Activated B-cell Type Diffuse Large B-cell Lymphoma Research Articles

GRK2, an Inhibitor of MALT1-Dependent Oncogenic Signaling, Is Downregulated By microRNA in ABC-DLBCL

Background: The “CBM” complex, composed of the scaffolding protein CARMA1, the adaptor protein BCL10, and the effector protein MALT1, performs multiple pivotal functions as a mediator of antigen receptor-dependent induction of the NF-κB transcription factor and subsequent lymphocyte activation. The effector molecule of the CBM complex, MALT1, regulates downstream signaling via two important functions: first, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of NF-κB signaling. Deregulated/constitutive activation of MALT1, which can result from gain-of-function mutations in either B cell receptor (BCR) or CARMA1 or from chromosomal translocation involving the MALT1 gene, underlies the pathogenesis of a variety of lymphoid malignancies including activated B-cell type-diffuse large B-cell lymphoma (ABC-DLBCL) and mucosa associated lymphoid tissue (MALT) lymphoma. We recently discovered that G-protein-coupled receptor kinase 2 (GRK2) binds to MALT1 and inhibits MALT1 scaffold and proteolytic activities. Further, we found that knockdown of GRK2 in ABC-DLBCL enhances tumor growth in vivo, suggesting that GRK2 may act as a tumor suppressor in MALT1-dependent lymphomas. Interestingly, we found that GRK2 mRNA levels are markedly lower in a subset of DLBCL cases in comparison to normal B cell controls and that lower GRK2 level is associated with reduced survival in ABC-DLBCL patients. We thus sought to investigate how GRK2 expression is regulated in ABC-DLBCL. Methods and Results: We surveyed 6 published DLBCL sequencing datasets (334 cases total) and identified only one case with a mutation/deletion in the ADRBK1 (GRK2) gene. Thus, we concluded that mutation/deletion could not account for the lower levels of GRK2 in a subset of DLBCL cases. We next investigated other potential mechanisms that could play a role in downregulating GRK2 in ABC-DLBCL, including the impact of microRNA (miRNA). Using DICER-knock-down (KD) and knock-out (KO) HEK 293T cells, we demonstrated that GRK2 protein level significantly increases when miRNA processing is impaired. Next, we performed a bioinformatic analysis which identified two candidate microRNAs, miR-148b and miR-331, whose levels inversely correlate with GRK2 mRNA in ABC-DLBCL patient tumor specimens. These two microRNAs were also identified among the top four candidate hits (miR-125b, miR-148b, miR-125a and miR-331) using mirDIP, a microRNA Data Integration Portal which we used to screen for candidate miRNAs predicted to target the 3'UTR of GRK2. We confirmed direct targeting of GRK2 by three top candidate miRNAs, miR-148b, miR-125a and miR-125b, using a GRK2 3'UTR reporter assay. Notably, we found that expression of these three microRNAs is higher in ABC-DLBCL cells (OCI-Ly3 and TMD8), which demonstrate lower GRK2 mRNA expression in comparison to primary B cells. We showed that overexpression of either miR-125a, miR-125b or miR-148 in OCI-Ly3 and TMD8 cells leads to reduced GRK2 protein level, increased MALT1 scaffolding and proteolytic activities and enhanced ABC-DLBCL cell proliferation. Conversely, anti-miRNA Locked Nucleic Acids (LNA) inhibitors of miR-125a, miR-125b or miR-148 each inhibited ABC-DLBCL cell proliferation and MALT1 activity. Further evaluation of the effect of anti-miRNA LNA inhibitors on tumor progression is under way, using an ABC-DLBCL xenograft animal model. Conclusions: Together, our data show that specific miRNAs (miR-148b, miR-125a and miR-125b) down-regulate GRK2 expression in ABC-DLBCL, which in turn enhances MALT1 scaffolding and proteolytic activities, leading to increased tumor cell proliferation. Future studies investigating miRNA inhibitors which enhance GRK2 expression and thereby suppress MALT1 activity may inform novel strategies for inhibiting MALT1-dependent lymphomagenesis.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain–in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Abstract A18: GRK2, an inhibitor of MALT1-dependent oncogenic signaling, is downregulated by microRNA in ABC-DLBCL

Abstract Introduction and Background: The “CBM” complex is composed of the scaffolding protein CARMA1, the adaptor protein BCL10, and the effector protein MALT1. This complex performs multiple pivotal functions as a mediator of antigen receptor-dependent induction of the NF-κB transcription factor and subsequent lymphocyte activation. The key downstream effector of the CBM complex, MALT1, regulates downstream signaling via both scaffolding and proteolytic functions. Inappropriate activation of MALT1, which can result from somatic gain-of-function mutation in upstream regulators of MALT1 or chromosomal translocation involving the MALT1 gene, underlies the pathogenesis of a variety of lymphoid malignancies including activated B cell type-diffuse large B cell lymphoma (ABC-DLBCL) and mucosa associated lymphoid tissue (MALT) lymphoma. We recently discovered that G-protein-coupled receptor kinase 2 (GRK2) binds to MALT1 and inhibits MALT1 scaffold and proteolytic activities. Further, we found that knockdown of GRK2 in ABC-DLBCL enhances tumor growth in vivo, suggesting that GRK2 may act as a tumor suppressor in MALT1-dependent lymphomas. Interestingly, we found that GRK2 mRNA levels are markedly lower in a subset of DLBCL cases in comparison to normal B-cell controls and that lower GRK2 level is associated with reduced survival in ABC-DLBCL patients. We thus sought to investigate how GRK2 expression is regulated in ABC-DLBCL. Methods and Results: We first investigated whether mutations/deletions in the ADRBK1 gene (GRK2) account for the lower levels of GRK2 expression in a subset of DLBCL cases. Only one mutation among 6 published DLBCL sequencing datasets (334 cases total) was identified. We next considered that other mechanisms, such as regulation by microRNA (miRNA), could play a role in downregulating GRK2. Using DICER-deficient HEK 293T cells, we demonstrated that GRK2 protein level significantly increases when miRNA processing is impaired. We then used mirDIP, a microRNA Data Integration Portal, to screen for candidate miRNAs predicted to target the 3’UTR of GRK2. We identified 4 top candidate hits, 2 of which were also identified in bioinformatic analysis as miRNA whose level inversely correlates with GRK2 mRNA level in ABC-DLBCL patient tumor specimens. Using a GRK2 3’ UTR reporter system, we confirmed direct targeting of GRK2 by these miRNAs. Next, we generated stable ABC-DLBCL cell lines (TMD8, OCI-Ly3) overexpressing candidate miRNAs. Our results demonstrate that candidate miRNA overexpression results in reduced GRK2 expression and increased MALT1 scaffolding and proteolytic activities, NF-κB transcriptional activity and ABC-DLBCL cell proliferation. Conclusions: Together, our data suggest that miRNAs down-regulate expression of GRK2 in ABC-DLBCL which in turn enhances MALT1 scaffolding and proteolytic activities, leading to increased tumor cell proliferation. Future studies are aimed at identifying potential miRNA inhibitors which enhance GRK2 expression and thereby suppress MALT1 pro-tumorigenic activity in ABC-DLBCL. Citation Format: Jing Cheng, Mei Smyers, Matt Trotta, Neil M Carleton, Lisa M Maurer, Peter C Lucas, Linda M McAllister-Lucas. GRK2, an inhibitor of MALT1-dependent oncogenic signaling, is downregulated by microRNA in ABC-DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A18.

Diffuse large B-cell lymphoma and new insights into its pathobiology and implication in treatment.

The most common non-Hodgkin lymphoma (NHL) subtype is diffuse large B-cell lymphoma (DLBCL). It accounts for roughly 30% of all cases of NHL affecting both nodal and extra nodal sites. There are molecular subtypes of DLBCL, germinal centre subtype (GCB), and activated B-cell (ABC), based on gene expression profiling (GEP), in accumulation to distinct morphological and clinicopathological subtypes. To prognosticate patients, the International Prognostication Index (IPI) and its variants are used. In ABC type DLBCL, limited stage disease is treated with a combination of abbreviated systemic chemotherapy (three cycles) and field radiation therapy. Although advanced stage disease is treated with a full course of chemotherapy as well as novel agents (Bortezomib, Ibrutinib, Lenalidomide). In this review study, we looked at the role of multiple aspects of genetic and microenvironment changes which have effects in DLBCL tumours.

Detection of Recurrent Genetic Mutations in Intravascular Large B-Cell Lymphoma By Comprehensive Genetic Analyses Using Peripheral Blood Circulating Cell-Free DNA

Abstract Background: Intravascular large B-cell lymphoma (IVLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) characterized by the selective growth of tumor cells in the lumina of small vessels. The characteristics of the disease, such as the lack of remarkable lymphadenopathy and the presence of non-specific clinical symptoms, including documented fever of unknown origin, make precise diagnosis difficult; random organ biopsies from bone marrow, skin, and lung are still performed to detect tumor cells. Recently, many recurrent genetic mutations have been detected, especially in DLBCL, but the genetic background in IVLBCL has not been fully elucidated because of the difficulty in obtaining sufficient live tumor cells from diagnostic specimens for subsequent genetic analyses. Previously, we demonstrated that peripheral blood circulating cell-free DNA (cfDNA) in DLBCL is an alternative tumor DNA (tDNA) source for comprehensive genetic analyses (Suzuki Y, et al. ASH 2014 #1658). In this report, we demonstrate that the concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and that efficiently accumulating the tDNA in peripheral blood is useful for comprehensive genetic analyses to detect recurrent genetic mutations in IVLBCL. Aims: Confirmation of recurrent genetic mutations in IVLBCL using plasma cfDNA. Methods: The study population included 54 B-cell lymphoma patients (IVLBCL, N=21; DLBCL, N=21; follicular lymphoma, N=12) and healthy volunteer donors (N=14). In IVLBCL patients, primary bone marrow (BM) cells containing tumor cells were transplanted into NOD/Shi- scid IL2Rγnull mice to generate a xenograft model (Shimada K, et al. Leukemia 2016), and the tumor cells from tumors formed in the spleen of the mouse model were purified using anti-CD19 antibody-conjugated magnetic beads to obtain tDNA. For genetic analyses using cfDNA and DNAs from purified tumor cells, whole-exome sequencing (WES) using the HiSeq 2000 (Illumina) and Sanger sequencing for reconfirmation of those mutations were performed. Results: The cfDNA concentrations were significantly higher in DLBCL and IVLBCL than in normal donors (p=0.037 and 0.036, respectively), and the concentration was significantly higher in IVLBCL than in DLBCL (p Discussion: The concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and it appeared to be sufficient to carry out comprehensive genetic analyses to accumulate tDNA. Moreover, the WES analysis using cfDNA could detect genetic mutations more efficiently than that using corresponding BM-DNA. The profiling of recurrent genetic mutations in IVLBCL is partly similar to that in activated B-cell type DLBCL with mutations in NF-κB pathway-related factors, and, intriguingly, they resemble those in B-cell lymphomas that infiltrate into immunologically sanctuary organs, e.g., primary CNS, testis, and breast lymphomas. The present data suggest that genetic analysis using cfDNA in addition to random organ biopsies may become a powerful, safer, and painless strategy to predict the presence of IVLBCL in the clinical setting. Disclosures Shimada: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Kusumoto: Chugai: Honoraria, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing support, under the direction of Shigeru Kusumoto, was provided by Cheryl Wright of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd, Research Funding. Masaki: MSD: Research Funding; Eisai: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Research Funding; Daiichi Sankyo: Research Funding; Taisho Toyama Pharmaceuticals: Research Funding; Taiho Pharmaceutical: Research Funding; Takeda Pharmaceuticals: Research Funding; Mitsubishi Tanabe Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Teijin Pharmaceutical: Research Funding; Japan Blood Products Organization: Research Funding; Pfizer: Research Funding; Asahi Kasei Pharmaceutical: Research Funding; Ono Pharmaceutical: Research Funding; Shinogi Pharmaceutical: Research Funding; Meiji Seika Pharma: Research Funding; Mochida Pharmaceutical: Research Funding. Kiyoi: Meiji Seika Pharma Co.,Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; MSD K.K.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Novartis Pharma K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Plasmablastic Lymphoma: Synergism betweenWnt/B-Cateninand theRAS Signalling Molecules May Identify Potential Targets for Therapy

Background:Plasmablastic lymphoma (PBL), is a rare aggressive B-cell lymphoma that shares many overlapping characteristics with activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) and multiple myeloma (MM). High expression ofWnt/β-cateninpathway molecules has been linked with several aspects of tumour biology in ABC-DLBCL and MM. In MMWnt/β-cateninplay critical role in chemoresistance, while high FOXP1 in ABC-DLBCL exert poor prognosis through up-regulation of theWnt/β-cateninsignalling pathway. There is strong evidence that enhanced crosstalk between the Wnt/β-catenin andRASpathways impact tumorigenesis and metastasis of cancerous stem cells in various cancers. In breast cancer; targeting theWnt/β-cateninand RAS pathways with small molecular inhibitors have shown effective results. The role of the Wnt/β-catenin signalling pathway and its corresponding linkage toRASsignalling molecules remained unknown in PBL. This pilot study provides preliminary data in relation to expressionof Wnt/β-cateninandRASpathways molecules in PBL in contrast to ABC-DLBCL. Method:FFPE RNA from diagnostic tissue samples in PBL patients (n=31) were compared with ABC-DLBCLs (n=18) patients for keyWnt/β-cateninandRASpathway molecules expression, utilizing nCounter (NanoString Technologies) platform. Qlucore Omics Explorer software was employed with defined criteria (fold change >2.0; p<0.01 and q <0.05) for statistical analysis. Gene Set Enrichment Analysis (GSEA) from 5 publicly available gene data sets was used to analyze the expression of other accompanying pathways. Result:We identified significant differential expression of mRNA related toWnt/β-cateninsignalling between ABC-DLBCL and PBL (Figure 1). Expression ofWnt/β-cateninsignalling inhibitors (CXXC4, SFRP2, and DKK1) were significantly higher among PBL compared to ABC-DLBCL (8.12-3.22 log fold difference). In divergence, molecules linked withWnt/β-cateninsignalling activation were elevated in PBL when compared to ABC-DLBCL (FZD3andWNT10B). The GESA analysis proved that the RAS pathway was significantly up-regulated in PBL patients compared to ABC-DLBCL. In particular, the expression of crucial RAS pathway genes such asNRAS, RAF1, SHC1, andSOS1was significantly up-regulated in PBL patients when compared to ABC-DLBCL patients (Figure 2). Conclusion:Our data suggest that the expression ofWnt/B-catenintarget genes and ligands are enhanced in PBL patients along with the up-regulation of theRASsignalling pathway molecules as compared to ABC-DLBCL. The heightened expression of crucialWnt/B-catenininhibitors does not down-regulate the Wnt/β-catenin signalling. We anticipate that the combined down-regulation of theWnt/ β-cateninandRASpathways by targeting its key members (RAF1, NRAS, FZD3) may serve to contain tumor progression in PBL, hence impacting prognosis. Disclosures Stewart: Gilead:Honoraria;Sandoz:Honoraria;Teva:Honoraria;Amgen:Honoraria;Celgene:Honoraria;Abbvie:Honoraria;Roche:Honoraria;Janssen:Honoraria;Novartis:Honoraria;AstraZeneca:Honoraria.

GRK2 suppresses lymphomagenesis by inhibiting the MALT1 proto-oncoprotein.

Antigen receptor-dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein-coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.

Paics Inhibition Is a Potential Therapeutic Strategy for MYC-Positive DLBCL

Diffuse large B-cell lymphoma (DLBCL) is among the most common hematological malignancies with varying prognosis. As many as forty percent of patients eventually experience relapsed/refractory disease after combinatorial chemo-immunotherapies, R-CHOP, and prognosis after relapse is dismal. MYC is among the most established prognostic factors and associated with clinically-distinct subsets of DLBCL with poor prognosis: double-expressor lymphoma (DEL) and double-hit lymphoma (DHL). MYC is co-expressed with BCL2 in DEL, which consists of 60% of activated B-cell type DLBCL (ABC-DLBCL) cases, while DHL, defined by coexistence of MYC and BCL2/BCL6 rearrangements, were reportedly observed in 15% of germinal center B-cell like DLBCL (GCB-DLBCL). Considering that MYC-positive DLBCLs exhibit dismal outcomes, pharmacological inhibition of MYC activity is highly demanded; however, direct targeting of MYC has been proven challenging. Here we show that PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), which catalyzes a critical step in de novo purine synthesis, functions downstream of MYC in DLBCL cells. We further show MRT252040, a newly-developed PAICS inhibitor, effectively suppresses proliferation of MYC-driven DLBCL cells in vitro and in vivo. Through the nCounter-based transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) tissues from 170 untreated DLBCL patients, we found that MYC and PAICS were co-expressed and their mRNA levels were among the most predictive for poor prognosis after standard R-CHOP therapy. Their expression levels were particularly high in a subset of ABC-DLBCL and extranodal DLBCL, namely in DEL and DHL cases. Importantly, these findings were validated using three independent cohorts (Schmitz et al. NEJM, 2018). MYC and PAICS expression levels were high in most DLBCL lines and low in normal B cells in the lymph nodes, while they were variable in primary DLBCL tissues, revealed by nCounter and immunofluorescence. This trend was more evident in PAICS due presumably to active de novo purine biosynthesis in highly-proliferative cell lines and a subset of DLBCLs, including MYC-positive DLBCLs. These findings were also validated using the DepMap, a publicly-available genome-wide CRISPR/Cas9 dropout screen datasets. PAICS was among the top-ranked essential genes for the survival of DLBCL cell lines. Since co-expression of MYC and PAICS in a subset of DLBCL were indicative of a functional relationship between the two factors, we explored publicly-available ChIP-seq datasets to see if MYC directly regulates PAICS expression. As expected, MYC ChIP-seq signals were highly enriched near the PAICS promoter in a series of cancer cell lines. Furthermore, shRNA-mediated MYC knockdown led to reduced levels of PAICS mRNA in MYC-positive DLBCL cells and significantly slowed their growth. Collectively, these data suggest that PAICS is a direct transcriptional target of MYC, playing a key role in proliferation of MYC-positive DLBCL cells. To assess the feasibility of PAICS-inhibition as a therapeutic option for MYC-positive DLBCLs, we tested MRT252040 for its anti-lymphoma activity in vitro and in vivo. To do so, we first assessed cell cycle status and Annexin positivity upon MRT252040 treatment using a series of DLBCL cell lines. As expected, MRT252040-mediated PAICS inhibition induced cell cycle arrest and apoptosis. Furthermore, MRT252040 treatment significantly delayed proliferation of DLBCL cell lines, namely those harboring MYC rearrangements. Finally, to assess anti-lymphoma activity of MRT252040 in vivo, we tested MRT252040 efficacy using patient-derived xenograft DLBCL. After xenotransplantation, proportions of lymphoma cells per total mononuclear cells in peripheral blood were examined over time by FACS, and MRT252040 (or vehicle) treatment was initiated once lymphoma cells constituted >0.1%. MRT252040-treated mice survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of MRT252040 monotherapy against DLBCL in vivo. Our data suggest that MYC regulates the de novo purine synthesis pathway via directly transactivating PAICS expression. We propose that MRT252040, a newly-developed PAICS inhibitor, warrants attention as a novel therapeutic approach for MYC-positive DLBCLs, which otherwise exhibit poor clinical outcomes. Disclosures Ohshima: SRL, Inc.: Consultancy; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding. Akashi:Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

MALT1 Phosphorylation Controls Activation of T Lymphocytes and Survival of ABC-DLBCL Tumor Cells

The CARMA1/CARD11-BCL10-MALT1 (CBM) complex bridges T and B cell antigen receptor (TCR/BCR) ligation to MALT1 protease activation and canonical nuclear factor κB (NF-κB) signaling. Using unbiased mass spectrometry, we discover multiple serine phosphorylation sites in the MALT1 C terminus after Tcell activation. Phospho-specific antibodies reveal that CBM-associated MALT1 is transiently hyper-phosphorylated upon TCR/CD28 co-stimulation. We identify a dual role for CK1α as a kinase that is essential for CBM signalosome assembly as well as MALT1 phosphorylation. Although MALT1 phosphorylation is largely dispensable for protease activity, it fosters canonical NF-κB signaling in Jurkat and murine CD4 Tcells. Moreover, constitutive MALT1 phosphorylation promotes survival of activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) cells addicted to chronic BCR signaling. Thus, MALT1 phosphorylation triggers optimal NF-κB activation in lymphocytes and survival of lymphoma cells.

Diffuse large B-Cell lymphoma-review

Diffuse large B cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma (NHL) accounting for approximately 30 percent of all NHL cases, involving both nodal and extra-nodal sites. Apart from distinct morphological and clinicopathological subtypes, DLBCL can be dived into molecular subtypes, Germimal Centre Subtype (GCB) and Activated B-Cell (ABC) based on gene expression profiling. IPI (International Prognostication Index) and its variants are used to prognosticate the patients. Limited stage DLBCL is primarily treated with combined modality therapy consisting of abbreviated systemic chemotherapy (three cycles), and involved field radiation therapy, whereas advanced stage disease is treated with full course of chemotherapy with recommendation of addition of novel agents ( Bortezomib, Ibrutinib, Lenalidomide) in ABC type DLBCL.

GRK2 Is a Novel Modulator of the MALT1 Oncoprotein

Background and Significance: Lymphocyte antigen receptor-dependent stimulation of the NF-κB transcription factor is a key event during the normal adaptive immune response. However, dysregulated activation of these signaling pathways can lead to lymphoma. Stimulation of the B-or T-cell receptor promotes assembly of the CARMA1-Bcl10-MALT1 (CBM) signaling complex, where MALT1 functions as the essential effector molecule by carrying out two important functions: First, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, acts as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of canonical NF-κB signaling. Activating mutations in either BCR or CARMA1 promote constitutive and exaggerated MALT1 activity and are seen as driver mutations in diffuse large B cell lymphoma (DLBCL). Aberrant MALT1 activity also underlies the pathogenesis of MALT lymphoma, another subtype of B-cell malignancy. As such, MALT1 has emerged as a potential new target for pharmaceutical inhibition in the treatment of lymphoma. Here, we describe our discovery of a novel MALT1-binding partner, G protein-coupled receptor kinase 2 (GRK2). We hypothesize that GRK2 plays a critical role in the pathogenesis of lymphoma by modulating MALT1 activity.Methods and Results: Using co-immunoprecipitation and liquid chromatography mass spectrometry we identified GRK2 as a novel MALT1-interacting protein. We validated our results by showing that endogenous GRK2 and MALT1 co-immunoprecipitate (CO-IP) in both B cells and T cells. We next utilized GST-tagged purified proteins to demonstrate a direct interaction between GRK2 and MALT1. We then performed a number of studies to investigate the functional consequences of GRK2 interaction with MALT1. A CO-IP assay demonstrated that GRK2 inhibits Bcl10-dependent binding to MALT1, and a luciferase reporter assay revealed that GRK2 inhibits Bcl10/MALT1-dependent NF-κB activity. In addition, we demonstrated that GRK2 also inhibits MALT1 proteolytic activity using Western blot analysis of two known MALT1 substrates, the deubiquitinase cylindromatosis (CYLD) and the NF-kB family member RelB. Moreover, in Jurkat T cells, overexpression of GRK2 impeded AgR-induced NF-κB activation while stable GRK2 knockdown resulted in enhanced AgR-induced phosphorylation of inhibitor of kappaB (IκB) and secretion of interleukin2 (IL-2).We next began to investigate the potential of GRK2 to function as a tumor suppressor by blocking MALT1 activity. Of note, utilizing a published data set to compare GRK2 mRNA expression in 44 DLBCL patient samples to that of 20 samples of healthy B-cell controls, we found that GRK2 mRNA levels are markedly lower in DLBCL cases. We also compared GRK2 mRNA levels among patients diagnosed with DLBCL grouped by vital status, using data from the Cancer Genome Atlas (TCGA). We found that tumors from patients who died of disease within three years expressed significantly lower levels of GRK2 as compared to tumors from patients alive after three years. We also wished to investigate the impact of GRK2 expression on activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) cells, which require MALT1 activity for growth and survival. We were successful in engineering GRK2 overexpression in control germinal center B-cell like (GCB) DLBCL cells, which do not depend on MALT1 activity for growth. In contrast, we found that in ABC-DLBCL cells, which do depend on MALT1 activity, GRK2 overexpression greatly impairs cell proliferation, making it difficult to grow these engineered cells. These data provide support for the concept that GRK2 may have a potential tumor suppressor role in MALT1-dependent lymphoma.Conclusions: Our study represents the first report of a negative modulator of MALT1 and provides critical new insight into mechanisms regulating its activity. We hope to harness this information in developing novel strategies for inhibiting MALT1-dependent lymphomagenesis. DisclosuresNo relevant conflicts of interest to declare.

B-cell–specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice

AbstractThe adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell–type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2. Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88p.L252P (the orthologous position of the human MYD88p.L265P mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2. Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL. Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2, and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures:No relevant conflicts of interest to declare.

Elevated Monoclonal Free Light Chains Are a Serum Marker of ABC Type Diffuse Large B-Cell Lymphoma

Abstract 1591 Background:The serum free light chain (FLC) assay quantitates free kappa and free lambda immunoglobulin light chains. We previously showed elevated FLC are present in approximately 1 in 3 diffuse large B cell lymphoma (DLBCL) patients from both a phase II cooperative group clinical trial and a large clinic based epidemiology cohort. Approximately 10% of DLBCL patients had elevated FLC that was monoclonal in these series. Patients with monoclonal elevated FLC have very poor outcome with almost 50% of patients progressing in the first 15 months following initial treatment with immunochemotherapy. Unlike patients with polyclonal FLC elevation, where a variety of host effects may cause a rise in both kappa and lambda, it is likely the excess FLC in monoclonal patients is directly related to the tumor. DLBCL are a heterogenous group of tumors that can be classified into germinal center (GCB) or activated B cell (ABC) groupings using immunohistochemistry (IHC) staining algorithms. Here we examine the association of monoclonal free light chains with ABC vs. GCB DLBCL tumors. Methods:Newly diagnosed DLBCL patients were prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) or NCCTG clinical trial N0489. Serum FLC was quantitated from enrollment research serum draws using the Freelite FLC assay (The Binding Site, Ltd., Birmingham, UK). Monoclonal elevated FLC was defined as an elevated kappa or lambda with a corresponding abnormal FLC ratio. Immunohistochemistry (IHC) staining was performed on paraffin tissue from research tissue microarrays (TMAs). Results:IHC tissue staining was available from 12 patients with monoclonal elevated FLC unrelated to renal failure. Germinal center markers were predominantly negative: 2 of 11 were LMO2 positive (18%), 1 of 12 was CD10 positive (8%), and zero cases out of 11 were GCET1 positive. FoxP1 was positive in 5 of 9 cases (56%) and MUM1 was positive in 4 of 12 cases (33%). By the Tally algorithm, 10 of 11 cases (91%) were ABC type DLBCL The percentage of ABC patients by the Tally algorithm in the corresponding DLBCL patients without monoclonal FLC was 45% (N=108, p=0.004). Conclusions:Approximately 10% of DLBCL patients have elevated monoclonal FLC and these patients have poor prognosis. Elevated monoclonal FLC may be a serum marker for ABC-type lymphoma, as the tumors from patients are predominantly negative for germinal center stains and ABC by the Tally algorithm. The secretion of light chains by the tumor suggests an activated B cell clone and the poor prognosis is characteristic of ABC type DLBCL and. Given the likelihood of the excess monoclonal FLC being tumor related, the potential exists for tracking of tumor response and progression via serum FLC in these patients. Replication of these findings in an additional cohort of DLBCL patients is planned.Table 1CaseAGEGenderIPISerum KappaSerum LambdaSerum FLC RatioTumor Light Chain RestrictionCD10GCET1MUM1FOXP1LMO2BCL6TallyNCCTG157M13.382.021.67−−+−++ABCNCCTG277M35.570.4313.1−−−+−−ABCNCCTG373M43.681.612.29+−++−−ABCMER176F34.872.112.31kappa−−−−ABCMER242M14.511.572.87−−−−−−ABCMER374M20.935.50.17−−−−+−GCBMER460F143.61.2335.4−−++−−ABCMER580F3130.7916.4kappa−−−+−−ABCMER680M22.831.471.93−−−+−+ABCMER758F25.160.3614.3kappa−−−−MER878M38.34.851.71−−+−−−ABCMER961M31.269.670.13lambda−−−−−ABC Disclosures:No relevant conflicts of interest to declare.

The t(14;18)(q32;q21) Confers Resistance to Apoptosis-Induced by Smoothened Inhibitors In Diffuse Large B-Cell Lymphoma That Can Be Restored by Functional Inhibition of BCL2

Abstract 2479Generally in cancers, high expression of anti-apoptotic proteins induced by oncogenic signaling pathways is a major cause for treatment failure. Previously, we have demonstrated that Hedgehog (Hh) signaling is activated and functional in diffuse large B-cell lymphoma (DLBCL) and that its inhibition, using the Smoothened (SMO) inhibitor cyclopamine-KAAD, induces apoptosis in cell lines of DLBCL of activated B-cell (ABC) subtype and cell cycle arrest (G1-phase) in those of germinal center (GC) subtype. Induction of apoptosis in ABC-DLBCL was due to decrease of BCL2 expression (protein and mRNA levels), a direct target of Hh signaling (Leukemia 2010;24:1025–36). We also found that BCL2 is upregulated in the tumor cells in the presence of stroma and that Hh inhibition abrogates this upregulation (Oncogene 2011, in press). Here, we explored the possibility of overcoming resistance to apoptosis-induced by Hh inhibition in GC-DLBCL using a combination of the SMO inhibitor “Hh antagonist” (Genentech Inc) with the BH3 mimetic ABT-737 (Abbot laboratories) in a panel of 5 GC type DLBCL cell lines, 4 of them with the t(14:18)(q32;q21) (DOHH2, SUDHL4, Toledo and OCI-LY19) and one without the translocation (HT). ABT-737 is a functional inhibitor of three major anti-apoptotic proteins of the BCL2 family (BCL2, BCL-xL and BCL-W). Combinatorial treatments were performed with increasing concentrations of the Hh antagonist (2.5, 5.0 and 7.5 μM) and with variable but minimal lethal doses of ABT-737 (inducing cell death less than 20%). Using cell viability, apoptosis (annexin-V) and cell cycle based assays, we found that the combined treatments resulted in a additive/synergistic decrease of cell viability and increase of apoptosis in comparison to the treatment with the Hh antagonist alone. In contrast to ABC type DLBCL cell lines, in DLBCL cells with t(14;18), Hh antagonist did not result in decrease expression of BCL2, BCL-xL and BCL-W indicating that the additive effect of ABT-737 was due to their functional inhibition and not by suppressing expression levels of these proteins. On the other hand, treatments with the Hh antagonist resulted in a concentration dependent decrease of BCL2 protein levels in HT cells. In summary, our data confirm that in the presence the t(14;18), inhibition of SMO does not result in decrease of BCL2 expression. In these cases, concomitant inhibition of BCL2 function and SMO is needed to induce apoptosis. Combined inhibition of Hh signaling and BCL2 function may have a therapeutic value for lymphomas with the t(14;18)(q32;q21). Disclosures:No relevant conflicts of interest to declare.

Immunohistochemical Methods for Predicting Cell of Origin and Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With Rituximab

Patients with diffuse large B-cell lymphoma (DLBCL) can be divided into prognostic groups based on the cell of origin of the tumor as determined by microarray analysis. Various immunohistochemical algorithms have been developed to replicate these microarray results and/or stratify patients according to survival. This study compares some of those algorithms and also proposes some modifications. Two-hundred and sixty-two cases of de novo DLBCL treated with rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like therapy were examined. The Choi algorithm and Hans algorithm had high concordance with the microarray results. Modifications of the Choi and Hans algorithms for ease of use still retained high concordance with the microarray results. Although the Nyman and Muris algorithms had high concordance with the microarray results, each had a low value for either sensitivity or specificity. The use of LMO2 alone showed the lowest concordance with the microarray results. A new algorithm (Tally) using a combination of antibodies, but without regard to the order of examination, showed the greatest concordance with microarray results. All of the algorithms divided patients into groups with significantly different overall and event-free survivals, but with different hazard ratios. With the exception of the Nyman algorithm, this survival prediction was independent of the International Prognostic Index. Although the Muris algorithm had prognostic significance, it misclassified a large number of cases with activated B-cell type DLBCL. The Tally algorithm showed the best concordance with the microarray data while maintaining prognostic significance and ease of use.