Tumor Microenvironment Molecular Signatures That Define Therapeutic Resistance in Mantle Cell Lymphoma

Introduction The tumor microenvironment of mantle cell lymphoma (MCL), an aggressive and incurable subtype of B-cell lymphoma, is dynamic and complex. The MCL microenvironment provides a niche for the tumor by promoting survival, therapeutic resistance, and immune evasion. Although the intrinsic mechanisms underlying MCL pathogenesis have been well-studied, as demonstrated by our understanding of the important roles that B-cell receptor signaling, the PI3K/AKT/mTOR axis, and OXPHOS play in MCL survival and the development of therapeutic resistance, the extrinsic mechanisms regulated by the lymphoma microenvironment are less well-known. Methods Whole exome sequencing (WES; n = 42) and RNA-seq (n = 76) were performed on fresh peripheral blood or apheresis patient primary samples with an extremely high MCL tumor percentage as determined by flow cytometry versus biopsies with a more diverse cellular mixture, including MCL tumor microenvironment components such as macrophages, T-cells, NK cells, and monocytes. Our previously published MCL cohort was also analyzed in this study (Zhang et al., Science Translational Medicine, 2019). Joint WES and RNAseq mutation calling, expression analysis, and cell type deconvolution from the transcriptome were performed using the BostonGene automated pipeline. Results To characterize the cellular composition and functional state of the MCL tumor microenvironment as well as tumor properties, we created 26 separate molecular signatures related to various functional processes such as anti-tumor immune infiltration, immune checkpoint inhibition, immunosuppression, and stromal compartment represented by angiogenesis and mesenchymal stromal cells. We also utilized these 26 immune and stromal signatures in conjunction with PROGENy (Pathway RespOnsive GENes) analysis to create a signature-based model associated with sensitivity or resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. In this model, increased T-cell, NK cell, and B-cell processes, in addition to p53 pathway activity, were associated with sensitivity to ibrutinib, demonstrating that the tumor microenvironment plays a critical role in the MCL response to this widely used FDA-approved agent. Moreover, initial analysis only identified one BTKC481S mutation in an ibrutinib-resistant MCL sample, again demonstrating that mutations in BTK are rare in ibrutinib-resistant MCL and that diverse mechanisms underlie the development of this resistance. For more in-depth analysis, we performed concurrent analysis of only the biopsy samples in conjunction with an additional previously published cohort (n = 123; Scott et al., JCO, 2017). Unsupervised clustering based on the activities of the proposed signatures produced 4 MCL types as follows: immune infiltration combined with increased stromal signatures (type MCL-A), high immune and checkpoint molecules expression with low stromal expression (type MCL-B), non-immune with increased stromal signature and tumor-promoting cytokines (type MCL-C), and lacking immune infiltration and stromal expression with highest content of malignant B cells (type MCL-D). Interestingly, the ibrutinib-resistant MCL samples primarily belonged to the MCL-C subtype (80%), whereas most of the ibrutinib-sensitive samples (70%) were assigned to subtype MCL-B (Chi-square test p-value = 0.01), which is prominent in anti-tumor immune infiltration without tumor-promoting stromal context, suggesting that ibrutinib may promote immune microenvironment effective action against MCL or work more effectively in an active immune environment. Conclusion The identified enriched immune cell signatures suggest that MCL cells may be sensitive to specific and novel immune checkpoint inhibitors and other immune activators. Ibrutinib sensitivity and resistance are clearly separated based on their tumor microenvironment portraits, suggesting that the tumor microenvironment has a prominent role in regulating ibrutinib activity and response. Furthermore, ibrutinib may alter the tumor microenvironment to promote anti-tumor activity, which requires further investigation. Disclosures Wang: MoreHealth: Consultancy, Equity Ownership; Celgene: Consultancy, Research Funding; Juno Therapeutics: Research Funding; Kite Pharma: Consultancy, Research Funding; Dava Oncology: Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Aviara: Research Funding; BeiGene: Research Funding; BioInvent: Consultancy, Research Funding; VelosBio: Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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Targeting Transcription Checkpoint Using a Novel CDK9 Inhibitor in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140865 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Junwei Lian ◽

Yu Xue ◽

Alexa A Jordan ◽

Joseph McIntosh ◽

Yang Liu ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Mouse Models ◽

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mantle Cell ◽

Cdk9 Inhibitor

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.

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Unravelling the Heterogeneity of Mantle Cell Lymphoma Ecosystem By Single Cell RNA Sequencing

Blood ◽

10.1182/blood-2018-99-110540 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4118-4118

Author(s):

Makhdum Ahmed ◽

Hui Guo ◽

Shaojun Zhang ◽

Lalit Sehgal ◽

Preetesh Jain ◽

...

Keyword(s):

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Single Cell ◽

Research Funding ◽

Cell Lymphoma ◽

Clonal Evolution ◽

Advisory Committees ◽

Cell Clones ◽

Mantle Cell

Abstract Background: Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma that is incurable. MCL has a complex ecosystem of malignant B-cells and stromal and immune cells that play a supporting role for tumor growth, ultimately leading to the potential re-emergence of the disease. The tumor microenvironment has been reported as a crucial factor in MCL pathogenesis and progression. Thus, if we can identify the tumor microenvironment components and define the characteristics of malignant and non-malignant cells, this will pave the way for studying clonal evolution of MCL in vivo. Methods: Both pre- and post-treatment, fresh tumor biopsy samples of MCL were obtained. The cells were dissociated and re-suspended in PBS with >10% serum. A final concentration of 1,200 cells/uL were used for single cell sorting in the chromium system (10X Genomics, California). We sequenced the mRNA in the NextSeq 500 platform. All analysis was conducted using R-programming language (version 3.4). Results: From four MCL patients (L1-L4), we obtained 9,400 cells. Three of the four samples were collected through apheresis (i.e., L1-L3), and one sample (i.e., L4) from surgical biopsy of the involved lymph node. One patient had known TP53 mutated status (i.e., L1) and another patient had CCND1 translocation (i.e., L2). From the apheresis samples (L1-L3), the proportion of lymphocytes was 87%, 68% and 65%. We identified 10 defined clusters of cells based upon their gene expression from all four samples. Six of the 10 clusters were clonal B-cells with strong expression of CCND1, CD79A and CD79B. We also identified clonal T-cells (both CD8+ and CD4+) and monocyte/macrophage clusters. SOX11 expression was absent in one B-cell clone, indicating this clone may be SOX11-negative MCL. The monocyte-macrophage cluster demonstrated strong BCL2 expression, which was not expressed by the B-cells clones. CD19 expression was ubiquitous among the B-cell clones but weaker as compared with other B-cell markers. When the signaling was compared among the four samples, the chemo-resistant cells (sample L1) demonstrated upregulation of NOTCH1 signaling, DNA-damage repair, interferon-alpha response, MYC targets and the HIF1A pathway. Two cell clones did not express any canonical markers and were not identified as a defined cluster. Conclusions: We identified meaningful sub-populations of MCL that define the tumor microenvironment. There is considerable inter-tumor and intra-tumor heterogeneity of MCL at a single cell resolution, which indicates that developing a uniform treatment regimen may prove to be difficult. Ubiquitous expression of CD79A or CD79B may help guiding precision medicine such as the development of novel CAR-T cell therapy. Longitudinal follow up of the same patients may define clonal evolution of MCL and unravel the spatio-temporal interplay. Figure. Figure. Disclosures Wang: AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Novartis: Research Funding; Acerta Pharma: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding.

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FOXO1 Dependent Transcription Network Is a Targetable Vulnerability of Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-153378 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 30-30

Author(s):

Ja-Young Jang ◽

Inah Hwang ◽

Heng Pan ◽

Michael Kluk ◽

Jun Yao ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Research Funding ◽

Cell Lymphoma ◽

Cell Lineage ◽

Mantle Cell ◽

Transcription Program ◽

Privately Held

Abstract Mantle cell lymphoma (MCL) often has an adverse prognosis and despite aggressive multimodal treatment with conventional and targeted therapies, the median survival of MCL patients remains approximately 4 years. Thus, there is a significant unmet need to find novel targets and rational combination treatments. Targeting lineage vulnerabilities driven by specific transcription factors has been broadly confirmed as an effective intervention in many human cancers. To identify the transcription program dependency of MCL cells, we conducted an unbiased domain-focused CRISPR-Cas9 screening against a library of 8,750 sgRNAs targeting 1,434 transcription factors in MCL cell lines (JEKO1, MAVER1, UPN1, and CCMCL1). We identified Forkhead Box O1 (FOXO1), EBF1, PAX5, and IRF4 as 4 transcription factors that are specifically required for MCL survival and growth. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis further revealed that the four transcription factors act together to orchestrate B cell lineage transcriptional program and MCL cell survival. Despite its well-recognized role as a tumor suppressor, FOXO1 has been implicated as a lineage specific transcription factor involved in mature B cell development. Genetic studies revealed a critical role of FOXO1 in germinal center dark zone formation and lymphomagenesis, raising the possibility that FOXO1 acts as a master transcription factor for lineage survival transcription program of MCL. Indeed, hierarchical interaction analysis revealed that FOXO1 functions as a pioneer factor that facilitate the chromatin access of other B cell lineage transcription factors. We demonstrated that interaction of FOXO1 to its cognate motif stabilizes B cell transcription factor complex and supports MCL progression. Along this line, we show that enforced expression of FOXO1 in myeloid leukemia cells induces transdifferentiation and B-cell specific gene expression. Mechanistically, we demonstrate through tiling CRISPR scanning screen that forkhead DNA binding and c-terminal transactivation domains of FOXO1 are specifically required for the viability of MCL cells. Given our finding of FOXO1 as a lineage-specific oncogene in MCL, we next explored the possibility of developing FOXO1-tageted inhibitors. We screened a library of potential small molecule inhibitors of FOXO1 (Forkhead BioTherapeutics) and identified cpd10 as one of the most potent and selective FOXO1 inhibitors (IC 50=76 ×/÷ 1.7. nM) Through the CCMCL1 MCL-NSG preclinical model, we found that cpd10 (100 mg/kg/daily) was well tolerated without overt toxicities. Importantly, prolonged treatment induced a robust cytotoxic response of MCL cells and suppressed MCL progression in vivo. Altogether, our findings identified FOXO1 as a MCL lineage-survival oncogene that can be exploited as a therapeutic target of future drug development. Disclosures Cantley: Petra Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Elemento: Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Janssen: Research Funding; Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy. Belvedere: Forkhead BioTherapeutics: Current Employment. Paik: Forkhead BioTherapeutics: Research Funding.

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Structural Basis of Feedback Control of Oncogenic Signaling in B-Lymphoid Malignancies

Blood ◽

10.1182/blood-2021-149189 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 355-355

Author(s):

Jaewoong Lee ◽

Mark E. Robinson ◽

Ning Ma ◽

Kohei Kume ◽

Dewan Artadji ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Lipid Rafts ◽

Research Funding ◽

Cell Lymphoma ◽

Structural Elements ◽

Oncogenic Signaling ◽

Site Specific ◽

Mantle Cell ◽

Substrate Motif

Abstract Introduction: Multiple structural elements are known that recruit kinases to sites of active signal transduction (e.g. lipid rafts) within the plasma membrane. For instance, pleckstrin homology (PH) domains and the conserved intracellular loop (CIL) motifs recently discovered by us (Lee et al., Nature 2021) direct kinase molecules (Src, BTK, AKT and PI3K) to large signaling clusters in lipid rafts to amplify normal or oncogenic B-cell signaling. While concepts of site-specific recruitment of kinases to initiate signaling are well established, little is known about spatial control of inhibitory phosphatases and how they are directed to sites of maximal signaling strength. Results: Here we discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balances oncogenic signaling strength and is essential for cell-survival in B-cell malignancies, including B-ALL, CLL and mantle cell lymphoma. This was unexpected because CD25 (IL2RA) is known as one of three chains of the Interleukin-2 (IL2) receptor on T- and NK-cells. Studying clinical outcome and gene expression in multiple clinical cohorts, we found CD25 as a top-ranking predictor of poor clinical outcome in patients with B-ALL, mantle cell lymphoma and CLL. Our mechanistic experiments revealed that CD25 expressed on B-cells is monomeric. Unlike T- and NK-cells, CD25 expressed on activated or transformed B-cells does not form a heterotrimeric IL2 receptor with the IL2Rb and common g-chain. Instead of contributing to IL2-signaling, CD25 showed dynamic recruitment either to the B-cell receptor (BCR) or transforming oncogenes within lipid rafts. Conditional deletion of CD25 at earliest stages of B-cell development in mice resulted in profound B-cell defects. However, these defects were not replicated by IL2-deficiency and B-cell development in Il2-/- mice was normal. Inducible deletion of CD25 in genetic models for B-ALL and mantle cell lymphoma revealed an essential function of CD25 in maintaining homeostasis of signaling strength. In the absence of CD25, B-ALL and mantle cell lymphoma cells showed autonomous Ca 2+ oscillations, constitutive activation of Src-family kinases, BTK and ERK as well as the AMPK energy-stress sensor. CD25-deficient B-ALL and mantle cell lymphoma cells initially showed increased proliferation but rapidly died from exhaustion. Consistent with massive accumulation of p53, CD25-deficient B-ALL cells failed to form colonies and to initiate leukemia in transplant-recipient mice. Our mechanistic studies focused on the short (13 residues) cytoplasmic tale of CD25, which features a prominent serine/threonine kinase substrate motif. In vitro kinase assays for 62 candidate serine/threonine kinases revealed PKCd as top-ranking kinase and the serine residue 268 in the tail of CD25 as its principal substrate. The proximity-based labeling assays (BioID) revealed that the CD25 tail recruits PKCd and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts (see Schematic). Consistent with these results, phospho-proteomic studies revealed that inducible deletion of CD25 resulted in acute inactivation of SHIP1 and SHP1 as well as the inhibitory RACK1 scaffold. Multiscale molecular dynamics (MD) simulations revealed that PKCd-mediated phosphorylation of the CD25-tail (S268) tightened interactions with RACK1 and SHIP1 and SHP1 phosphatases, whereas these interactions were destabilized when the CD25-tail was mutated (S268A). Conclusions: Mechanisms of site-specific recruitment of kinases for signal amplification have been extensively studied. However, structural elements that direct inhibitory phosphatases to sites of maximal signaling activity for feedback control remained largely unknown. Here we discovered a PKCd substrate motif in the cytoplasmic tail of CD25 (S268) as a central element to recruit inhibitory phosphatases, scaffolded by RACK1, to curb excessive signaling activity and energy expenditure. High expression levels of CD25 enable higher baseline activity of oncogenic signaling and are associated with poor patient-outcome. Our preclinical studies based on CD25-antibody-drug-conjugates (ADC) revealed that CD25 as a novel target in refractory B-ALL and mantle cell lymphoma. Figure 1 Figure 1. Disclosures Weinstock: SecuraBio: Consultancy; ASELL: Consultancy; Bantam: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

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Mutations Affecting RNA Binding Proteins Are a Novel Feature of Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-129136 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1478-1478

Author(s):

Krysta M Coyle ◽

Prasath Pararajalingam ◽

Sarah E Arthur ◽

Nicole Thomas ◽

Miguel Alcaide ◽

...

Keyword(s):

Alternative Splicing ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Board Of Directors ◽

Research Funding ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cell Lymphoma ◽

Advisory Committees ◽

Mantle Cell

Objectives Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. We sought to extend our understanding of the molecular etiology of this malignancy using an integrative genomic analysis of diagnostic biopsies. Methods We performed exome sequencing on 51 frozen MCL tumors and analyzed these alongside previously published exome cohorts. We sequenced tumour genomes and matched constitutional DNA from 34 frozen MCLs, along with matched constitutional DNA, to more broadly identify the pattern of non-coding mutations. Based on mutations identified in this discovery cohort, we re-sequenced 18 recurrently-mutated genes in 212 archival MCLs, each having clinical follow-up data. We also performed RNA-seq on 110 of these cases and analyzed these data for alternative splicing and differential expression, including the differential splicing of HNRNPH1 in the context of recurrent intronic mutations. We investigated the functional and phenotypic effect of mutations and deregulated HNRNPH1 protein through ectopic expression of full-length HNRNPH1 and a mini-gene containing the exons and introns affected by mutations. Using custom droplet digital PCR (ddPCR) assays, we validated alternative splicing patterns in HNRNPH1 itself and other targets identified through re-analysis of available CLIP-seq data. Results In addition to confirming the prognostic association of TP53 and NOTCH1 mutations in MCL, we identified two additional genes associated with outcome: EWSR1 with poor outcome (HR = 5.6) and MEF2B with good outcome (HR = 0.2). By comparing mutation patterns to diffuse large B-cell lymphoma (DLBCL), we identified an MCL-specific missense hot spot in MEF2B, non-specific truncating mutations in EWSR1, and truncating mutations affecting the DAZAP1 C-terminus in both MCL and DLBCL. The DAZAP1 mutations are predicted to alter protein sub-cellular localization and disrupt protein-protein interactions. We also identified the focal recurrence of non-coding mutations surrounding a single exon of the HNRNPH1 gene that were largely restricted to MCL. These mutations affected a region bound by HNRNPH1 protein and disrupted the preferred binding motif of this protein. Intronic mutations were significantly associated with alternative splicing of the HNRNPH1 mRNA and appear to disrupt a negative regulatory loop that normally limits the level of HNRNPH1. Using cell-based assays, we have evaluated the role of HNRNPH1 in cell survival and proliferation. Our interrogation of alternative splicing events in downstream targets implicate HNRNPH1 as a master splicing regulator which may broadly perturb the transcriptome and proteome to favor lymphomagenesis in MCL. Conclusions We discovered three novel MCL-related genes with roles in RNA trafficking or splicing, namely EWSR1, DAZAP1, and HNRNPH1. Mutations in these RNA-binding proteins were identified in 49 of 291 (17%) samples analyzed. Our results improve the current understanding of the MCL mutational landscape, highlight the similarities and differences between MCL and DLBCL, and strongly implicate a role for aberrant regulation of RNA metabolism in MCL pathobiology. We elucidated a functional role for recurrent non-coding HNRNPH1 mutations specific to MCL and identified multiple downstream targets. We continue to explore putative trans targets of HNRNPH1, a novel oncoprotein in MCL. Disclosures Steidl: Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Bayer: Consultancy; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding. Connors:Bristol-Myers Squibb: Consultancy; Seattle Genetics: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Johnson:Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding. Scott:Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding.

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CD5 Negative Mantle Cell Lymphoma: Clinicopathologic Correlations and Outcome in 58 Cases

Blood ◽

10.1182/blood.v128.22.2964.2964 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2964-2964

Author(s):

Annapurna Saksena ◽

Yuan Miao ◽

Pei Lin ◽

Michael Wang ◽

C. Cameron Yin ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

P Value ◽

Clinicopathologic Features ◽

Advisory Committees ◽

Mantle Cell

Abstract Introduction: Mantle cell lymphoma (MCL) is a B-cell neoplasm that has a characteristic immunophenotype of being positive for CD5, B-cell antigens and cyclin D1. A small subset of cases of MCL can be negative for CD5, approximately 5% in the literature. The clinicopathologic features and prognosis of patients with CD5-negative MCL are poorly characterized. Here, we study a group of patients with CD5- MCL and compare them with a group patients with CD5+ MCL. Methods: From a total of 270 cases of MCL accessioned from 2004-2015, 58 CD5- cases (study group) and 212 CD5+ cases (control group) were identified. All cases of MCL were positive for cyclin D1 by immunohistochemistry and, in most patients, CCND1-IGH was shown FISH. Cases negative for CD5 were assessed by flow cytometry and/or immunohistochemistry. Fisher exact test was utilized to analyze differences between the CD5- and CD5+ groups. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox proportional hazards model analyses for OS and PFS were performed (SPSS 22 software). A P-value of less than 0.05 was considered statistically significant. Results: The CD5- group included 39 men and 19 women with a median age of 66 years (range, 36- 88 years) at time of diagnosis. The CD5- and CD5+ groups shared overlapping clinicopathological features, but CD5- cases showed a lower percentage of men (P=0.006) than CD5+ cases. Treatment information was available for 50 patients. Twenty-nine (58%) patients were treated initially with R-Hyper CVAD therapy (rituximab, fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with high dose methotrexate and cytarabine). Seventeen (34%) patients were treated initially with less aggressive therapy: 7 with R-CHOP; 8 had other rituximab-based chemotherapy regimens; 2 received rituximab as a single agent. Four patients (8%) were observed without therapy. After induction, 34 patients achieved complete remission (CR), 5 patients achieved partial remission (PR), 6 patients showed no response (NR) or progressive disease (PD), and 5 patients lost follow-up. Ten patients also underwent stem cell transplantation (SCT): 5 patients received allogeneic SCT, the other 5 autologous SCT. With a median follow-up of 45.7 months (range, 2.0-174.3 months), 13 of 56 (23.2%) patients died, 43 of 56 (76.8%) patients were alive at last follow-up, and the rest of 2 patients lost follow up. The induction chemotherapy regimens and CR and PR rate were not significantly different between the CD5- and CD5+ groups (p>0.05). Survival analysis showed patients with CD5- MCL had a tendency for longer OS (Figure 1A, P=0.078). Further analysis showed that lack of CD5 expression predicted a superior OS in a few subsets of MCL patients defined with 1) normal WBC count (p=0.049); 2) Stage I/II disease (p=0.046); 3) Low/intermediate MIPI (p=0.041) and 4) Ki67≥30% (at a borderline p value of 0.05). Patients with CD5- MCL also showed a significantly longer progression-free survival (PFS) (Figure 1B, P=0.01). Absence of CD5 expression was associated with a better PFS in MCL patients with advanced disease (stage III-IV) (P=0.035), a normal leukocyte count (P=0.018), a normal serum lactate dehydrogenase level (P=0.046), classical morphology (P=0.029), and low/intermediate MIPI (p=0.0004). Multivariate Cox regression analysis revealed that MIPI was the only independent prognostic factor for both OS and PFS (P=0.026 and P=0.001 respectively) and CR/PR also predict a better OS (P=0.004) in CD5- MCL patients. Conclusion: The clinicopathologic features were similar between patients with CD5- MCL and those with CD5+ MCL, except that less men in the CD5- MCL group. Lack of CD5 expression was associated with a favorable PFS in MCL patients. Recognizing this subgroup of CD5- MCL has not only a diagnostic significance, but also a prognostic significance. Figure Figure. Disclosures Wang: Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; BeiGene: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Onyx: Research Funding.

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Activity of Bruton's Tyrosine Kinase (Btk) Inhibitor PCI-32765 in Mantle Cell Lymphoma (MCL) Identifies Btk As a Novel Therapeutic Target,

Blood ◽

10.1182/blood.v118.21.3688.3688 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3688-3688 ◽

Cited By ~ 3

Author(s):

Sabine Ponader ◽

Sriram Balasubramanian ◽

Lan V Pham ◽

Jun Chen ◽

Archito T. Tamayo ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Research Funding ◽

Actin Polymerization ◽

Cell Lymphoma ◽

B Cell Malignancies ◽

Mantle Cell ◽

Bcr Signaling ◽

Btk Inhibitor

Abstract Abstract 3688 B cell receptor (BCR) signaling is critically involved in the progression of several B cell malignancies, but its role in mantle cell lymphoma (MCL) remains incompletely defined. Bruton's tyrosine kinase (Btk) is a central regulator of BCR signaling and can be selectively and irreversibly inhibited by PCI-32765, which is emerging as a new, molecularly targeted therapy for patients with B cell malignancies. In this study, we explored the role of Btk and the activity of PCI-32765 on BCR signaling in several MCL lines, including Granta-519, Jeko-1, JVM-2, JVM-13, Maver-1, Mino, NCEB-1, Rec-1 and Z-138. Btk and surface IgM protein expression was detected in all MCL lines at variable levels. In a 3-day proliferation assay, JVM-2 & MINO emerged as the most sensitive lines to PCI-32765 (GI50: 1.75–4.4uM). Rec-1 was resistant to PCI-32765 alone (11.3uM) but became much more sensitive (1.45uM) upon BCR stimulation using anti-IgM (10ug/mL). Other lines such as Maver-1, Granta-519 & Jeko-1 all required >10uM of PCI-32765 for inhibition and BCR stimulation did not make much difference. When signaling pathways downstream of BCR activation were studied, intracellular calcium flux following stimulation with IgM was observed in all lines (except JVM-2) and was inhibited at <100nM PCI-32765 in most of them, but no correlation between this and growth inhibition was observed. Constitutive BTK autophosphorylation was observed in all lines and was completely abolished by PCI-32765. BCR stimulation increased p-BTK which was also blocked by PCI-32765 in all lines. Mino and JVM-2 showed constitutive p-ERK activity, which was slightly increased upon BCR stimulation and could be blocked with PCI-32765, whereas the more resistant lines such as Maver-1 and Rec-1 had low endogeneous levels of p-ERK, but which was increased by BCR stimulation and only partially or not reversed by PCI-32765 at 5uM. Little change was observed in levels of p-PLCg1 or p-NF-kB p65. Additionally, in all cell lines stimulation with anti-IgM led to an increased secretion of the chemokines CCL3 and CCL4, which are surrogate biomarkers for BCR-derived activation of neoplastic B cells (Burger JA et al., Blood 113:3050–8, 2009) with greatest increase in JVM-13 and Rec-1 cells. Pre-treatment of these two MCL lines with PCI-32765 significantly inhibited CCL3 and CCL4 secretion in a dose dependent fashion with total abrogation of chemokine secretion at concentrations of 10 mM PCI-32765 (see Figure). Early clinical data indicate that PCI-32765 induces a rapid reduction in lymphadenopathy accompanied by a transient lymphocytosis (in CLL, but also in MCL patients), presumably due to mobilization of the malignant B cells from the tissue compartments into the peripheral blood. Therefore, we analyzed the effect of PCI-32765 (conc. 0.5 and 1 mM) on MCL responses to a lymph node homing chemokine, CXCL13. We found that CXCL13-induced actin polymerization in Rec-1 cells was significantly reduced by PCI-32765, even at lower concentration. We conclude that MCL cells express functional Btk, which is involved in BCR signaling in MCL cells. Blockade of Btk function using PCI-32765 inhibits MCL cell proliferation, BCR signaling, chemokine secretion, and interferes with MCL cell actin polymerization. These findings highlight the importance of BCR signaling and Btk in MCL, help explain the activity of the Btk inhibitor PCI-32765 in MCL patients, and provide biomarkers that may be of value in the clinic. Disclosures: Balasubramanian: Pharmacyclics: Employment. Chen:Pharmacyclics: Employment. Wang:Pharmacyclics: Research Funding. O'Brien:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Burger:Cellgene: Consultancy; Pharmacyclics: Consultancy, Research Funding; Genzyme: Consultancy; Calistoga: Research Funding; Noxxon: Consultancy, Research Funding.

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Transcriptomic Heterogeneity and Clonal Evolution Associated with Therapeutic Resistance in Mantle Cell Lymphoma Revealed By Single Cell RNA-Seq

Blood ◽

10.1182/blood-2019-129134 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5217-5217

Author(s):

Changying Jiang ◽

Shaojun Zhang ◽

Yuanxin Wang ◽

Rongjia Zhang ◽

Yang Liu ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Single Cell ◽

Research Funding ◽

Tumor Heterogeneity ◽

Cell Lymphoma ◽

Clonal Evolution ◽

Therapeutic Resistance ◽

Transcriptomic Profiling ◽

Pdx Model ◽

Mantle Cell

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

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Indolent Mantle Cell Lymphoma: A Distinct Subgroup Characterized by Leukemic Phase Disease without Lymphadenopathy.

Blood ◽

10.1182/blood.v114.22.3937.3937 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3937-3937 ◽

Cited By ~ 3

Author(s):

Dong Chen ◽

David S. Viswanatha ◽

Clive S. Zent ◽

Tait D. Shanafelt ◽

Timothy G. Call ◽

...

Keyword(s):

Bone Marrow ◽

Lymph Node ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

Advisory Committees ◽

Mantle Cell ◽

Leukemic Phase

Abstract Abstract 3937 Poster Board III-873 Introduction Mantle cell lymphoma (MCL), defined by the pathognomic CCND1/IGH translocation, commonly presents with lymphadenopathy and has an aggressive clinical course with poor survival. However, a minority of patients with MCL present with lymphocytosis without substantial adenopathy. The clinical and pathologic features of this group of patients are not currently well understood. Methods Patients with monoclonal B-cell lymphocytosis, positive CCND1/IGH translocation status by FISH analysis and absence of lymphadenopathy were identified in our institutional B-cell lymphoproliferative disorder database. A comprehensive review of clinical, laboratory, and histopathologic features was conducted. Results of ancillary immunophenotypic, molecular genetic, and cytogenetic studies were reviewed. Univariate survival analysis was performed using the method of Kaplan and Meier. Result We identified a total of 35 patients meeting the selection criteria (10 female and 25 male; median age 66 yrs, range 41-86). The median absolute lymphocyte count at presentation was 8.7 × 109/L (range 2.7-180). Eleven patients subsequently developed adenopathy (MCL-LN) while 24 patients had persistent lymphocytosis without adenopathy (MCL-PB). There were no significant differences in age, gender, absolute leukocyte count, percent bone marrow involvement by lymphoma, or immunophenotypic features of the neoplastic B-cells between these 2 subgroups at initial evaluation. All 11 MCL-LN (100%) and 14 of the 24 MCL-PB patients (58%) had splenomegaly at presentation. Morphologic and immunophenotypic features in peripheral blood (n=35), bone marrow (n=34), lymph node (n=7), spleen (n=7) and other involved tissues (n=4) were typical of MCL in 34 patients. One patient had an unusual disease recurrence resembling hairy cell leukemia, but was retained in the analysis given the persistence of the CCND1/IGH abnormality and molecular evidence of clonal identity with previous specimens. All patients with MCL-LN and 12/24 (50%) of patients with MCL-PB received therapy including R-CHOP, cladribine, or rituximab. The remaining 12 MCL-PB patients (50%) were managed expectantly. Patients with MCL-PB (median survival of 156 months from diagnosis) had a significantly better survival than patients with MCL-LN (13 months) regardless of splenic involvement (P=0.0002) (Figure 1). Conclusion Among patients with MCL presenting in leukemic phase without adenopathy, we have identified 2 subgroups with distinct clinical outcomes. Patients who do not develop subsequent lymph node involvement (MCL-PB) have a generally more favorable clinical outcome. These findings imply fundamental differences in the pathobiology and molecular oncogenesis between these subgroups, suggesting the presence of distinct MCL entities. Disclosures: Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Gnentch: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospire: Research Funding; Polyphenon Pharma: Research Funding; Sanofi-Aventis: Research Funding; Biogenc-Ides: Membership on an entity's Board of Directors or advisory committees; Genmab: Membership on an entity's Board of Directors or advisory committees. Witzig:Novartis: Research Funding.

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Biweekly Bendamustine Monotherapy for Relapsed or Refractory Indolent B-Cell Non-Hodgkin Lymphoma and Mantle Cell Lymphoma: A Rabbit-14 Trial

Blood ◽

10.1182/blood.v128.22.5325.5325 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5325-5325

Author(s):

Tadahiko Igarashi ◽

Hisashi Wakita ◽

Hideki Tsujimura ◽

Nobuyuki Aotsuka ◽

Shinichi Masuda ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Research Funding ◽

Cell Lymphoma ◽

Complete Response ◽

Median Number ◽

Treatment Schedule ◽

Random Allocation ◽

Significant Difference ◽

Mantle Cell

Abstract [Background] Bendamustine has demonstrated high response rates in non-Hodgkin lymphomas (NHL). However, standard administration schedule frequently shows delayed hematological recovery resulting in the discontinuation of treatment. Here we designed a novel treatment schedule that could be more tolerable and conducted randomized phase II study (UMIN000008702). [Methods] Patients (pts) with relapsed/refractory(R/R) indolent B-cell NHL and mantle cell lymphoma were randomly assigned to standard arm (120mg/m2 on day1 and 2, every 3 weeks) or Benda-14 arm (120 mg/m2 on day1 and 15, every 4 weeks) of bendamustine monotherapy. Each arm was repeated to 6 cycles and the accomplishment rate (AR) of all scheduled treatment was analyzed as primary end point. [Results] A total of 46 pts were enrolled into the study. Baseline characteristics were: median age 64 years (range 46-78); 48% male; 76% follicular lymphoma; 33% ECOG PS ≥1; 50% having one previous regimen. 65% stage III/IV. 24% bone marrow positive. 33% with bulky mass (>6cm). Using random allocation, twenty two and 24 pts were assigned to standard and Benda-14 arm and the AR of 6 cycles in each arm was 41 and 38%, respectively. The median number of cycles was 4.5 in both arms. Eleven (50%) in standard and 10 (42%) in Benda-14 arm withdrew from protocol due to mainly prolonged hematological toxicities. Three withdrew due to disease progression. Two withdrew due to adverse events (AE). Grade 4 non-hematological AE was observed in one. Overall response rate (ORR) in the standard arm was 77% (95% confidence interval [CI], 59 to 95), including a 50% complete response (CR) compared with 83% (95% CI, 68-99), including a 46% CR in Benda-14 arm.　After a median follow-up time of 16 months, the median event-free survival (EFS) in the standard arm and Benda-14 arm was 14.6 months (95% CI, 8-26) and 15 months (95% CI, 11 - not reached), respectively. There was no significant difference between two arms in AR and in EFS (p=0.431). The overall survival (OS) in both arms was the same of 89% at 15 months. [Conclusions] Although this study did not confirm the superiority of Benda-14 to complete 6 cycles of treatment, Benda-14 arm appears to be equally tolerable and active as the present standard therapy. Benda-14 could be a study arm of next trial that determines better practical strategy for this disease population. Disclosures Igarashi: Zenyaku-Kogyo Inc.: Research Funding. Tsukasaki:Daiichi Sankyo Co., Ltd.: Consultancy; Takeda: Research Funding.

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