role in tumor progression, metastases, and therapy resistance. We present iTALK, a computational approach to characterize and illustrate intercellular communication signals in the multicellular tumor ecosystem using single-cell RNA sequencing data. iTALK can in principle be used to dissect the complexity, diversity, and dynamics of cell-cell communication from a wide range of cellular processes.

ROR1 is a transmembrane receptor with tightly controlled expression during embryonic development. While it is expressed on multiple tumor types it is not expressed in normal adult tissues. ROR1-positive B cell non-Hodgkin's lymphomas (B-NHL) include mantle cell lymphoma (MCL), and diffuse large B cell lymphoma. Given its unique expression pattern, ROR1 represents a tumor-specific therapeutic target with little or no normal tissue toxicity. VLS-101, utilizes the UC-961 anti-ROR1 antibody which is conjugated to monomethyl auristatin E (MMAE) via a cleavable linker. VLS-101/ROR1 complex induce internalization facilitates the release of a cytotoxic agent resulting in killing of ROR1-expressing cancer cells. With the rise of novel targeted therapies, resistance has been a major challenge among MCL patients. MCL is one of the most therapeutically resistant and aggressive forms of NHL-B, making it a challenging cancer to treat. The advancement of ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, has been a beneficial change; however, ibrutinib-resistant MCL remains an urgent unmet clinical need. Furthermore, MCLs are either intrinsically resistant to ibrutinib, as well as to venetoclax (Bcl-2 inhibitor) and CAR T-cell therapy, or they may acquire resistance after a short response to these therapies. While many studies have looked into overcoming each of these different types of resistance, no studies have been conducted to overcome a multiple-resistance phenotype simultaneously. Therefore, identifying effective therapies that target these resistance mechanisms is crucial. In this study, we assessed the efficacy of VLS-101 in preclinical models of MCL with intrinsic or aquired resistance to ibrutinib, venetoclax, and CD19 CAR T therapies. By using MCL cell lines, primary patient samples and our established MCL patient-derived xenografts, we explored the expression of ROR1 in MCL and investigated the effects of in vitro and in vivo effects ROR1 targeting using VLS-101. To assess total and cell surface ROR1 expression we used flow cytometry (FACS), western blot analysis, and reverse-transcriptase-polymerase chain reaction (RT-PCR). Cell viability and apoptosis assays were performed to evaluate the in vitro efficacy of VLS-101 in MCL cell lines and primary patient MCL samples. To assess the apoptosis and cell cycle arrest in cells treated with VLS-101, we used annexin V/propidium iodide-staining followed by flow cytometry analyses. In vivo efficacy was tested using various PDX models with various multiple resistances. All 5 cell lines and all 9 PDX models of MCL tested showed high ROR1 mRNA and protein expression levels, whereas 3 out of 4 primary human samples expressed cell surface ROR1. VLS-101 treatment showed anti-MCL activity at the concentrations of 0.3-9 μg/ml in most of cell models tested, which correlated with a significant G2/M cell cycle arrest. Furthermore, VLS-101 induced a time- and dose-dependent apoptosis, as shown by increases in annexin V/propidium iodide-staining. VLS-101 treatment of ibrutinib-venetoclax dual-resistant ROR1+ PDX model resulted in significant regressions of the tumor bearing spleens and livers when compared to vehicle controls (p=0.0001 and p=0.002 respectively). In the ibrutinib-CD19 CAR T dual-resistant ROR1+ model, vehicle-treated animals showed s.c. tumor masses with a mean volume of 1440 mm3, whereas tumor masses were barely palpable (mean = 160 mm3) or had completely regressed in animals treated with VLS-101 at 1.0 mg/kg or 2.0 mg/kg, respectively. VLS-101 showed no adverse effects as monitored by animal clinical observations and weekly body weight measurements. Collectively, these results indicate that ROR1 expression on MCL cells can be utilized for selective targeting. These preclinical data document that the MMAE-containing ADC, VLS-101, can cause cell cycle arrest and induce apoptosis in vitro and safely induce tumor regressions in highly resistant in vivo PDX models of MCL derived from patient tumors. Importantly, our study revealed that even the heavyly pretreated tumors in the clinical setting may express targetable levels of ROR1 and that targeting ROR1 using ADC is a promising approach for the treatment of MCL. Building on these types of results, a Phase 1 clinical trial on VLS-101 (NCT03833180) is ongoing in patients with lymphoid cancers. Disclosures Jessen: VelosBio: Current Employment, Current equity holder in private company; eFFECTOR: Current equity holder in private company. Lannutti:VelosBio: Current Employment, Current equity holder in private company; Gilead Sciences: Current equity holder in publicly-traded company. Wang:Dava Oncology: Honoraria; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; Lu Daopei Medical Group: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; InnoCare: Consultancy; Pulse Biosciences: Consultancy; Juno: Consultancy, Research Funding; Beijing Medical Award Foundation: Honoraria; Nobel Insights: Consultancy; Verastem: Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Guidepoint Global: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria.

Introduction: Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin's lymphoma. It is an incurable disease with frequent relapse from chemotherapies, targeted therapies, and cell therapies. Dysregulated expression of BCL-2 family members resulting in enhanced cell survival frequently occurs in many cancer types and often contributes to the development of therapeutic resistance. The BCL-2 inhibitor venetoclax has been shown to be effective in treating refractory/relapsed MCL patients. However, resistance often occurs and one of the underlying mechanisms of this resistance is the increased expression of other anti-apoptotic BCL-2 family members, such as BCL-XL and MCL-1. In this study, we assessed the in vitro and in vivo efficacy of a novel and highly potent BCL-2/XL dual inhibitor AZD4320 in preclinical models. Methods: Cell viability assay was tested after 72-hour treatment with AZD4320 in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines by CellTiter-Glo (Promega). The assay was also done after a 24-hour treatment in primary PDX cells. Cell apoptosis assay was performed to determine if AZD4320 induces cell apoptosis in MCL cell lines. Furthermore, the in vivo efficacy of AZD4320 was assessed in a CAR-T resistant MCL patient-derived xenograft (PDX) model. Results: AZD4320 significantly inhibited cell proliferation in all tested MCL cell lines, including both ibrutinib/venetoclax-sensitive and -resistant cell lines. It had an IC50 value at a low nanomolar range between 0.59 nM to 18 nM. Consistently, AZD4320 was effective in targeting primary PDX cells ex vivo. AZD4320 induced cell apoptosis in a dose-dependent manner. AZD0466, the nanomedicine formulation of AZD4320 (30mg/kg, weekly, IV), dramatically inhibited tumor growth and prolonged mouse survival in an ibrutinib-CAR-T dual-resistant PDX mouse model. All mice tolerated the treatment dose without any body weight loss. Conclusion: The novel BCL-2/XL dual inhibitor AZD4320 demonstrated excellent anti-MCL activity in both ibrutinib/venetoclax-sensitive and -resistant MCL cells in vitro. This was further validated in vivo in a ibrutinib-CAR-T dual-resistant PDX model. These findings provide evidence that dual targeting of BCL-2 and BCL-XL by AZD4320 is promising as it may overcome therapeutic resistance in relapsed/refractory MCL. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang:OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Loxo Oncology: Consultancy, Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Lu Daopei Medical Group: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; Pulse Biosciences: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Guidepoint Global: Consultancy; Targeted Oncology: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; InnoCare: Consultancy.

Background: Mantle cell lymphoma (MCL) is a distinctive B-cell non-Hodgkin's lymphoma characterized by poor prognosis. Despite clinical success of the covalent Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, a subset of patients need to discontinue ibrutinib therapy due to treatment related adverse events, which are primarily caused by off-target effects. Furthermore, primary or acquired resistance to ibrutinib continues to emerge and often leads to dismal clinical outcomes. Therefore, exploration of more target-specific BTK inhibitors is crucial to minimize the adverse events and provide clinical benefit. CAR T therapy has achieved unprecedented response in patients with relapsed or refractory MCL. However, the development of resistant phenotypes is a new emerging medical challenge in MCL patients with unknown mechanisms. Here, we characterize the therapeutic efficacy of LOXO-305, a next generation non-covalent small molecule inhibitor with high selectivity for BTK. Preclinical efficacy of LOXO-305 alone or in combination with venetoclax (ABT199), a selective Bcl-2 inhibitor, was evaluated in MCL using in vitro and in vivo CAR T-resistant PDX models. Methods : In vitro cell viability was measured after 72 hour treatment with LOXO-305 alone and in combination with ABT-199 in MCL cell lines using Cell Titer Glo luminescent cell viability assay (Promega). To determine whether LOXO-305 induces cell death through cell apoptosis, we used annexin V/PI staining followed by flow cytometry analysis. To evaulate in vivo drug efficacy we used patient-derived xenograft (PDX) models established from primary patient samples. Results: LOXO-305 treatment, as a single agent, resulted in effective MCL cell growth inhibition in a panel of MCL cell lines including ibrutinib and/or ABT-199-resistant cell lines (IC50=6.6-24.4μM), except for JeKo BTK KD cells with BTK knockdown (KD) via CRISPR/Cas9 technology (IC50>30 μM). To improve the efficacy, we decided to investigate the potential of LOXO-305 in combination with ABT199, since the combo of ibrutinib and ABT199 is clinically beneficial in MCL patients. Indeed, LOXO-305 significantly improved the inhibitory effect of ABT-199 in the ABT-199 resistant Mino-R and JeKo BTK KD cells, suggesting that this combination could be further explored in overcoming ABT-199 resistance in MCL. The compelling synergistic effect was further confirmed by annexin V/PI apoptosis assay. Next, we assessed the in vivo efficacy of LOXO-305 in an ibrutinib-CAR T dual-resistant PDX model. LOXO-305 effectively reduced tumor size after 40 days of treatment as a single agent. Moreover, LOXO-305 treatment showed significant anti-tumor effects in an ibrutinib-ABT199-CAR T triple-resistant PDX model that recapitulates the most aggressive human MCL variants invivo. In this model, LOXO-305 treatment effectively decreased the tumor load in mice spleen and liver (p Conclusions: By using various in vitro and in vivo multiple resistant MCL models we determined that LOXO-305 holds great promise for an effective single agent or combined treatment of the most eggressive forms of MCL, and that a continued investigation of the rationale for a combined therapy with ABT-199 is imperative to understand its role in overcoming ibrutinib-ABT199-CAR T triple resistance. Disclosures Wang: OMI: Honoraria, Other: Travel, accommodation, expenses; MoreHealth: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Targeted Oncology: Honoraria.

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 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.