Activated B-cell-like Diffuse Large B-cell Lymphoma Cell Lines Research Articles

First-in-class inhibitor of HSP110 blocks BCR activation through SYK phosphorylation in diffuse large B-cell lymphoma.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is driven by aberrant activation of the B-cell receptor (BCR) and the TLR/MyD88 signaling pathways. The heat-shock protein HSP110 is a candidate for their regulation as it stabilizes MyD88. However, its role in overall BCR signaling remains unknown. Here, we used first-in-class HSP110 inhibitors to address this question. HSP110 inhibitors decreased the survival of several ABC-DLBCL cell lines in vitro and in vivo, and reduced the phosphorylation of BCR signaling kinases, including BTK and SYK. We identified an interaction between HSP110 and SYK and demonstrated that HSP110 promotes SYK phosphorylation. Finally, the combination of the HSP110 inhibitor with the PI3K inhibitor copanlisib decreases SYK/BTK and AKT phosphorylation synergistically, leading to suppression of tumor growth in cell line xenografts and strong reduction in patient-derived xenografts. In conclusion, by regulating the BCR/TLR signaling pathway, HSP110 inhibitors are potential drug candidates for ABC-DLBCL patients.

Abstract 463: The small nucleolar RNA SNORD46 and SNORD72 have tumor suppressor activity in diffuse large B cell lymphoma

Abstract Background. Following the identification of an 11q24.3 gain upregulating the ETS factors ETS1 and FLI1 in diffuse large B cell lymphoma (DLBCL) (Bonetti et al. Blood 2013), we observed the anti-lymphoma activity of blocking the protein-protein interaction of ETS factors with RNA helicases, using YK-4-279 and its clinical derivative TK-216 (Spriano, Chung et al. CCR 2019). DLBCL cells exposed to the two small molecule inhibitors presented a significant upregulation of small nucleolar RNAs (snoRNAs). Besides their primary functions in rRNA processing, snoRNAs are often deregulated in cancers, with microRNA-like actions and direct interaction with proteins. Here, we characterized SNORD46 and SNORD72, the two snoRNAs most upregulated cells after YK-4-279 and TK-216 and mapped to the 1p34.1 locus recurrently lost in DLBCL. Methods. Real-time PCR, immunoblotting, and shRNA silencing were conducted based on established protocols. RNA-Protein Interaction Prediction (RPISeq) was used to calculate the interaction probabilities of snoRNAs with proteins of interest. The prediction was made using both the Random Forest (RF) and the Support Vector Machine (SVM) classifiers, using a stringent threshold of >0.6. Results. The time-dependent changes of SNORD46 and 74 were validated with real-time PCR in the U2932 activated B cell-like (ABC) DLBCL cell line exposed to YK- 4-279 for 4 or 8 h. Upregulation of SNORD46 and 74 were significantly upregulated compared to their host genes upon YK-4-279 treatment, suggesting these snoRNAs have different functions from the genes in which they are encoded. Across 48 lymphoma cell lines, SNORD74 was more expressed in ABC DLBCL than in the remaining cell lines (P 0.03) or GCB DLBCL (P 0.05), while SNORD46 did not differ. Stable lentiviral-based overexpression of SNORD46 and 74, U2932 ABC DLBCL demonstrated that both SNORD46 and SNORD74 overexpression impaired growth while SNORD92, the control did not SNORD74 overexpression inhibited pAKT-Ser473, suggesting it could exert its anti-tumor effect by negatively impacting BCR signaling. Since constitutive BCR is the main driver for the pathogenesis in ABC DLBCL, we studied the impact of BCR signaling on SNORD46 and 74 expressions. IgM stimulation downregulated the snoRNAs in U2932 and TMD8 ABC DLBCL cell lines at 1 and 2.5 h poststimulation, with a more substantial effect for SNORD74. Furthermore, the snoRNAs were upregulated in four ABCs and four GCB DLBCLs cell lines exposed to the PI3K/mTOR inhibitor bimiralisib. Bioinformatic analysis indicated the possible binding of SNORD46 and especially of SNORD74 to BCR signaling molecules (SYK, LYN, BTK, PI3Kδ, IRAK4, IRAK1, CARD11), RNA helicases (RHA, DDX21, DDX5) and essential DLBCL proteins (BCL6, IRF4). Conclusions. SNORD46 and SNORD74 exert tumor suppressor activity in DLBCL cells, possibly as negative regulators of the BCR signaling. Citation Format: Elaine YL Chung, Filippo Spriano, Sara Napoli, Giulio Sartori, Luciano Cascione, Francesco Bertoni. The small nucleolar RNA SNORD46 and SNORD72 have tumor suppressor activity in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 463.

Sgr-1505 Is a Potent MALT1 Protease Inhibitor with a Potential Best-in-Class Profile

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Previously, we described the discovery of novel MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells and the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models as well as combination potential with agents including standard-of-care (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). When administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, SGR-1505 demonstrated tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential for patients with selected B-cell lymphomas. Here we further characterized SGR-1505, in a series of in vitro and ex vivo assays, as well as RNA-seq analysis to examine changes in gene expression from in vivo tumor samples. We also compared SGR-1505 with a competitor Phase I candidate, JNJ-67856633 (JNJ-6633) (ref 3, 4). Results: SGR1505 potency and downstream effects were evaluated in a series of biochemical and cell based assays. SGR-1505 showed excellent potency in the biochemical assay and strong anti-proliferative effects on ABC-DLBCL cells. SGR-1505 was more potent than JNJ-6633 in all assays tested (Table 1). These results are also consistent with the result from a human primary T-cell based assay, where SGR-1505 showed at least ten-fold better potency than JNJ-6633. RNA-seq analysis was conducted to examine changes in gene expression from in vivo tumor samples. Greater modulation of BIOCARTA NF-kB pathway genes was seen with SGR-1505 compared to JNJ-6633, as measured by mean absolute change in gene expression. At 6 hr and later timepoints, we also observed a trend of increases in genes related to cell cycle pathways, such as cell cycle, DNA damage, and apoptosis. SGR-1505 is being evaluated in the SGR-1505-102 phase 1 study, which is an ongoing first-in-human, single center, dose escalation study to evaluate the safety, tolerability, PK and PD of SGR-1505 tablets in healthy participants (ACTRN12623000358640p). Preliminary data showed changes in target engagement markers at concentrations predicted by the in vitro and ex vivo assays, consistent with MALT1 protease inhibition. Conclusions: SGR-1505, a MALT1 protease small molecule inhibitor, consistently demonstrated better potency in in vitro and ex vivo assays when compared to the clinical-stage JNJ-6633 compound and greater effects on NF-kB pathway gene expression in in vivo tumor samples based on RNA-seq analysis. Changes in biological pathways mediated by MALT1 were also observed at relevant doses in the ongoing SGR-1505 healthy volunteer study. Currently, a phase 1 clinical trial in patients with mature B cell neoplasms is also ongoing (NCT05544019). The data presented suggests SGR-1505 has a potential best-in-class profile and supports advancing the ongoing clinical development of SGR-1505.

A Phase 1, Open-Label, Multicenter, Dose-Escalation Study of Sgr-1505 As Monotherapy in Subjects with Mature B-Cell Malignancies

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.

SGR‐1505‐101: A PHASE 1, OPEN‐LABEL, MULTICENTER, DOSE‐ESCALATION STUDY OF SGR‐1505 AS MONOTHERAPY IN SUBJECTS WITH MATURE B‐CELL MALIGNANCIES

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of the nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. In particular, constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity, and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. Aims: These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential and may expand therapeutic options for patients with selected B-cell lymphomas supporting further evaluation of SGR-1505 in clinical trials. Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies. At present, the study has been activated in 3 sites in the United States. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1–6 subjects, and at higher dose levels using a conventional 3+3 design. Encore Abstract—previously submitted to EHA 2023 The research was funded by: Schrodinger Keywords: aggressive B-cell non-Hodgkin lymphoma, molecular targeted therapies, ongoing trials Conflicts of interests pertinent to the abstract. B. Yoo Employment or leadership position: Schrodinger Stock ownership: Schrodinger Z. Nie Employment or leadership position: Schrodinger Stock ownership: Schrodinger G. Krilov Employment or leadership position: Schrodinger Stock ownership: Schrodinger J. Tan Employment or leadership position: Schrodinger Stock ownership: Schrodinger H. Wright Employment or leadership position: Schrodinger Stock ownership: Schrodinger D. Weiss Employment or leadership position: Schrodinger Stock ownership: Schrodinger W. Yin Employment or leadership position: Schrodinger Stock ownership: Schrodinger K. Akinsanya Employment or leadership position: Schrodinger Stock ownership: Schrodinger

Targeting EGR1-Medited Metabolic Reprogramming to Overcome Ibrutinib Resistance in Aggressive B Cell Lymphomas

Targeting specific signaling pathways in malignant cells through the use of small-molecule kinase inhibitors has revolutionized the treatment of malignancies. Bruton tyrosine kinase (BTK), a key component of the B cell receptor (BCR) signaling pathway, is a promising molecular target in several types of lymphoma, including diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Ibrutinib, a selective BTK inhibitor, has achieved initial response rates as high as 70% in these lymphomas when used as a single agent. Acquired drug resistance, however, is significant and together with primary resistance makes ibrutinib by itself unsuitable if the goal of treatment is long-term survival. Therefore, understanding and targeting ibrutinib resistance mechanisms is an unmet clinical need. DLBCL, which represents 30% to 40% of newly diagnosed lymphomas, comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). The BCR/BTK signaling pathway is essential for the growth of ABC DLBCL, which is more aggressive and less curable than GCB DLBCL with current regimens. We established two representative ABC DLBCL cell lines (TMD8 and OCI-Ly10) with ibrutinib resistance by gradually increasing the concentration of ibrutinib during passage in culture. RNA-seq analysis demonstrated that the BCR pathway gene signature is enriched in resistant cell lines when compared to parental cells. The most upregulated gene is EGR1, a transcription factor that activates multiple oncogenic pathways including MYC and E2F. Elevated EGR1 expression is also observed in ibrutinib-resistant primary mantle cell lymphoma cells when treated with ibrutinib. Using multiple metabolic and genetic approaches, we discovered that overexpression of EGR1 causes metabolic reprogramming to oxidative phosphorylation (OXPHOS) and ibrutinib resistance. Mechanistically, EGR1 mediates metabolic reprogramming through transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation accelerates intracellular ATP production via the mitochondrial tricarboxylic acid (TCA) cycle, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in patient-derived xenograft mouse models. These findings suggest that targeting EGR1-medited metabolic reprogramming to OXPHOS with IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

TPL2, a New Prognostic Factor and a Potential Therapeutic Target in ABC-DLBCL

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype of DLBCL, characterized by the constitutive activation of NF-κB and JAK/STAT3 pathways on the background of the chronic active BCR signaling. Being featured by mutations in MYD88 and/or CD79B, it potentially comprises the MCD/C5 subtype in the recent genetics-based categorization. In light of the inferior prognosis of this subtype to germinal center B-cell (GCB) like DLBCL, identification of subtype-specific strategies has been sought. Tumor progression locus 2 (TPL2) is an intracellular serine/threonine kinase that is reported to be activated downstream of TLRs/MYD88 signaling and to play roles in oncogenesis. However, the involvement of TPL2 in B-cell malignancies is less defined despite relatively higher transcript expression than in solid tumors. Results: To address the impact of TPL2 protein levels on DLBCL phenotypes, we performed an IHC analysis on 79 FFPE samples of DLBCL diagnosed and treated at our institution (48 non-GCB and 31 GCB according to Hans algorithm). When divided into two groups, one with strong TPL2 expression (TPL-high) and the other with no/weak expression (TPL2-low), the ratio of TPL2-high did not differ between non-GCB and GCB (67% vs 64%). However, TPL2-high had a significantly higher percentage of positive B symptom (42% vs 11%, p=0.005) and extra-nodal involvement (75% vs 52%, p=0.047) compared to TPL2-low, and these trends were observed more specifically in non-GCB. Moreover, it is noteworthy that TPL2-high showed a significantly lower overall survival in non-GCB (p=0.037), but did not in GCB (p=0.999) (Figure 1). Intriguingly, the analysis combined with pSTAT3 (Y705) staining revealed that TPL2-high accompanied by positive pSTAT3 tended to display particularly poor prognosis in non-GCB. Then, we analyzed the mechanism by which TPL2 is involved in the tumorigenesis of ABC-DLBCL using TMD8, a well-described ABC-DLBCL cell line with MYD88L265P and CD79BY196 mutations. A specific TPL2 inhibitor significantly affected the cell proliferation of TMD8 cells, whereas did not on the cells with GCB phenotype. Of note, TPL2 inhibitor induced G1 cell-cycle arrest via downregulation of CyclinE and CDK2 and upregulation of p27 in western blot analysis, which was confirmed by quantitative PCR. Next, we investigated the downstream signaling of TPL2 in ABC-DLBCL. Western blot analysis revealed that TPL2 inhibitor as well as genetic TPL2 knock down suppressed the activation of NF-κB components and STAT3, and the expression of c-MYC. These results implied the involvement of TPL2 in IL-6/IL-10 autocrine loop and resultant JAK/STAT3 activation. As expected, treatment with TPL2 inhibitor downregulated IL-6/IL-10 mRNA by quantitative PCR and accordingly decreased the levels of IL-6/IL-10 in the cell culture supernatant of treated TMD8 cells. Therefore, we speculated that TPL2 promoted the oncogenic activation of NF-κB and STAT3 pathways via IL-6/IL-10 upregulation in ABC-subtype. Lastly, we evaluated the physiological function of TPL2 by western blot analysis using 293T. Although an overexpression TPL2 alone did not elicit NF-κB and STAT3 activation, when co-expressed with MYD88L265P TPL2 obviously activated these pathways, whereas dominant negative TPL2 failed to demonstrate those effects even with MYD88L265P co-expression. These findings indicated that TPL2 could possibly exert its tumorigenicity in MYD88L265P-dependent manner in ABC-DLBCL. Conclusion: In association with the importance as an unfavorable prognostic factor, our results suggested that TPL2 might mediate the crosstalk between the oncogenic MYD88L265P and the activation of NF-κB and STAT3 pathways, coordinately contributing to the tumorigenesis of ABC-DLBCL. From this standpoint, TPL2 could emerge as a new therapeutic target in ABC-DLBCL. Figure legend Overall survival of non-GCB (A) and GCB (B) DLBCL patients, with regard to the TPL2 expression: TPL2-low (red line) or TPL2-high (black line). Overall survival was determined by the Kaplan-Meier method and compared by using the log-rank test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification BCL6 and miR-30 family associating with Ibrutinib resistance in activated B-cell-like diffuse large B-cell lymphoma

Ibrutinib has clear efficacy for activated B-cell-like diffuse large B cell lymphoma (ABC-DLBCL) in previous clinical researches. However, the resistance of Ibrutinib has limited its therapeutic benefit and the potential mechanism remains unclear. This study was aimed to identify potential candidate genes and miRNA targets to overcome Ibrutinib resistance in ABC-DLBCL. First, two expression profiles were downloaded from the GEO database, which used to identify the DEGs related to Ibrutinib resistance in ABC-DLBCL cell lines by GEO2R analysis separately. And the common DEGs were obtained though Venn diagram. Then Gene ontology (GO) and pathway enrichment analysis were conducted by DAVID database. From STRING database, BCL6, IL10, IL2RB, IRF4, CD80, PRDM1and GZMB were determined to be the hub genes by protein–protein interaction (PPI) network. Through miRNA-mRNA targeting network, we found that BCL6, IRF4, CD80, and PRDM1 were common target genes of miR-30 family. The cBioPortal database showed that BCL6 had the highest level of genetic alterations among DLBCL. In addition, another expression profile from GEO database showed that BCL6 was significantly high expression in no responsive patients after Ibrutinib treatment, and the receiver operating characteristic (ROC) curve which was used to evaluate the relationship between BCL6 expression and its effect was 0.67. MTT assay showed that treatment with FX1 (a BCL6 inhibitor) can enhance the sensitivity of Ibrutinib in C481S BTK HBL-1 cells. The results suggested that BCL6 and miR-30 family maybe associate with Ibrutinib resistance in ABC-DLBCL.

Naquotinib exerts antitumor activity in activated B-cell-like diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (NHL), is categorized into two major subtypes, activated B-cell-like (ABC) and germinal center B-cell-like (GCB). The ABC subtype is associated with worse prognosis than the GCB subtype using currently available therapies such as combination treatment with rituximab plus standard cytotoxic chemotherapy. The B-cell receptor (BCR) pathway is activated in ABC DLBCL, suggesting that inhibition of this pathway could provide an alternative strategy for treatment. Naquotinib is an irreversible tyrosine kinase inhibitor (TKI) originally designed to target the epidermal growth factor receptor (EGFR). As sequence alignment analysis indicates that irreversible EGFR-TKIs also inhibit Bruton's tyrosine kinase (BTK), here, we characterized the inhibitory effects of naquotinib against BTK in comparison to ibrutinib, acalabrutinib, tirabrutinib and spebrutinib. Naquotinib inhibited BTK kinase activity with similar potency to that for EGFR activating mutations. In vivo, naquotinib induced tumor regression and suppressed tumor recurrence in TMD8 and OCI-Ly10, ABC DLBCL cell line xenograft models, at a lower dose than the clinically relevant dose. Compared to other BTK inhibitors, naquotinib showed faster onset and comparable inhibition of BTK following incubation with cell lines for 3 and 20 h. In addition, naquotinib showed longer continuous inhibition of BTK following removal of the compound, lasting for at least 26 h after removal. Pharmacokinetics studies in the TMD8 xenograft model showed higher concentration and slower elimination of naquotinib in tumors than other BTK inhibitors. These data suggest that naquotinib may have therapeutic potential in ABC DLBCL patients.

Oncogenic Cooperation between EGR1 and BRD4 in Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) grows rapidly and represents 30% to 40% of newly diagnosed lymphomas, and comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive with a current cure rate of only ~40%. The B cell antigen receptor (BCR) signaling pathway is known for its critical role in the survival and proliferation of ABC DLBCL cells. Our recent studies revealed that IL6- and IL10-mediated JAK1/STAT3 activation enhances the survival and proliferation of ABC DLBCL cells. Furthermore, we discovered that EGR1, a transcription factor known to be a downstream effector of BCR signaling, is also a downstream target of JAK1 signaling in DLBCL. While EGR1 has been characterized as a tumor suppressor in leukemia, the role of EGR1 in human lymphoma remains unclear. Initial immunohistochemical analysis of 102 DLBCL cases revealed that not only is the level of EGR1 elevated in DLBCL as compared to normal human tonsils, lymph nodes and mantle cell lymphomas, but its level is higher in ABC DLBCL (non-GCB) than GCB DLBCL, indicating functional significance of EGR1 in DLBCL. In addition, we found that 7 out of 8 ABC DLBCL cell lines are sensitive to EGR1 knockdown, whereas only half of 8 GCB DLBCL cell lines are affected. Our genomics studies discovered dual functions of EGR1 in regulating three signaling pathways. EGR1 mediates transcriptional activation associated with two hallmark oncogenic pathways, JAK1/STAT3 and BCR signaling. Our EGR1 ChIP-seq results revealed a large overlap of EGR1 genomic occupancy with that of BRD4, a highly expressed bromo and extraterminal (BET) protein in DLBCL known to induce MYC expression and activation of MYC target genes. This agrees with the RNA-seq results showing that multiple oncogenic pathways including MYC are activated by EGR1 in ABC DLBCL. The synergistic inhibition of cell growth was observed between EGR1 shRNA and JQ1, an inhibitor of BRD4. Surprisingly, the RNA-seq data and pathway analyses also revealed EGR1-mediated suppression of the type I interferon pathway, which otherwise causes cancer cell death. The majority of the interferon pathway genes are up-regulated upon EGR1 knockdown. EGR1-modulated suppression of the interferon pathway appears to be functionally significant as EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. EGR1 is known to bind the promoter of p300 and activate p300 expression. P300 catalyzes H3K27 acetylation (H3K27ac), an active histone mark that recruits BRD4 for transcription of MYC and MYC pathway genes, among others. Indeed, immunoblot analysis revealed that EGR1 knockdown reduces H3K27ac expression in ABC DLBCL cells, leading to reduced expression of MYC and MYC target genes based on H3K27ac ChIP qPCR analysis. The results provide a molecular basis for oncogenic cooperation between EGR1 and BRD4 in ABC DLBCL. In summary, our study demonstrates that EGR1 is an oncogenic driver orchestrating multiple important signaling pathways. Notably, EGR1 creates therapeutic vulnerability in ABC DLBCL because both EGR1-mediated transcriptional activation and repression provide growth advantage to the cancer cells. Disclosures No relevant conflicts of interest to declare.

KLHL14 Is a Tumor Suppressor in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer of aberrant B-lymphocytes. Although a portion of DLBCL is curable with standard immunochemotherapy, patients who fail this treatment have a poor prognosis. Recently, cancer genomics has paved the way for better understanding of the genetic basis of lymphoma pathogenesis. Characterization of point mutations and structural alterations has uncovered novel molecular targets for lymphoma therapy and provided a comprehensive view of lymphoma development. By performing multiplatform genomic analysis of DLBCL biopsy samples, we have identified KLHL14 as a recurrent target of somatic mutations in activated B-cell-like (ABC) DLBCL biopsies (10.8% of patients). KLHL14 contains a BTB (broad complex, tramtrack, and bric a brac) domain that can potentially mediate dimerization and binding to Cullin3 (CUL3)-a essential scaffold component of the Cullin-RING-based E3 ubiquitin ligase complexes. KLHL14 also contains kelch repeats that can form a B-propeller tertiary structure that can serve as a substrate-binding domain. KLHL14 is highly expressed in B-cells but is found at low levels in non-immune tissues. Deficiency of KLHL14 in mice leads to embryonic lethality while KLHL14 heterozygous mice show reduction of B-1a cells, suggesting a role for KLHL14 in B-cell homeostasis. Importantly, KLHL14 mutations are highly enriched in tumors belonging to the recently defined MCD (MYD88L265P/CD79B mutation) genetic subtype of DLBCL, the subset of ABC DLBCLs. Somatic mutations primarily localize to the N-terminus of the protein in the BTB domain and BACK (BTB and C-terminal Kelch) domain. However, the impact of these mutations as well as the molecular function of KLHL14 is largely unknown. To investigate the biological effect of KLHL14 loss of function, we used an inducible CRISPR/Cas9 system to delete KLHL14 in ABC DLBCL cell lines and monitored cell growth. Ablation of KLHL14 resulted in an increase in cell proliferation and survival, supporting a role for KLHL14 as a tumor suppressor. Next, we performed a multiplatform -omic analysis (proteomics, phosphoproteomics, ubiquitinomics, high-throughput sequencing) to explore the signaling networks and interactome of KLHL14. Whereas ectopic expression of wild-type KLHL14 altered the dynamics of tyrosine phosphorylation and ubiquitylation events in ABC DLBCL lines, KLHL14 lymphoma-associated mutant alleles had little if any effect, suggesting that they are loss-of-function variants. Gene expression profiling by RNA-sequencing revealed that KLHL14-inactivated cells have a higher NF-kB target gene expression than wild-type cells. Thus, tumor-associated inactivating mutations of KLHL14 depend on a subset of essential NF-kB-related oncoproteins for their survival and this might contribute to the proliferative advantage of DLBCL. In summary, we have uncovered a tumor suppressive function of KLHL14 and found that KLHL14 mutants promote ABC DLBCL survival by increasing NF-kB activity. These findings suggest that tumors with KLHL14 inactivating mutations may serve as a marker of resistance to anti-NF-kB treatment and provide the basis for treating MCD subtype patients with downstream NF-kB pathway inhibitors in the clinical settings. Disclosures Staudt: Nanostring: Patents & Royalties.

BTK Inhibitors Induce ABC-DLBCL Cell Apoptosis By Inhibiting CYLD Phosphorylation

BTK inhibitors induce ABC-DLBCL cell apoptosis by inhibiting CYLD phosphorylation Xin Xu1, Zhigang Zhu1, Feng Liu1 , Weijie Zhong1,Huabao Xiong2, Tiefeng Feng3, and Qingshan Li3 1Geriatrics Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. 2Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, U.S.A. 3Hemotology Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. Activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) is a common aggressive diffuse large B cell lymphoma (DLBCL). Even though CD20 antibody (Rituximab) combination therapy improved the total prognosis of DLBCL, there are still many relapsed and refractory ABC-DLBCL cases. The new treatment options are urgently needed. Pathogenesis of ABC-DLBCL is different from germinal center B cell-like diffuse large B cell lymphoma (GCB-DLBCL), which is related to activation of B cell antigen receptor(BCR) signaling. The Bruton tyrosine kinase(BTK) plays a central role in BCR signaling. Inhibition of BTK function will affect BCR signaling activity, which is involved in maintaining the malignant phenotype in B-cell lymphoma. Now in clinic, BTK inhibitors like Ibrutinib or Acalabrutinib are used extensively in chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma(MCL) as first line therapy or second line therapy. But in DLBCL its role is unclear now.Some DLBCL clinic trials got the better PFS and OS in BTK inhibitor combination therapy group(combined with Rituximab or RCHOP) but some got the negative results. Whether BTK inhibitors can definitely improve ABC-DLBCL survival and the mechanism how BTK inhibitors inducing cell death are needed further investigations. CYLD (cylindromatosis) is a tumor suppressor, its activity is inhibited by phosphorylation, which is related to apoptosis regulation pathway NF-kB in many kinds of tumors.In this study, we found that CYLD to be constitutively phosphorylated in ABC-DLBCL cell line models (HBL-1 and OCI-Ly10) as well as primary samples from patients with non-GCB DLBCL. We subsequently examined the effect of BTK inhibitors Ibrutinib (PCI-32765) and Acalabrutinib (ACP-196) on CYLD phosphorylation. We found that BTK inhibitors Ibrutinib and Acalabrutinib both induced ABC-DLBCL apoptosis by down-regulating CYLD phosphorylation in vivo and in vitro, even in the Rituximab resistant ABC-DLBCL cell line.Combination of BTK inhibitors and Rituximab would enhance this effect. Knockdown of CYLD in ABC-DLBCL cells reduced the level of cell death induced by BTK inhibitors, which showed that this cell death induced by BTK inhibitors was CYLD dependent. Through this study, we conclude that BTK inhibitors induce CYLD inactivation by regulating CYLD phosphorylation is clinical beneficial in ABC-DLBCL therapy, especially the relapsed or refractory ABC-DLBCL cases after Rituximab therapy. Disclosures No relevant conflicts of interest to declare.

Abstract LB-303: Substrate-mimetic covalent inhibitor of MALT1 is most effective against CARD11 mutant ABC-DLBCL

Abstract Diffuse Large B-cell lymphoma is the most common subtype of B-cell non Hodgkin Lymphoma (B-NHL) representing 30% of all B-NHL. There are three molecular subtypes: germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma (PMBL). Of those, Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL) represents a third of the patients and is characterized by constitutive NF-κB activity due to mutations in various proteins of the B-cell receptor (BCR) as well as Toll-like receptor (TLR) pathways. Mutations in the BCR pathway include: activating mutations of CD79A/B (20% of ABC-DLBCLs) and CARD11 (10%) and deletion or inactivating mutation of A20 (20-30%). In the TLR pathway, MYD88 is mutated in 37% of ABC-DLBCL patients. Given this scenario, numerous therapeutic strategies targeting proteins signaling downstream of the BCR pathway have been proposed for ABC-DLBCL -including inhibitors targeting kinases SYK, BTK, PI3K, PKC, MAPKs and AKT or the protease MALT1. MALT1 inhibition provides advantages to other targets because it is downstream of most of the BCR pathway mutations present in patients, including CARD11. Patients with CARD11 mutations do not respond to BTK inhibitors. However, none of the MALT1 inhibitors reported to date are good candidates for clinical use. Here we report a new class of substrate mimetic MALT1 inhibitors based on Z-VRPR-fmk. Our lead compound, SCM-02-138 displayed nanomolar potency in biochemical and cellular MALT1 protease reporter assays. Crystallography shows covalent binding of the compound to MALT1 active site Cys residue. SCM-02-138 was highly selective for MALT1 and showed over 100-fold differential killing in sensitive vs resistant cell lines. MALT1 inhibition was most effective in CARD11 mutant cells. SCM-02-138 was active in vivo, as assessed by hIL10 inhibition in xenografted models of ABC-DLBCL cell lines. Moreover it was effective against ABC-DLBCL xenografts and PDX DLBCL ex vivo. Combination of SCM-02-138 with other BCR inhibitors revealed PI3K delta inhibition as the most synergistic combination. Combination of SCM-02-138 and CAL-101 potentiated both proliferation inhibition and apoptosis. In summary, we have developed a new class of MALT1 peptidic inhibitor characterized by nanomolar potency, irreversibility and high specificity. This inhibitor will be particularly useful against CARD11 mutant ABC-DLBCL, which are resistant to BTK inhibitors. Citation Format: Lorena Fontan, David Scott, John Hatcher, Qi Qiao, Ilkay Us, Gabriella Casalena, Mariette Bekkers, Ulrike Philippar, Matthew Durant, Spandan Chennamadhavuni, Hao Wu, Nathaniel Gray, Ari Melnick. Substrate-mimetic covalent inhibitor of MALT1 is most effective against CARD11 mutant ABC-DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2017-LB-303

RNA Interference Screen Implicates TNFAIP3 and FOXO1 in MALT1 Inhibition Resistance

Recent studies have identified small molecule inhibitors of the paracaspase activity of MALT1, a protease and scaffolding protein involved in the B-cell receptor (BCR) signaling pathway, that are effective killing lymphomas in vitro and in vivo in xenograft models of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL). ABC-DLBCL is characterized by constitutive NF-κB activity. This activation has been attributed to mutations in various protein components of the B-cell receptor (BCR) as well as Toll-like receptor (TLR) pathways. However, not all ABC-DLBCL cell lines and primary patient samples were equally sensitive to MALT1 inhibitors in vitro.In order to discover genetic modifiers of response to MALT1 inhibition we used an shRNA library screening approach. MALT1 inhibition sensitive cell line HBL-1 was infected with DECIPHER barcoded shRNA library Module 1 and cells were treated with vehicle or 300 nM of MALT1 inhibitor MI-2 for 22 days. At this time cells were harvested and genomic DNA extracted. PCR was used to amplify barcodes and gel purified bands were extracted and sequenced. Cellecta's Deconvoluter software was used to quantify the number of reads per shRNA, reads were normalized to total number of reads and fold change between vehicle and MI-2 treated cells was calculated. Among the top positively and negatively enriched hairpins, we found a significant number of genes involved in the BCR pathway including: positively regulated shRNAs against TNFAIP3 and FOXO1 and negatively regulated hairpins against BTK, CD79B and PI3K genes PIK3C2A and PIK3C2D. Interestingly, TNFAIP3 and FOXO1 are negative regulators of the BCR pathway while BTK, CD79B and PI3K genes are positive regulators of this pathway.In order to validate these results and given the abundance of inhibitors of different proteins in the BCR pathway, we run a focused combination screen using MALT1 inhibitor MI-2 and inhibitors against other proteins in the pathway in 4 MALT1 sensitive cell lines. Combinations with PI3K inhibitors were most synergistic (combination index (CI) ranging 0.12-0.67), while BTK and PKC inhibitors showed an additive effect (CI ranging 0.7-0.9). These results were confirmed using a second MALT1 inhibitor, mepazine. In order to characterize the molecular mechanism by which MALT1 inhibition cooperates with PI3K, we focused on the FDA approved drug Idelalisib. In vitro treatment of cells with MI-2 and Idelalisib showed that effect on cell growth was a combination of decreased proliferation and increased apoptosis. Moreover, we found a decrease in AKT phosphorylation followed by a decrease in FOXO1 T24 phosphorylation and an accumulation of FOXO1 protein. This result correlates with our finding that FOXO1 knockdown favors MALT1 inhibition resistance. In vivo treatment of TMD8 xenografts with a combination of MI-2 and Idelalisib showed a stronger effect than either drug used as a single agent or vehicle, confirming the increased efficacy of the combination over either drug alone.In summary, we have used an shRNA library screening in order to determine which proteins and pathways cooperate with MALT1 inhibition to kill ABC-DLBCL and to evaluate combinatorial treatments in an unbiased manner. This same approach has pointed out TNFAIP3 and FOXO1 as possible biomarkers of response. This is especially interesting since these two proteins are mutated in a proportion of ABC-DLBCL patients and could affect response to treatment not only against MALT1 inhibitors but potentially any BCR targeted therapy. DisclosuresMelnick:Janssen: Research Funding.

Abstract C177: TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway

Abstract BACKGROUND: NEDD8 activating enzyme (NAE) regulates NEDD8 conjugation in neddylation pathway, a part of the ubiquitin-proteasome system, and controls cell cycle progression, DNA replication, and signal transduction via activation of cullin-RING ligases (CRLs) and subsequent degradation of their substrates. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of some types of hematological cancer. Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) remains the least curable form of DLBCL despite recent therapeutic advances. Because either phosphatidylinositol-3 kinase (PI3K)/AKT signaling or NF-κB signaling constitutively activated by B cell receptor signaling and MYD88 are reported to promote cancer cell growth and survival in ABC-DLBCL, the key kinases in these pathways [PI3Kδ, Bruton's tyrosine kinase (BTK), and Interleukin-1 receptor-associated kinases (IRAK)] are considered to be potential targets for ABC-DLBCL therapy. We previously reported an NAE inhibitor, TAS4464, that could inactivate NF-κB by accumulating phosphorylated IκBα (p-IκBα). Here, we report on the possible application of TAS4464 to treat ABC-DLBCL that is insensitive to kinase inhibitors. MATERIALS AND METHODS: Human ABC-DLBCL cell lines, OCI-LY10, SU-DHL-2, and TMD8 were used to evaluate the biological activity of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation and levels of CRLs substrates were evaluated by Western blot analysis. The levels of NF-κB-targeted gene transcripts were assessed by qRT-PCR. The antitumor activities of TAS4464 were evaluated in both subcutaneous and systemic xenograft model of TMD8. RESULTS: TAS4464 led to growth arrest and death in ABC-DLBCL cell lines in the nanomolar range through inhibition of cullin neddylation. Similar effects were also observed in a cell line insensitive to the kinase inhibitors that inhibit divergent activator of NF-κB. qRT-PCR analysis revealed TAS4464 inhibited the NF-κB pathway in all ABC-DLBCL cell lines tested through accumulation of p-IκBα, which directly inhibit NF-κB activity. Intravenous administration of TAS4464 showed strong antitumor activity in a TMD8 xenograft model, with inhibited cullin neddylation and activation of caspases 3 and 8 in the tumors. Futhermore, in a TMD8 systemic model, intravenous administration of TAS4464 prolonged mice survival more than two-fold. CONCLUSION: TAS4464 leads to cell growth through inhibition of NF-κB pathway regardless of the activation signaling pathway of NF-κB pathway. Though NF-kB activation signaling is complicated, the downstream inhibition of NF-κB pathway by TAS4464 indicated the potential for treatment of ABC-DLBCL in which NF-kB was dominant for their survival. In addition, TAS4464 demonstrates marked antitumor activity and survival benefit in a human ABC-DLBCL model. Therefore, TAS4464 may be a valuable addition to current options for chemotherapy in ABC-DLBCL. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C177.

Abstract A19: MALT1 inhibition as an anchor for combinatorial therapy of ABC-DLBCL.

Abstract MALT1 (Mucossa Associated Lymphoid tissue Lymphoma Translocated protein 1) is critical for the proliferation and survival of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL), the most chemo-resistant form of DLBCL. MALT1 mediates activation of the B-cell receptor pathway (BCR) downstream of characteristic somatic mutations in CD79, CARD11 or MYD88 that lead to chronically activated NF-κB. MALT1 is a paracaspase and the effector enzyme of the CARD11/Bcl10/MALT1 signalosome, a high order assembly that functions as an amplifier of BCR signaling to NF-κB. MALT1 constitutes a compelling therapeutic target because: i) it is the only paracaspase in humans, ii) MALT1 knockout mice are viable, and iii) ABC-DLBCLs are biologically dependent on MALT1 paracaspase activity. MALT1 is only active when forming multimeric complexes. In order to identify potential MALT1 inhibitors we engineered a leucine zipper-MALT1, obliged and enzymatically active dimer, and established a paracaspase enzymatic assay for high throughput screening. Screening a ~50,000 compound chemical diversity library allowed us the identification and validation of 19 distinct chemical scaffolds that inhibited MALT1 with an IC50<20 μM. Three compounds significantly induced selective dose-dependent suppression of MALT1-dependent ABC-DLBCL cells (MI-2, p<0.0001; MI-4, p=0.006; MI-11, p<0.0001). The most potent compound in cell-based assays was MI-2 with a GI25 in the low nanomolar range. MI-2 analogs also displayed nanomolar activity. In depth analysis using NMR and LC-MS revealed that MI-2 binds covalently to the active site of MALT1. In DLBCL cells, MI-2 inhibited MALT1 cleavage of its targets TNFAIP3, BCL10 and RelB, as well as nuclear translocation of c-REL and overall NF-κB activation. MI-2 inhibited proliferation by inducing G1 arrest and ultimately promoted apoptosis in ABC-DLBCLs including those with mutations that bypass BTK inhibitors, like CARD11 activating mutation. MI-2 was non toxic in vivo and potently and specifically inhibited the growth of xenotransplanted ABC-DLBCLs (p=0.014, t-test) but not GCB-DLBCLs. Moreover, MI-2 selectively killed primary human ABC-DLBCL specimens ex vivo. Given that multiple pathways contribute to ABC-DLBCL pathogenesis, we hypothesized that MALT1 inhibitors would be most effective within combinatorial therapy regimens. Along these lines MI-2 strongly enhanced the activity of CHOP chemotherapy drugs against ABC-DLBCL cells. The addition of MI-2 to doxorubicin allowed for 2.5 to 13-fold reduction in the doxorubicin dose as determined by the dose-reduction index that was specific for the doxorubicin resistant cell lines (GI50 > 200 nM). Because the BCR pathway constitutes a complex network of signaling molecules beyond NF-κB activation, we tested combination of MI-2 with inhibitors of other proteins in this pathway affecting other branches of this pathway. Combination of MI-2 with the pan PI3K inhibitor BKM120, that was in our hands the most effective against a broad group of ABC-DLBCL cell lines, resulted in synergistic cell killing of OCI-Ly10 and Rc-K8 and had an additive effect in HBL-1 while it was less than additive for OCI-Ly3 and TMD8. These cell lines harbor mutations in different proteins of the pathway, which may contribute to the differences in response to the combination. Finally MI-2 strongly synergized with BH3 mimetics (most notably ABT-737) that target fundamental complementary survival pathways to BCR signaling in ABC-DLBCLs. Synergistic killing was at least partially due to induction of apoptosis, as concurrent administration of the two drugs induced increased apoptosis assessed by Caspase-7/3 activity and Annexin V+ DAPI- flow cytometry. In summary, we identified the first specific MALT1 inhibitor drug and demonstrated a promising role for MALT1 targeted therapy as an anchor of rational combinatorial therapy against ABC-DLBCL. Citation Format: Lorena Fontan, Chenghua Yang, Himaly Shinglot, Venkataraman Kabaleeswaran, Volpon Laurent, Michael Osborne, Elena Beltran, Monica Rosen, Rita Shaknovich, Shao N. Yang, Randy D. Gascoyne, Leandro Cerchietti, Jose A. MArtinez-Climent, J Fraser Glickman, KAtherine Borden, Hao Wu, Ari Melnick. MALT1 inhibition as an anchor for combinatorial therapy of ABC-DLBCL. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A19.

Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models

Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis.Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. [Display omitted] Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

A Virtual B Cell Lymphoma Model to Predict Effective Combination Therapy

Aberrant activation of the B cell receptor (BCR) signaling network drives survival and proliferation of many B cell malignancies, such as activated B cell like diffuse large B cell lymphoma (ABC-DLBCL). A number of small molecule inhibitors targeting various kinases in the BCR signaling network have been developed. However, clinical application of these targeted agents is facing several challenges such as low response rate and acquired drug resistance. The limited efficacy of single agent targeted therapy is at least partially due to pathway reactivation through crosstalks and compensatory circuits. By simultaneously repressing multiple nodes in the signaling network, combination therapy has the potential to completely extinguish signaling and induce more potent and durable response. The complexity of BCR signaling network makes it difficult to infer which combinations will be effective and synergistic. Given the large number of possible drug combinations, comprehensive experimental screening – including exploration of multiple dosages – is not practically feasible.Computational models of signaling networks that can accurately reconstruct signaling dynamics in silico may represent a useful alternative to experimental screening and trial-and-error experimental investigation. Here, we developed a computational model that integrates signal transduction, tumor growth, and drug kinetics to accurately simulate BCR signaling dynamics and the effect of drug-induced perturbation on signaling output and cell viability. We used this model to predict effective combinatorial therapy in silicoand validated some of these predictions using in vitro experiments.Based on experimentally verified protein-protein interactions, we constructed the first detailed kinetic model of the BCR signaling network covering three major signaling pathways downstream of BCR, namely NFkB, PI3K/AKT and MAPK. The model captured complex crosstalk between these three pathways and multiple feedback loops. Simulated kinase activation time courses under temporal antigen stimulus successfully recapitulated normal BCR signaling dynamics as reported in literature.Using published drug response data in the BCR signaling-dependent ABC-DLBCL cell line TMD8, we trained a tumor growth model which in combination with the kinetic model enabled reliable prediction of viability response of many drug combinations at various dosages. For example, predicted viability response of BTK inhibitor ibrutinib in combination with inhibitors targeting other kinases in the network, e.g. BKM-120 against PI3K, sotrastaurin against PKC-beta closely matched previously published experimental data in TMD8 (r>0.86,p<1e-11).We then sought to identify synergistic drug combinations by simulating viability response at 10x10 virtual dosages for each drug combination and estimating synergism using Bliss independence model. Computational screening predicted dual blockage of LYN and SYK as the most synergistic combination, which we confirmed experimentally by treating TMD8 cells with LYN inhibitor Dasatinib and SYK inhibitor R406 at multiple doses.Finally we sought to use our model to predict biomarkers of sensitivity and resistance to specific treatment strategies. By integrating expression levels of BCR signaling network components assessed by published primary DLBCL RNA-seq data, we simulated patient-specific drug responses and computed the correlation between expression level of specific components with viability response under specific treatments. We found that overexpression of PTP1B, which dephosphorylates BTK substrate PLCg2, predicts relative sensitivity to BTK inhibition. Supporting this prediction, we observed increased PTP1B expression in DLBCL cell lines sensitive to ibrutinib treatment, suggesting PTP1B as potential biomarker for ibrutinib sensitivity.In summary, this study provides a novel approach to computationally optimize combinatorial targeted therapy against aberrant BCR signaling and paves the way for the discovery of effective patient-specific drug combinations. DisclosuresMelnick:Bioreference: Scientific Advisory Board, Scientific Advisory Board Other; Calgene: Consultancy; Janssen: Research Funding; Genentech: Speakers Bureau.

BET Bromodomain Inhibitor OTX015 Affects the Expression of Micrornas Involved in the Pathogenesis of Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, accounting for 30%-40% of all cases. Despite a major improvement in the cure rate, a large number of DLBCL patients lack therapeutic options. Aberrant changes in histone modifications, DNA methylation and expression levels of non-coding RNA, including microRNA (miRNA), contribute to DLBCL pathogenesis and represent potential therapeutic targets. OTX015 targets bromodomain and extra-terminal (BET) proteins, which are epigenetic readers contributing to gene transcription. It has shown preclinical activity in hematologic and solid tumor models (Gaudio et al, AACR 2014; Noel et al, EORTC-NCI-AACR 2013) and promising early results in an ongoing phase I study (Herait et al, AACR 2014; NCT01713582). To better understand the mechanism of action of OTX015, we studied molecular changes induced by this compound in DLBCL cell lines.Methods. Total RNA was extracted from 2 DLBCL cell lines, the germinal center B-cell (GCB) type DOHH2 and activated B-cell-like (ABC)-type SU-DHL-2, following treatment with 500 nM OTX015 or DMSO for 4h or 8h. RNA samples were labeled with cyanine-3 dye using the Agilent microRNA Complete Labeling System & Hyb Kit and hybridized to the Agilent Human microRNA microarray v.3. Raw expression values were obtained with Agilent Feature Extraction Software, log-transformed and normalized by the quantile method. Data were filtered to exclude relatively invariant features and those below the detection threshold. Limma (Linear Models for Microarray data analysis) was employed using R/Bioconductor and the filtered dataset. Baseline miRNA profiling was obtained from 22 DLBCL cell lines with the Nanostring nCounter Human v2 miRNA Expression Assay kit. Baseline gene expression profiling (GEP) was obtained in these cell lines with the Illumina HumanHT-12 v4 Expression BeadChip. Selected miRNA changes were validated by real-time PCR. Validated miRNA targets were retrieved using the miRWalk database (Dweep et al, 2011). Gene Set Enrichment Analysis (GSEA) software was used to assess enrichment of miRNA targets in the GEP datasets.Results. miRNA profiling of the GCB and ABC DLBCL cell lines exposed to OTX015 identified four downregulated miRNAs and eight which were upregulated. Among them, the oncomirs miR-92a-1-5p (log2 FC, -2.01; P=0.004) and miR-21-3p (log2 FC, -0.37; P=0.0045) were downregulated, while the tumor suppressor miR-96-5p (log2 FC, 0.39; P=0.041) was upregulated. Interestingly, changes of these miRNAs matched GEP variations of validated target genes (e.g., miR-92a-1-5p: CDKN1A, log2 FC, 0.81, CDKN2A, log2 FC, 0.81; miR-96-5p: MYC, log2 FC, -0.57, MYD88, log2 FC, -0.35). We then evaluated if these three miRNAs play a role in OTX015-sensitivity by obtaining baseline miRNA and GEP profiling data in 22 DLBCL cell lines. Compared to 8 cell lines with lower sensitivity to OTX015 (IC50 >500 nM), the 14 sensitive cell lines (IC50 <500 nM) presented lower miR-96-5p expression levels (log ratio, 2.12; P=0.026) and their GEPs were significantly enriched for validated miR-96-5p targets (normalized enrichment score, 1.4; P=0.026), suggesting miR-96-5p levels may predict response to OTX015.Conclusions. Changes in the expression levels of biologically relevant miRNAs may contribute to response to OTX015. miR-92a-1-5p, the oncomir which was most strongly downregulated by OTX015, is a member of the MYC target MIR17HG (mir-17-92 cluster), involved in the pathogenesis and chemo-resistance of lymphomas, mainly contributing to PI3K/AKT/mTOR pathway activation. Since the cell cycle transcriptional regulator E2F1 is targeted by mir-17-92, OTX015 may contribute to cell cycle arrest and to downregulation of the E2F1 target gene reported with BRD inhibitors in DLBCL cell lines. miR-21-3p, also downregulated by OTX015, is a well-known oncomir, and forced miR-21-3p expression in transgenic mice results in the development of leukemias and lymphomas. miR-96-5p, upregulated by OTX015, targets oncogenes such as RAS or MYC, and low expression has been reported in mantle cell lymphoma. Interestingly, low miR-96-5p baseline levels were associated with higher sensitivity to OTX015, an observation meriting validation in other tumor models and evaluation in clinical studies. DisclosuresStathis:Oncoethix SA: Consultancy, Research Funding. Riveiro:Oncoethix SA: Consultancy, Research Funding; Oncology Therapeutic Development: Employment. Bertoni:Oncoethix SA: Research Funding.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.