MCL Cell Research Articles

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

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.

Targeting the Synthetic Lethality Interaction of MTAP and PRMT5 to Overcome Drug Resistance and Enhance Anti-Cancer Immunity in Mantle Cell Lymphoma

Background: Complex genetic, epigenetic alterations and metabolic reprogramming are among the hallmarks of mantle cell lymphoma, an aggressive B-cell type of non-Hodgkin's lymphoma. CDKN2A and MTAP are frequently co-deleted in human cancers including MCL. Deletion of MTAP gene results in the accumulation of methylthioadenosine (MTA), a selective inhibitor of protein arginine methyltransferase 5 (PRMT5), signifying a dependence of MTAP deleted tumors on PRMT5. PRMT5 plays an important role in normal cellular processes via epigenetic and post-translational modification of RNA splicing, DNA repair and cell cycle regulators. These pathways may be activated in response to targeted therapies to maintain tumor growth and survive chemotherapy. Sustained PRMT5 expression and activity could be therapeutically targeted in MTAP-deficient MCL. Mutations in RNA splicing factors are common in lymphoma and increase the susceptibility to perturbations in splicing events. Deregulated PRMT5 might induce alternative splicing, leading to loss of expression of proteins or epitopes, and subsequent immune evasion and failure of tumor specific immune therapy. Multiple mechanisms may co-exist. Results: CDKN2A and MTAP were commonly co-deleted in ibrutinib-resistant MCL tumors (71%, p = 0.01). To corroborate with this finding, we found that PRMT5 was overexpressed in both primary ibrutinib-resistant MCL and cell lines, with its expression levels inversely correlated with the patient responses to ibrutinib treatment (p = 0.004). Metabolomics analysis revealed elevated S-Adenosyl-methionine (SAM) and S-Adenosyl-homocysteine (SAH) (t-test p <0.001 and p <0.05), PRMT5 cofactor and product, respectively, in ibrutinib-resistant MCL cell lines. PRMT5 plays an important role in the regulation of cell cycle progression. Consistently, treatment with PRMT5 inhibitors induced cell cycle arrest in MCL cell lines (20% and 47% reduction in S phase for JVM13 and JVM2 respectively). Interestingly, treatment with the catalytic inhibitors (GSK3226595 and EPZ015666) led to S-phase stasis in which MCL cells seem incapable of replication. Meanwhile, PRMT5 inhibition with these inhibitors prevented the growth of MCL cell lines as indicated by remarkable decreases in the cell viability (IC50 = 1-1.4 µM and 0.58-0.95µM, respectively). PRMT5 enzymatic activity is required for its function in promoting cell cycle progression and proliferation, as treatment with its inhibitors leads to dramatic reduction in the level of SDMA-containing proteins, especially that of H4R3me2s. More importantly, pharmacological blockade of PRMT5 with single agent GSK3226595 significantly inhibited tumor growth in ibrutinib-resistant MCL PDX models (n = 5, p <0.05), implicating PRMT5 as an actionable target in MCL. PRMT5-mediated alternative splicing may contribute to immune escape and immune suppression. Indeed, our preliminary results revealed that T cells from MCL specimen with high expression of PRMT5 displayed decreased glucose uptake (fold MFI <1-1.23 vs. 2.2), mitochondria mass (p <0.01) and ΔΨm (fold MFI <1-1.26 vs. 2.3), by flow cytometry. Strikingly, the same patient-derived cytotoxic T-lymphocytes (CTL) exhibit reduced cytotoxicity towards autologous lymphoma cells, compared to those derived from healthy donors (p <0.05), strongly implicating impaired T cell metabolic fitness plus immune escape and suppression diminished T cell effector function. Conclusion: We presented evidence that MCL with MTAP deletion confers synthetic lethal dependence on PRMT5, making it an attractive therapeutic target. As an important metabolic and epigenetic regulator, loss of PRMT5 leads to malfunction of the constitutive splicing machinery that induces alternative splicing events, genetic instability and cell cycle arrest. Sustained PRMT5 expression and activity confers tumor aggressiveness and therapeutic resistance in MTAP-deficient MCL. Pharmacological blockade of PRMT5 may dramatically enhance T-cell immunity, while suppressing MCL cell proliferation and tumor growth in vivo. Further successful mechanistic studies would facilitate the identification of novel therapeutics and strategies to overcome resistance to targeted therapies and significantly improve the efficacy of immunotherapy for much better clinical outcomes of MCL. Disclosures Wang: Oncternal: Consultancy, Research Funding; Lu Daopei Medical Group: Honoraria; Juno: Consultancy, Research Funding; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; MoreHealth: Consultancy; Targeted Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; Verastem: Research Funding; OncLive: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; Molecular Templates: Research Funding; Nobel Insights: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; InnoCare: Consultancy.

CDK9 As a New Therapeutic Vulnerability for Ibrutinib Resistance Mantle Cell Lymphoma (MCL)

MCL is an aggressive B-cell lymphoma with poor prognosis due to emergence of drug resistant populations and lymphoma progression. Ibrutinib is a bruton's tyrosine kinase (BTK) inhibitor that was shown to have high response rates in MCL patients. However, as the use of this drug continues to grow in MCL and other B-cell lymphomas, emergence of resistance and fatal progression are of increasing clinical concern. Currently the mechanisms driving IR are poorly understood and no recurrent driver mutations have been identified in MCL. We have modeled acquired resistance to ibrutinib and implemented chemical proteomics and a cell-based drug screen approaches in IR MCL lines and primary samples, we have shown that MCL cells become resistant to ibrutinib through a kinome-adaptive reprogramming mechanism that lead to constitutive activation of the PI3K/AKT/mTOR pathway with increased levels of Myc and sustained transcription activation. The acquired IR MCL cells have increased rates of growth and augmented adhesion to stromal cells. Collectively, our published and preliminary studies indicate that IR MCL relies on global transcriptome remodeling and subsequent kinome reprogramming, leading to molecularly and clinically aggressive phenotype and resistance to ibrutinib therapy. Rather than there being a single mechanism of acquired IR, kinase networks are rewired in a plethora of ways in MCL cells as they become resistant to ibrutinib treatment. Adaptive remodeling of the kinome creates therapeutic challenges, where even combination targeted kinase inhibitor treatments are unlikely to be successful. Here, using genomic, chemical proteomic and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally and importantly, we provided that a novel and facile ex vivo image-based functional drug screening platform could predict clinical therapeutic responses of IR MCL and identified vulnerabilities that can be targeted to disable the evolution of IR. Disclosures Shain: Amgen: Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding; Karyopharm: Research Funding, Speakers Bureau. Shah:Kite/Gilead, Jazz, Incyte: Research Funding; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria; NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Moffitt Cancer Center: Current Employment.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI < 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI > 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Perturbations in HNRNPH1 Splicing and Abundance Affect Global Splicing and Proliferation in Mantle Cell Lymphoma

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 previously discovered non-coding and silent mutations in HNRNPH1 that affect its splicing and contribute to poor outcomes for patients with MCL. We sought to extend our understanding of the mechanisms by which HNRNPH1 contributes to MCL pathology using a combination of in vitro models and integrative analysis of RNA sequencing from MCL tumors. Methods We previously sequenced ribosomal RNA-depleted RNA from 130 MCL tumors. Based on our earlier identification of mutations in HNRNPH1 and altered splicing of this gene, we performed differential splicing analyses using rMATS and leafcutter. We investigated the functional and phenotypic effect of deregulated hnRNP H1 protein through siRNA knockdown. Results Our previous work identified that splicing of HNRNPH1, and not total mRNA expression, correlated with protein abundance in MCL tumors. As a result, our analysis of alternative splicing focused on events associated with altered splicing of HNRNPH1. We identified 155 unique alternative splicing events (ΔPSI > 0.1, FDR < 0.1). Gene ontology analysis identified various aspects of RNA processing which are significantly enriched within this gene list, including mRNA splicing, transport, and metabolic process. This nominates HNRNPH1 as part of the complex network controlling alternative splicing within MCL. Available CLIP-seq in HeLa cells provides evidence for direct interactions between hnRNP H1 and transcripts identified by our analysis (e.g. RBM25, EIF4A1, HNRNPA2B1). Of the 155 events we identified, more than half involved retained introns. Generally, retained introns result in non-productive RNA species, which indicates that this program of intron retention in MCL is a mechanism by which protein abundance can be regulated by hnRNP H1. For all cases with available Mantle Cell Lymphoma International Prognostic Indicator (MIPI) classification, we determined the splicing ratio for HNRNPH1 and observed a general association between high MIPI scores and a lower ratio of non-productive HNRNPH1 transcripts. This suggested that the increased hnRNP H1 abundance we observed in HNRNPH1-mutant tumors contributes to increased proliferation of MCL cells. We verified this in vitro with siRNA knockdown of HNRNPH1 in HEK cells, which resulted in a significant decrease in cell proliferation. Conclusions We have described a pattern of alternative splicing in MCL that is associated with alterations in HNRNPH1 splicing and related protein abundance. The prevalence of retained introns suggests that hnRNP H1 regulates the abundance of protein-coding transcripts via alternative splicing coupled to nonsense-mediated decay. We continue to explore targets of hnRNP H1, a novel oncoprotein in MCL. Disclosures Morin: Celgene: Consultancy.

Abstract 2205: TG-1701, a novel irreversible Bruton's kinase (BTK) inhibitor, cooperates with ublituximab-driven ADCC and ADCP in in vitro and in vivo models of ibrutinib-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare subtype of aggressive B-cell non-Hodgkin lymphoma (NHL) that remains incurable with standard therapy. The first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has proven to be an effective agent for patients with relapsed/refractory MCL, although development of a cysteine to serine mutation at the BTK catalytic site (BTKC481S) or over-activation of the NF-kB pathway can impair MCL response to most BTK inhibitors (BTKis). TG-1701 is a novel irreversible inhibitor highly specific to BTK, with improved selectivity when compared to ibrutinib, currently being evaluated in a phase 1 clinical trial in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) patients. Here we show that, when compared to irreversible and reversible BTKis including ibrutinib, acalabrutinib, and ARQ-531, TG-1701 showed similar cytotoxic activity either in ibrutinib-sensitive or in ibrutinib-resistant MCL cells. We then evaluated the activity of TG-1701 and ibrutinib on anti-CD20 antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), as ibrutinib has previously been show to block rituximab-mediated ADCC due to off-target inhibition of ITK (Kohrt et al., Blood 2014). In sharp contrast with ibrutinib, which partially antagonized rituximab activity, we found that TG-1701 did not impair the anti-CD20-mediated ADCC and ADCP in a multicellular co-culture system that associated MCL to effector cells. In another set of cell-based assays, we showed that the treatment with TG-1701 led to a strong decrease in IL-10 expression, together with a significant increase in IL-2, IL-6 and IL1RN gene transcription, thus associating TG-1701 exposure with the promotion of an immune response within the tumor microenvironment (TME). Accordingly, TG-1701 cooperated with the novel anti-CD20 antibody ublituximab and the novel PI3Kδ-CK1ϵ dual inhibitor umbralisib in reducing the tumor growth in both ibrutinib-sensitive (REC1) and ibrutinib-resistant (UPN-1res) mouse models of MCL. In accordance with in vitro data, the antitumor activity of the drug combination was associated with increased production of IL-2, IL-6 and TNFα, and with an increased infiltration of mouse macrophages and NK cells. Altogether, these data warrant further clinical investigation of TG-1701-mediated engagement of TME-related anti-tumor effect, and clinical evaluation of the triple combination of TG-1701, ublituximab, and umbralisib, is ongoing. Citation Format: Marcelo L. Ribeiro, Diana Reyes-Garau, Meritxell Vinyoles, Marc Armengol, Hari Miskin, Francesc Bosch, Pablo Menendez, Emmanuel Normant, Gael Roue. TG-1701, a novel irreversible Bruton's kinase (BTK) inhibitor, cooperates with ublituximab-driven ADCC and ADCP in in vitro and in vivo models of ibrutinib-resistant mantle cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2205.

CircCDYL Serves as a New Biomarker in Mantle Cell Lymphoma and Promotes Cell Proliferation.

IntroductionMantle cell lymphoma (MCL) is a rare subtype of B-cell lymphoma. Circular (circ) RNA is a member of the non-coding RNA family. However, clinical references to circRNAs in MCL are not clear.MethodsIn this study, we detected the expression level of circCDYL in the plasma of MCL patients compared to healthy donors by the quantitative reverse transcription polymerase chain reaction. The diagnostic value of circCDYL was determined using a receiver operating characteristic (ROC) curve. We constructed a circCDYL short hairpin RNA plasmid and infected the MCL cell line, Z138, to detect its effect on cell proliferation.ResultsCircCDYL was high expressed in the plasma of MCL patients. The ROC curve showed that circCDYL had diagnostic value (area under the curve (AUC) = 0.856). Functionally, circCDYL knockdown inhibited MCL cell proliferation. We conducted bioinformatics analyses and identified a circCDYL-micro (mi)RNA–mRNA/long non-coding (lnc)RNA network, highlighted by five miRNAs (hsa-miR-129-5p, hsa-miR-3163, hsa-miR-4662a-5p, hsa-miR-101-3p, and hsa-miR-186-5p), three lncRNAs (MALAT1, NEAT1, and XIST), and five mRNAs (NOTCH1, FMR1, ABCB1, TWIST1, and VEGFA).ConclusionThese findings indicate that circ-CDYL might serve as a potential diagnostic biomarker in clinical practice.

ROR1 Small Molecule Inhibitor (KAN0441571C) Induced Significant Apoptosis of Mantle Cell Lymphoma (MCL) Cells

Background: ROR1 - a receptor tyrosine kinase (RTK) - is essential for normal embryonic development, but is absent on most normal adult tissues. ROR1 is of importance of cell proliferation, differentiation, survival and metabolism. However, ROR1 is overexpressed in several types of cancer (onco-fetal RTK). MCL is an aggressive and incurable non-Hodgkin lymphoma characterized by translocation (11;14) (q13;q32) and cyclin D overexpression. ROR1 has been described to be highly expressed in MCL cells. We have previously presented results on a small molecule ROR1 inhibitor in CLL (KAN0439834) (Leukemia 32(10):2291, 2018). A second generation ROR1 inhibitor, KAN0441571C, has been developed with improved killing of tumor cells and with longer half-life time (>10h) (PK studies in mice/rats/dogs) compared to KAN0439834. Aim: In this study we examined effects of the ROR1 small molecule inhibitor KAN0441571C in human MCL cells (Granta-519, Jeko-1, JVM-2, Z138, Mino) as part of a pre-clinical evaluation. Methods: ROR1 expression was evaluated by flow cytometry and WB. Cytotoxicity was analysed by MTT and apoptosis by Annexin V/P staining and Western Blot for apoptotic proteins. Cytotoxicity (MTT) was also analysed by combining the ROR1 inhibitor with ibrutinib, acalabrutinib, venetoclax and bendamustine. Effects of KAN0441571C on ROR1 inactivation (dephosphorylation) and signaling pathways were evaluated by WB. Results: All five cell-lines expressed phosphorylated ROR1 (130 kDa) with a varying intensity. Surface expression (flow-cytometry) varied from 0% (JVM-2) to 100% (Mino and JeKo-1). The data indicate expression of also splice variants lacking the extracellular domain. KAN0441571C induced time and dose dependent apoptosis of the five MCL cell-lines which was p53 independent. EC50 varied between 100-250 nM (24h). Apoptosis was confirmed by cleavage of caspase 3 and PARP as well as down-regulation of the MCL-1 and BCL-2 proteins. Moreover, ROR1 was dephosphorylated by KAN0441571C. Downstream of ROR1 both the Wnt canonical and non-canonical pathways were inactivated depending on the cell line. KAN0441571C had in most cell lines a similar cytotoxic effect as ibrutinib, acalabrutinib and venetoclax while bendamustine was inferior. KAN0441571C had an additive effect to ibrutinib, acalabrutinib and venetoclax respectively and KAN0441571C in combination with either of these three agents induced a complete killing of the cell lines. Conclusions: KAN0441571C is a second generation of a novel class of ROR1-tyrosine kinase inhibitor. This small molecule was effective in inducing apoptosis of MCL cells with other mechanisms of action than for drugs in clinical use for MCL. Combination of KAN0441571C with other MCL targeting drugs induced a complete killing of the tumor cell population in a preclinical in vitro model. Our results support the further development of ROR1 small molecule inhibitors as a new therapeutic principle in MCL as well as in other B-cell malignancies with an additive effect to existing targeted therapeutics. Disclosures Schultz: Kancera AB: Employment, Equity Ownership. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Janssen: Research Funding; Kancera AB: Research Funding; BeiGene: Research Funding; Abbvie: Research Funding; Gilead: Research Funding.

Targeting eIF4A Using MZ-735 Potently Induces Cell Death in Lymphoma Cells and Rapidly Represses mRNA Translation at the Global Level and in C-MYC and Other Oncogenes

Introduction: Dysregulated C-MYC signaling is associated with the pathogenesis and poor survival in aggressive lymphomas such as diffuse large B cell lymphoma (DLBCL). Despite intense investigation, direct inhibitors of C-MYC have not yet advanced to the clinic. Various structural elements, such as TOP (terminal oligopyrimidine tract) motif, guanine quartet (CGG) 4 motif, and polypurine sequences (AGAGAG), have been shown to impart high level of dependence on eukaryotic initiation factor 4F (eIF4F). The eIF4F is comprised of 3 subunits, including the mRNA 5ʹ-cap-binding subunit eIF4E, the large scaffolding subunit eIF4G, and the RNA helicase subunit eIF4A. In the current project, we have investigated targeting eIF4A as a novel therapeutic strategy in lymphoma. Pateamine A (PatA) is a natural product originally isolated from a New Zealand marine sponge, Mycale sp. DMDA-PatA, a first generation PatA analogue, has demonstrated in vivo activity in xenograft cancer models. DMDA-PatA acts by decreasing the interaction between eIF4A and eIF4G that specifically inhibits cap dependent translation. MZ-735 is a recently developed PatA analogue that is more potent than DMDA-PatA and less protein bound. Materials and Methods: MZ-735 was synthesized at the CPRIT Synthesis and Drug-Lead Discovery Laboratory at Baylor University. Cytotoxicity was evaluated in diffuse large B cell and Mantle cell lymphoma cell lines using CellTiter-Glo (Promega®). Western blot analysis was performed for the expressions of oncogenes. Global translation was determined using surface sensing of translation (SUnSET) assay and Polysome profile followed by Western blotting and qPCR. RNA sequencing (RNA-seq) was carried out on drug versus dimethyl sulfoxide (DMSO)-treated cells in DLBCL and MCL. An unbiased proteomics analysis was performed using TMT mass spectrometry. Omics data were analyzed by gene set enrichment analysis (GSEA) and Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis. Results: MZ-735 were studied in 13 lymphoma cell lines. MZ-735 exhibited concentration- and time-dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations and showed minimal toxicity in normal PBMC cells. MZ-735 potently inhibits polysome formation, global protein synthesis and inhibits oncogene such as C-Myc and Cyclin D1 in DLBCL and MCL cells. An unbiased quantitative proteomics analysis was carried out to identify the immediate consequence of MZ-735 during a short exposure time of 3 hours. More than 5000 proteins were detected, of which 132 proteins were upregualated and 119 proteins were down regulated by more than twofold after MZ-735 treatment (P < 0.05; fold change, >2). Enrichment analysis suggested that Cancer hallmark proteins such as E2F target proteins, G2/M check point proteins and mitotic spindle proteins were highly enriched among these depleted proteins and the most affected downregulated pathways are cell cycle, cell division and chromosome segregation pathway. We are currently conducting RNAseq to determine whether the mRNA level change in the same or opposite direction as the protein level for any given gene across the genome. Furthermore, we will conduct proteomics and RNAseq in samples treated for longer time points, as the short exposure (3h) favors discovery of depleted proteins with very short half-lives. Conclusion: These results demonstrate that the PatA analogue MZ-735 can potently inhibit the translational apparatus in lymphoma. MZ-735 inhibits metabolic pathways required for cancer progression, leading to growth arrest and apoptosis in the lymphoma cells. MZ-735 and other eIF4A inhibitors represent a promising new class of anti-neoplastic agents, which may be particularly useful for silencing undruggable oncogenes such as C-MYC. Deep insights into the translational responses to eIF4A inhibitors at the single gene level will inform future development of this class of anti-neoplastic agents. Disclosures O'Connor: Allos Therapeutics: Consultancy; Acetylon Pharma: Other: Travel expenses, Research Funding; Celgene: Research Funding; Millenium: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Consultancy, Honoraria; Roche: Research Funding; Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding; Spectrum Pharma: Consultancy, Other: Travel expenses, Research Funding.

Pdox Models Empower Preclinical Drug Evaluation and Mechanistic Studies Via Faithful Recapitulation of the Pathology, Complex Heterogeneity, Genetic-Transcriptomic Landscape, and Therapeutic Response of Mantle Cell Lymphoma

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

Richter-like Pleomorphic Mantle Cell Lymphoma Composed of Epstein-Barr Virus–Positive Hodgkin-like Cells, a Diagnostic Challenge

Abstract Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with distinctive clinicopathologic features including the presence of t(11;14)(q13;q32) in almost all cases. Histologically identifiable variants are well described. Most MCLs are the classic variant, although more aggressive variants including blastoid and pleomorphic exist. The pleomorphic variant is a morphologic subtype composed predominantly of large atypical lymphoid cells. This variant can arise de novo or occur in patients with previous history of MCL as result of disease progression and clonal evolution. Mantle cell lymphoma is characteristically Epstein-Barr virus (EBV) negative. Here, we present an extremely unusual case of pleomorphic MCL that arose in a 69-year-old man with a previous 10-year history of indolent chronic leukemia. This case was unusual and diagnostically challenging because the large and pleomorphic lymphoma cells were EBV positive and had Hodgkin-like morphologic features and only focal cyclin D1 expression. Fluorescence in situ hybridization studies confirmed the presence of the CCND1-IgH gene rearrangement. The disease was clinically aggressive, and the patient died 12 months after diagnosis. Epstein-Barr virus–associated MCL and large cell progressions of MCL are only rarely reported. The additional features we describe, including only focal expression of cyclin D1 and Hodgkin-like morphology, make this an even more unusual and therefore difficult to identify lymphoma. Importantly, this case raises the question as to whether MCL can have histopathologic progressions analogous to the well-established EBV-associated Hodgkin-like Richter transformations of chronic lymphocytic leukemia/small lymphocytic lymphoma.

Targeting EZH1/2 induces cell cycle arrest and inhibits cell proliferation through reactivation of p57CDKN1C and TP53INP1 in mantle cell lymphoma.

ObjectiveTo explore the effect of dysregulation of epigenetic regulator EZH1 and EZH2 on the proliferation in MCL and the underlying mechanisms.MethodsIn this study, we elucidated the role of EZH1 and EZH2 overexpression by immunohistochemistry and correlated them to clinical outcome in 41 MCL patients. Quantitative real-time PCR and Western blot were applied to confirm the level of EZH1 and EZH2 in well-characterized MCL cell lines which were compared to those of naïve B cells. Then we manipulated the expression of EZH1 and EZH2 in MCL cells using CRISPR/Cas9 system to directly investigate their functional roles in MCL. We also evaluated the effect of two small molecule selective inhibitors, EPZ005687 and UNC1999, on MCL cell proliferation, cell cycle distribution and apoptosis in vitro. Finally, we performed RNA-sequencing (RNA-Seq) and Chromatin immunoprecipitation (ChIP) assay to further gain insight into the underlying molecular mechanisms. ResultsWe found that EZH2 protein is overexpressed in approximately half of this cohort of MCL cases. More importantly, the overexpression of EZH2 is associated with poor OS in the patients. Nevertheless, simple EZH2 depletion in vitro has little impact on the viability of MCL cells, predominantly because of the consequent up-regulation of EZH1. Consistently, UNC1999, a dual EZH1/2 inhibitor, unlike the EZH2 selective inhibitor EPZ005687, exerts a potent inhibitory effect on MCL cells. Furthermore, we discover CDKN1C and TP53INP1 as the two important cell cycle regulators, the expression of which are repressed by EZH1/2 mediated epigenetic regulation and are restored by EZH1/2 dual inhibition. ConclusionsOur study suggests that EZH2 participates in the pathogenesis of MCL which may serve as a potential biomarker for prognosis prediction. The dual inhibition of EZH1/2 is a promising therapeutic strategy for MCL.

Abstract 274: The ATR inhibitor BAY 1895344 shows strong preclinical activity in lymphomas and appears associated with specific gene expression signatures

Abstract Introduction. Targeting the DNA repairing pathways represents an intriguing new approach for cancer treatment. BAY 1895344 is a potent, highly selective and orally available ATR inhibitor (Luecking et al, AACR 2017) in its early clinical development in patients with advanced solid tumors and lymphomas (NCT03188965). We performed evaluated its antitumor activity in a large panel of lymphoma cell lines. Methods. IC50s and caspase 3/7 activation were obtained in cell lines derived from human B (n=50) or T cell (n=9), murine (n=2) and canine (n=1) lymphomas exposed to increasing doses of BAY 1895344 for 72h. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation vs controls. Transcriptome data (Illumina HumanHT 12 Expression BeadChips, HTG Biomarker Panel) were analyzed with GSEA (statistical significance: absolute NES >1.5, FDR < 0.01). In vivo efficacy was evaluated in xenograft studies of human lymphoma in mice. Results. BAY 1895344 showed anti-tumor activity with a median IC50 of 60 nM (95%C.I.; 3-500 nM) across the 62 cell lines, comprising mainly germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL, n.=17), activated B-cell DLBCL (8), mantle cell lymphoma (MCL, 10), marginal zone lymphoma (6), T cell lymphomas (9). BAY 1895344 was mostly cytotoxic with apoptosis induction in 38/62 (61%) of the cell lines. Sensitivity was not affected by lymphoma histology or DLBCL cell of origin, TP53/BCL2/MYC status or ATM/ATR loss. A xenograft experiment (MCL Rec-1 cell line) demonstrated complete tumor remission with BAY 1895344 (50 mg/kg, twice daily, 3 days on and 4 days off). Comparing the gene expression profile of 4 less (IC50>200nM) vs 3 very sensitive (IC50<10nM) GCB-DLBCL cell lines, cell cycle regulation and DNA repair genes were positively associated with higher sensitivity (E2F targets; G2M checkpoint; DNA repair), while survival and inflammation transcripts with lower (TNFA signaling; IL2/STAT5 signaling). Similar results for E2F targets, G2M checkpoint, TNFA signaling and IL2/STAT5 signaling were confirmed in MCL cell lines. Finally, we compared the transcriptome obtained using an NGS-based platform in 30 B cell lymphoma cell lines divided by the median IC50. Genes involved in BRCA1 and/or ATM networks, RB1 and/or TP53 targets, WNT signaling were higher in the most sensitive cell lines, while the opposite was true for genes taking part to apoptosis, cytokine interaction pathway, inflammatory response and TNFA signaling via NFκB, and MAPK signaling. Conclusion. A strong anti-tumor activity, associated with specific gene expression signatures, was seen with BAY 1895344 in lymphoma models, providing further support for the on-going phase I study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Luciano Cascione, Alberto Arribas, Emanuele Zucca, Anastasios Stathis, Antje Margret Wengner, Francesco Bertoni. The ATR inhibitor BAY 1895344 shows strong preclinical activity in lymphomas and appears associated with specific gene expression signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 274.

PS1437 SETD2 NON‐GENOMIC LOSS OF FUNCTION IN ADVANCED SYSTEMIC MASTOCYTOSIS (SM): PATHOGENETIC AND THERAPEUTIC IMPLICATIONS

Background:We have recently found that advanced SM patients (pts) and the HMC‐1.1 and −1.2 MCL cell lines display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non‐genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity and homologous recombination (HR). Inhibition of proteasome‐mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3.Aims:In this study, we aimed to identify the different players involved in SETD2 loss of function in ASM and MCL and to verify if they may be considered as promising therapeutic targets. Methods:Western blotting (WB) and immunofluorescence (IF) were used to assess proteins expression. Co‐immunoprecipitation assays were performed to investigate proteins interactions. Apoptosis and clonogenic assays were performed to test drugs efficacy.Results:Compared to cells from healthy donors, SETD2‐ and H3K36Me3‐deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA‐seq in SETD2‐deficient pts showed evidence of transcription and splicing defects like transcription‐induced chimeras and intron retention not observed in healthy donors. Moreover we found that SETD2 co‐immunoprecipitates (co‐IP) with p53 in HMC‐1.1 and −1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with the MDM2 inhibitor SP‐141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP‐141 treatment of HMC‐1 cells at micromolar doses induced cytostatic but not cytotoxic effects. Clonogenic assays supported the cytostatic effects of SP141 in HMC‐1.1 and −1.2 cells. siRNA‐mediated knock‐down of MDM2 also rescued SETD2 expression and activity, supporting the hypothesis that SETD2 hyper‐ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co‐IP also showed that SETD2 interacts with Aurora Kinase A (AKA), that is overexpressed in advanced SM. We found that AKA phosphorylates SETD2 and this phosphorylation may be involved in SETD2 loss of function. In fact, both inhibition by Danusertib and siRNA‐mediated silencing rescued SETD2 expression and activity. Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses.Summary/Conclusion:AKA triggered MDM2‐mediated ubiquitination contributes to SETD2 non‐genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53‐mediated checkpoint without the need for TP53 gene mutations. Inhibiting AKA or MDM2 activity or proteasome‐mediated degradation are promising therapeutic strategies. Supported by AIRC (project 16996) and AIL.

Potent targeting of B cell lymphoma and plasma cell tumors by a tetravalent, Fc-engineered antibody directed against the glycoantigen CD75s.

e14004 Background: Monoclonal antibodies are established treatment options for B cell-derived malignancies, but relapse is still the major challenge. Novel target structures may open alternative avenues to develop effective antibody therapies. Here, we characterized the novel tetravalent antibody ‘EBU-141 Tetra’ and identified the glycoantigen CD75s (α-2,6-sialylated lactosamines) as suitable target structure for antibody-based therapy. CD75s was detected on most B cell lymphomas, including Burkitt’s lymphoma, FL, DLBCL, MCL, CLL, and plasma cell tumors. Classical Hodgkin lymphomas were consistently negative while reactivity on individual cases of peripheral T cell lymphoma was seen. To evaluate CD75s as a target for antibody therapy, we generated a tetravalent, Fc-engineered chEBU-141 IgG1 antibody with enhanced avidity for CD75s and potent effector functions. Methods: ‘EBU-141 Tetra’ was produced by transient transfection and purified by affinity chromatography. Direct anti-tumor effects and Fc-mediated effector functions were investigated in cell proliferation assays, by fluorescence microscopy and in 51Cr release experiments using lymphoma and myeloma cell lines and patient-derived tumor cells. Peripheral blood mononuclear cells and monocyte-derived macrophages of healthy donors were used as human effector cells in the experiments. Results: ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the conventional chimeric antibody chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of ‘EBU-141 Tetra’ against Daudi Burkitt’s lymphoma, U266 plasma cells and CLL patient-derived tumor cells with EC50 values in the low nanomolar range. In addition, ‘EBU-141 Tetra’ demonstrated efficient phagocytosis of Burkitt’s lymphoma and myeloma cell lines. Thus, the novel tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ efficiently recruits immune effector cells for tumor cell lysis. Conclusions: Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma.

Inhibition of PI3K promotes anti-tumor immune responses to Mantle Cell Lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma that is characterized by a chromosomal translocation resulting in overexpression of Cyclin D1, dysregulation of the cell cycle, and constitutive activation of phosphatidylinositol 3 kinase (PI3K). MCL patients also have decreased natural killer T (NKT) cells – an innate-like lymphocyte population involved in initiating the anti-tumor immune response. We hypothesize that constitutive activation of PI3K in MCL alters CD1d-mediated antigen processing and presentation, ultimately resulting in decreased anti-tumor NKT cell responses. To investigate the role of PI3K on MCL survival and immunogenicity in vitro and in vivo, we utilized a panel of PI3K inhibitors (PI3Ki). Pretreatment of human MCL cell lines with micromolar concentrations of PI3Ki resulted in increased apoptosis and enhanced immunogenicity. While dual PI3K/mTOR inhibitor Dactolisib (BEZ-235), completely abrogates NKT/T cell responses of in vitro, stimulated peripheral blood mononuclear cells, PI3K-specific inhibition allows immune cells to function. In a murine model of MCL, PI3Ki-treated animals had decreased tumor burden and increased NKT/T cell responses to ex vivo stimulation with the most significant effect seen in the Buparlisib (BKM-120)-treated group. To elucidate the mechanism, MCL cells were treated in vitro with BKM-120 and flow cytometric analysis indicated increased CD1d expression, but decreased MHC class I expression. These results suggest that selectively targeting PI3K can reduce toxicity, as well as have a significant impact on tumor burden and immune function.

CD200 Expression in Diagnostic and Prognostic Assessment of Mature B Cell Lymphophoproliferative Neoplasms

Background:Multiparameter flow cytometry is a useful tool for diagnostic evaluation of mature B-cell neoplasms (MBN). Recently, it has been shown that assessment of CD200 expression may improve the distinction between chronic lymphocytic leukemia (CLL; CD200 positive) and mantle cell lymphoma (MCL; CD200 negative), but any potential as a prognostic marker for CLL remains to be established.Materials and methods:This cross sectional study was conducted on sixty-seven patients newly diagnosed as having mature B-cell lymphoproliferative disorders Levels of CD 200 in lymphoma cells were assessed.Results:CD200 was consistently expressed in CLL and hairy cell leukemia B cells, but not in MCL cells. Heterogeneous expression was noted in other CD5 positive Non-Hodgkin lymphomas. High CD200 expression (≥50%) was associated with a higher CD5, 19 and CD23 expression, older age, higher TLC and absolute lymphocyte count, hepatomegaly, splenomegaly and a higher Rai stage. There were no significant correlations between CD200 expression and response to treatment.Conclusion:CD200 could be of high value in distinguishing CLL, MCL, and atypical CLL. CD200 expression can also be of prognostic and therapeutic value in CLL cases.

Co-Targeting of BCL2 with Venetoclax and MCL1 with S63845 Is Synthetically Lethal In Vivo in Relapsed Refractory Mantle Cell Lymphoma with Complex Karyotype Changes

IntroductionMantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphomas characterized by (over)expression of BCL2 and good sensitivity to a small molecule BCL2 inhibitor venetoclax. In the present study we analyzed molecular mechanisms of venetoclax resistance in MCL cells, and tested strategies to overcome it based on concurrent targeting of BCL2 a MCL1.MethodsCell death was determined by flow cytometry using Annexin-V/PI staining. Establishment of MCL cell clones with knock-down or transgenic overexpression of MCL1, BIM and NOXA, western blotting, immunohistochemistry of formalin-fixed paraffin-embedded tissue sections, and immunoprecipitation experiments were carried out as previously described (Klanova et al, Clin Cancer Res, 2016). All PDXs were derived in our laboratory from patients with relapsed MCL. All PDX were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells from which they were derived. Samples were sequenced using SureSelectXT Human All Exon V6+UTR (Agilent Technologies, Santa Clara, CA) on the NextSeq 500 (Illumina, San Diego, CA) instrument according to manufacturer's protocols. Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapy was initiated when all mice developed palpable subcutaneous tumors (= day 1, D1). Venetoclax (VTX) and S63845 were from MedchemExpress, carfilzomib (CFZ) was from Charles University General hospital pharmacy. Carfilzomib (4 mg / kg) was administered intravenously (IV) on days 1 and 6. Venetoclax (40 mg / kg) was given by oral gavage on days 1, 2, 3, 6 and 7. S63845 (25 mg / kg) was administered IV on days 1, 2, 3, 6 and 7. Tumor volumes were calculated using the following formula: π / 6 × tumor length × width × height.ResultsBy transgenic overexpression or shRNA-mediated knock-down we confirmed key roles of proapoptotic proteins BIM and NOXA in mediating venetoclax-induced cell death in MCL. We demonstrated that both BIM and NOXA are differentially expressed between MCL cell lines on one side, and primary MCL cells and patient-derived xenograft (PDX) cells on the other side. First, NOXA protein is significantly overexpressed in most MCL cell lines. Second, biallelic deletions of BIM harbored by three commonly used MCL cell lines (JEKO-1, MINO and Z138), and previously reported to be present in approx. 30% of MCL patients, were not found in primary MCL cells. As a consequence, vast majority of the in vitro data was implemented on venetoclax-sensitive cell lines HBL2 and MAVER-1, whose patterns of expression of BCL2, MCL1, BIM and NOXA are similar to primary MCL cells. We demonstrated that MCL1, another key anti-apoptotic protein, plays an essential role in mediating resistance to venetoclax. First, MCL1 functions as a buffer for BIM released from BCL2 upon binding of venetoclax thereby preventing activation of BAX and induction of apoptosis. Second, marked upregulation of MCL1 protein was associated with acquired resistance to venetoclax in two most sensitive MCL cell lines HBL2 and MAVER-1. Based on the in vitro data we proposed two experimental treatment strategies that co-targeted MCL1 (along with inhibition of BCL2 with venetoclax): a direct blockage with a highly specific small molecule MCL1 inhibitor S63845, and an indirect blockage achieved by proteasome inhibitor carfilzomib that upregulates the proapoptotic protein NOXA that specifically binds and blocks MCL1. The combination of venetoclax and S63845 demonstrated synthetic lethality in vivo inducing the longest “remissions” of MCL bearing mice (i.e. temporary disappearance of subcutaneous MCL tumors) using a panel of four different PDXs derived from patients with relapsed / refractory MCL with complex karyotype changes (Figure 1). The combination of carfilzomib and venetoclax was far less effective, and at the same time more toxic suggesting functional blockage of MCL1 induced by overexpressed NOXA is either incomplete or insufficient.ConclusionsOur data strongly support investigation of venetoclax in combination with S63845 as an innovative proapoptotic treatment strategy for chemoresistant MCL patients with adverse cytogenetics in the clinical grounds. [Display omitted] DisclosuresTrněný:Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Gilead: Honoraria; Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Abbvie: Honoraria, Research Funding; F. Hoffman-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Sandoz: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board.

SAMHD1 Is Variably Expressed in Mantle Cell Lymphoma and Correlated to SOX11 but Not to Survival

IntroductionMantle cell lymphoma (MCL) is an incurable disease with a median survival of 3-5 years. Most cases express SOX11, a transcription factor associated with MCL pathobiology, which serves as a diagnostic marker. Current standard first-line therapy for younger MCL patients is rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and cytarabine (Ara-C), consolidated with high dose chemotherapy with autologous stem cell transplantation (ASCT), which is associated with prolonged survival (Eskelund et al. Br J Haematol 2016). Ara-C metabolizes into its active triphosphate form Ara-CTP which inhibits proliferation of the malignant cells. Recent reports suggest that the expression of the mammalian dNTPs hydrolase SAMHD1 determines response to Ara-C in acute myeloid leukemia (AML) by hydrolyzing Ara-CTP and thus by diminishing the anti-proliferative properties of Ara-C. Consequently, in vitro downregulation of SAMHD1 resulted in sensitization of AML cells to Ara-C (Herold et al., Nat Med 2017). SAMHD1 also exhibits anti-tumor properties via regulation of dNTP pool and is recurrently mutated in CLL (Clifford et al. Blood 2014) and T-PLL (Johansson et al. Blood Cancer J. 2018). In CLL a role in DNA-repair has been suggested (Clifford et al. Blood 2014). Thus, SAMHD1 may function as a tumor suppressor. In this study, we investigated for the first time the expression patterns of SAMHD1 in MCL and its association to clinical outcome, especially in patients receiving Ara-C as part of induction for ASCT.MethodsSAMHD1 and SOX11 expression was investigated by qPCR, WB and IHC on whole tissue sections or tissue microarrays. MCL cell lines were treated with gene-specific siRNA for SAMHD1 or SOX11. Data on overall survival (OS) was retrieved from patient records. For patients included in the Nordic MCL2 and MCL3 trials data on progression free survival (PFS) and OS were retrieved.ResultsInitial IHC showed that expression of SAMHD1 is high in normal T-cells and macrophages and low in cells in the mantle zones of reactive tonsils. Co-staining with CD20 in a heterogeneously treated cohort of primary MCL (n=104) showed a large variation between cases (median 73.15%, range: 0.4%-99.6%) and the staining intensity was lower than in T-cells. Sixty two/104 samples were also evaluated for SOX11 expression and the percentage of SOX11 positive cells moderately correlated with SAMHD1 expression (Spearman correlation coefficient 0.27, p=0.036). Analysis of mRNA expression (normalized to mRNA levels of respective genes in Granta519 cell line) of SAMHD1 (median RFI: 2.09, range: 0.18-10.69) and SOX11 (median RFI: 2.00, range: 0.00-7.11) in flow cytometry sorted primary MCL cells (n=19) showed a trend for correlation (Spearman correlation coefficient 0.45, p=0.053). However, downregulation of SOX11 by siRNA did not alter the expression of SAMHD1 and neither did downregulation of SAMHD1 by siRNA change the expression of SOX11 suggesting that the observed correlation is not due to a mutual regulation. We investigated the relation of SAMHD1 expression to clinical and pathological parameters in this cohort and found no correlation to OS, blastoid morphology, proliferation (% Ki67+ tumor cells) nor p53 positivity (>20% positively stained cells). Further, a cohort of patients treated within the Nordic MCL2 and MCL3 protocols (n=51) were investigated for SAMHD1 expression by IHC. Interestingly the distribution of SAMHD1 expression was different in this cohort (median 34.9%, range: 2.2%-97.1%; Mann-Whitney p=0.0019). Survival analysis showed a trend for better PFS and OS in patients with high (>75% SAMHD1 positive cells) and low (<25% positive cells) SAMHD1, although the differences were not significant (Kaplan-Meier with log rank test, p=0.053).ConclusionsIn MCL the expression of SAMHD1 varies over a broad range and correlates to expression of SOX11. However, no significant difference in PFS or OS among patients receiving Ara-C containing induction chemotherapy is found in this study. DisclosuresNo relevant conflicts of interest to declare.

Ibrutinib-Resistant Mantle Cell Lymphoma Undergoes Metabolic Reprogramming Towards Oxphos

IntroductionMantle cell lymphoma (MCL) is an incurable B-cell lymphoma subtype and constitutive activation of the B-cell receptor pathway is a hallmark of B-cell lymphomas. Bruton's tyrosine kinase (BTK) is a critical component of the B-cell receptor pathway, and ibrutinib, a first-in-class, once-daily, and oral covalent inhibitor of BTK, was developed to reduce/silence B-cell receptor pathway activity, leading to clinically remarkable anti-tumor activity. In our prior multiple-center Phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68% (Wang et al., NEJM, 2013), surpassing the effectiveness of other therapies. Although ibrutinib is extremely efficacious in patients with relapsed/refractory MCL, the one-year overall survival rate of ibrutinib-exposed patients who relapse is only 22%.MethodsPatient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with reverse phase protein array (RPPA) analysis of ibrutinib-sensitive and -resistant MCL cell lines. Metabolic assays including the measurement of mitochondria respiration rates with the Seahorse analyzer and reactive oxygen species (ROS) levels, targeted metabolomics, and ATP analysis. Functional studies targeting this molecular pathway were conducted, including in vitro cell viability and apoptosis assays, as well as in vivo efficacy studies in an ibrutinib-resistant MCL patient-derived xenograft mouse model.ResultsWES data analysis identified frequent inactivating somatic alterations in ATM, KMT2D, and TP53 in both the ibrutinib-sensitive and -resistant tumors. CDKN2A (5/7, 71%) was frequently deleted, and the deletion was only observed in the ibrutinib-resistant tumors (p = 0.010). The RNA-seq analysis identified a total of 63 protein-coding genes as the most differentially expressed genes (DEGs) between the ibrutinib-resistant and -sensitive groups, with a fold change of ≥ 2 or ≤ -2 and the false discovery rate (FDR q-value) ≤ 0.01. Among the DEGs, 26 genes were upregulated in ibrutinib-resistant tumors. In addition, gene set enrichment analysis (GSEA) revealed the marked upregulation of oncogenic pathways including c-MYC, mTOR (mTORC1), Wnt, and NF-ĸb signaling, followed by cell cycle, apoptosis, BCR signaling and DNA repair in the ibrutinib-resistant tumors. Notably, in addition to these oncogenic pathways, the metabolic pathways, including oxidative phosphorylation (OXPHOS), were significantly enriched in the ibrutinib-resistant tumors (normalized enrichment score > 3 and FDR q-value < 1e-5). In further support of this finding, metabolomics analysis and the measurement of ATP production and mitochondrial respiration indicated that the OXPHOS pathway is the predominant metabolic pathway employed by ibrutinib-resistant MCL cells. To determine the effects of targeting these pathways, OXPHOS was inhibited with a novel electron transport complex I inhibitor (IACS-010759, developed by MD Anderson Cancer Center) in both MCL cell lines and ibrutinib-resistant MCL patient-derived xenograft (PDX) models. Single agent IACS-010759 treatment at 10 mg/kg oral gavage for 5 consecutive days/week completely prevented tumor growth compared with the vehicle control as shown by measuring tumor volume (n = 5, p < 0.0001) and human β2M levels (n = 5; p < 0.0001) throughout treatment. No apparent toxicities were observed in the IACS-010759-treated MCL PDX mice.ConclusionThis current study warrants the exploitation of active cancer metabolic pathways, especially OXPHOS, to improve the clinical outcomes of MCL and additional lymphoma, which is actively being investigated in a Phase I lymphoma clinical trial (NCT03291938). DisclosuresWang:AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria.