GCB Diffuse Large B-cell Lymphoma Cell Lines Research Articles

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.

Mivavotinib, a Syk Inhibitor, in Relapsed/Refractory (R/R) Non-GCB Diffuse Large B-Cell Lymphoma (DLBCL) with or without MYD88 and/or CD79 Mutations: A Phase 2 Study

Background: Spleen tyrosine kinase (Syk) is a key signaling protein that activates survival pathways in B-cell lymphomas. The non-germinal center B-cell like (non-GCB) or activated B-cell like (ABC) cell-of-origin subtype of DLBCL has greater reliance on the B-cell receptor pathway and Syk signaling for survival, compared with the GCB subtype. Multiple signaling pathways are activated by Syk in ABC DLBCL (NF-κB and phosphoinositide 3-kinase [PI3K]), whereas only the PI3K pathway is activated by Syk in GCB DLBCL. MYD88 and/or CD79b mutations are strongly enriched within ABC DLBCL and are associated with poor prognosis. Syk hyperactivation has been observed in ABC DLBCL cell lines harboring MYD88/CD79 mutations, with activation of NF-κB via Syk-mediated signaling through both the BCR and the toll-like-receptor (TLR)-MYD88 pathways. Mivavotinib is an orally dosed, potent, and selective Syk inhibitor, which showed potent single agent activity in preclinical studies in ABC and GCB DLBCL cell lines, with the strongest activity against those harboring MYD88 and/or CD79b mutations. In the clinical setting, heavily pre-treated all-comer R/R DLBCL patients receiving single agent mivavotinib at the recommended phase 2 dose (100 mg once daily [QD]) showed an overall response rate (ORR) of 33% (16% complete responses [CR]) with durable responses (median duration of response [DoR] not reached [95% CI: 7 months - NE]) and a favorable safety profile. Subgroups by cell-of-origin showed a substantially higher ORR in patients with the non-GCB subtype (53%; 95% CI: 27, 79) than GCB (22%; 95% CI: 0.11, 0.37), and a median DoR of 15.7 months (95% CI: 2.2, NE) in non-GCB responders. CX-659-401 (NCT05319028) is an ongoing study evaluating two dosing regimens of mivavotinib monotherapy in patients with R/R non-GCB DLBCL, in whom circulating tumor DNA-based next generation sequencing (NGS) will be used to determine MYD88/CD79b mutational status. The goals of this strategy are to confirm the activity of mivavotinib previously observed in non-GCB DLBCL, evaluate its activity in DLBCL according to mutational status of MyD88/CD79b, and optimize the dose and schedule in this biomarker-defined patient population. Methods: This multicenter, open-label, phase 2 study will enroll patients with histologically confirmed de novo or transformed DLBCL that is non-GCB by immunohistochemistry (IHC) per Hans algorithm or ABC by gene expression profiling, including primary mediastinal and primary extranodal DLBCL. Patients must have had at least 2 but no more than 5 prior lines of systemic anticancer therapy. Following standard first-line R-CHOP (or equivalent), patients must have received 2nd-line salvage therapy with or without autologous stem cell transplant (ASCT), and/or CAR-T therapy, unless ineligible for 2nd-line salvage, ASCT, or CAR-T. Additional key eligibility criteria include age >18 years, ECOG performance status 0-2, and FDG-PET-avid measurable disease per 2014 Lugano criteria. DLBCL patients with primary brain or secondary central nervous system involvement with active disease will be excluded. Patients will be randomized to 1 of 2 arms (n=25 planned per arm): (1) 100 mg QD; or (2) 120 mg QD for 2 weeks followed by 80 mg QD thereafter; in 28-day cycles until disease progression or unacceptable toxicity. The primary endpoints are ORR (by independent radiology review according to 2014 Lugano Criteria) and safety/tolerability. Secondary endpoints include DoR, progression-free survival (PFS), overall survival (OS), and CR rate. Exploratory endpoints include pharmacokinetic, biomarker assessments, and investigator-assessed ORR, DoR, PFS, and CR rate. Results of this study will help to define the optimal dose and provide confirmatory efficacy and safety data of mivavotinib in patients with non-GCB DLBCL, including MYD88/CD79b-mutated DLBCL to guide further expansion in these biomarker-defined cohorts.

Abstract 3720: TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in the US accounting for about 22% of newly diagnosed cases of B-cell NHL. It is considered to be more aggressive and to have worse prognosis due to low response to existing treatments. KDM4 is a family of histone demethylases that can drive tumor growth by regulating transcription, cell cycle, and DNA replication/repair. Overexpression of KDM4 alters post-translational histone modification and is associated with many types of cancer, including DLBCL. TACH101 is a novel, potent small molecule inhibitor of KDM4 that is being developed for treatment of advanced cancers, including DLBCL. Methods: TACH101 was evaluated in vitro and in vivo using cancer cell lines and patient-derived organoid (PDO) and xenograft (PDX) models of DLBCL. Results: TACH101 is a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values < 0.100 μM for all four isoforms. An initial screen using a 301-cell line panel showed that DLBCL cell lines are sensitive to TACH101 (IC50 < 10 nM). In an additional panel of DLBCL cell lines, TACH101 inhibited proliferation in a dose-dependent manner in all DLBCL cell lines, independent of molecular subtype, with mean IC50’s of 0.03 ± 0.01 μM in ABC DLBCL cell lines (n=6); 0.02 ± 0.01 μM in GCB DLBCL cell lines (n=7) and 0.02 ± 0.01 μM in PMBL DLBCL cell lines (n=2). In OCI-LY19 DLBCL xenografts in vivo, TACH101 was well tolerated and inhibited tumor growth by 55% to 100%, depending on dosing regimen (either QD or BID following a 3 days on/4 days off schedule). In PK studies, TACH101 exhibited low clearance, moderate volume of distribution, and good oral bioavailability in mouse, rat, and dog. Moreover, treatment with TACH101 resulted in little or no inhibitory effects on CYP enzymes. Toxicity studies in rats and dogs identified potential target tissues and provided safety guidance for the use of TACH101 in human studies. Conclusions: The KDM4 inhibitor, TACH101, had compelling activity in preclinical DLBCL models, suggesting that TACH101 could be an effective therapy for DLBCL. Preparations to advance the drug into clinical trials are underway. Citation Format: Frank Perabo, Chandtip Chandhasin, Sanghee Yoo, Joselyn Del Rosario, Young K. Chen, Ellen Filvaroff, Jeffrey A. Stafford, Stephen Quake, Michael F. Clarke. TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3720.

Xpo-1 Inhibition Synergizes with BCR Inhibition, Blocks Tumor Growth and Tumor-Associated Macrophages Infiltration and Prolongs Survival in a Bioluminescent Animal Model of Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is an infrequent and aggressive non-Hodgkin lymphoma usually classified as diffuse large B-cell lymphoma (DLBCL), being most of the cases more closely related to activated B-cell type (ABC). PCNSL is associated with dismal prognosis, due in part to the difficulty for many chemotherapeuticals to cross the blood brain barrier.Selinexor (KPT-330) is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inactivates XPO-1 protein and induces anti-tumor effects mainly due to forced nuclear retention and activation of tumor suppressor proteins. Selinexor, that has shown excellent brain penetrance and promising results in pre-clinical models of glioblastoma, can inhibit both BCR and NF-kB signaling in malignant B-cells.In order to provide a pre-clinical rationale for the design of new therapeutic strategies in the PCNSL, we assessed the role of XPO-1 inhibition using intracerebral xenograft murine models.To compare the sensitivity of DLBCL-derived cell lines to selinexor, we determined the inhibitory concentration 50 (IC50) in terms of survival and proliferation after 96 hours in 4 ABC and 5 GCB DLBCL cell lines. Analysis showed that DLBCL cell lines have equivalent sensitivity to selinexor, regardless the cell of origin. Since SINE compounds have been shown to inhibit BCR signaling, we next tested the potential synergy between ibrutinib and selinexor. In 3 out of 4 ABC-DLBCL cell lines there was a strong synergy. In contrast, none of the 3 GCB-DLBCL cell lines analyzed were sensitive to up to 100 µM single agent ibrutinib; interestingly, however, treatment with selinexor sensitized SUDHL4 (GCB) cells to ibrutinib and the combination index values indicated strong synergism between the two drugs.We then established an orthotopic xenograft model of PCNSL by stereotactic injection of OCI-Ly10 cells stably transfected with luciferase into the brain parenchyma of nude athymic mice. Eleven days after the injection of cells all animals had developed detectable tumors confined to the CNS. Tumor size was measured and animals were randomly distributed into drug or vehicle experimental group. At this time point mice were treated with 5mg/kg of selinexor or vehicle via oral gavage three times a week; subsequently, bioluminiscence was assessed twice a week. Treatment with selinexor significantly blocked tumoral growth (two-way ANOVA: p< 0.0001; figure 1A). Specific time-point analysis showed that differences were significant as soon as 12 days after treatment (2,255e+008 +/- 8,648e+007 vs 8,670e+006 +/- 1,981e+007). Also, treatment with selinexor increased mice survival, with a median survival of 48 days in the treatment group compared to 34 days in the vehicle group (p< 0.0001; figure 1B). At final point, histopathological analysis showed diffuse infiltration in meninges and cerebral parenquima of highly proliferative CD20-positive B-cells (virtually 100% Ki-67-positive malignant B cells). Since the infiltration of tumoral associated macrophages (TAMs) has been related to worse prognosis in different lymphomas, including PCNSL, we analyzed the infiltration by F4/80+ TAMs after two weeks of treatment with selinexor or vehicle. We observed that all tumors from vehicle-treated mice were infiltrated with TAMs, whereas treatment with selinexor significantly reduced the presence of TAMs as assessed by immunohistochemistry, especially in the parenquima. Next, we evaluated the potential synergy between ibrutinib and selinexor in vivo. For that 12 mice with equivalent tumor size were assigned to each of the following four groups: selinexor only (5mg/kg twice a week), ibrutinib only (25mg/kg daily), combination or vehicle. The combination of both drugs significantly increased survival compared to both treatments as single agent, whereas there was no significant difference between ibrutinib and selinexor treatments alone (figure 1C). Tumoral growth was equally blocked by all treatments (figure 1D). Currently, we are evaluating the effect of the combination in the infiltration and immunophenotype of TAMs, including M1 vs M2 phenotype and PD1 expression. Results will be available at the time of the meeting.Selinexor proved to be an active agent in blocking tumoral growth in DLBCL infiltrating CNS, this providing pre-clinical evidence for the development of selinexor as new therapeutic option for PCNSL or DLBCL with CNS involvement. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract PO-07: The FLI1 direct target ASB2 promotes NF-KB pathway activation in diffuse large B-cell lymphoma of the germinal center B-cell type

Abstract Introduction: Gains affecting chromosome 11 are recurrent events in lymphomas. The 11q24.3 gain occurs in 25% of diffuse large B-cell lymphoma (DLBCL), and it is associated with the overexpression of two ETS transcription factors, ETS1 and FLI1 (Blood 2013). Here, we have focused on the latter to identify the network of FLI1 regulated genes in GCB DLBCL. Methods: GCB and ABC cell lines. Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013, GSE10846, and EGAS00001002606. Anti-FLI1 antibody - ChIP Grade (ab15289). ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNA) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was analyzed using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. Results: The analysis of DLBCL cell lines showed that FLI1 protein levels were higher in GCB (n=12) than ABC (n=8) cell lines and was more commonly expressed at high levels in GCB (n= 414) than ABC (n= 518) DLBCL clinical specimens. Integration of identified binding sites from ChIP-Seq with RNA-Seq from GCB DLBCL cell lines (OCI-Ly1 and VAL) with genetically silenced FLI1 allowed the identification of putative FLI1 direct targets. The FLI1 negatively regulated genes included tumor-suppressor genes involved in negative regulation of cell cycle and p53 cascade. Among the FLI1 positively regulated targets we found genes annotated for immune response, MYC targets and B cell receptor, TNF-alpha and IL2 signaling pathways. Of note, direct targets of FLI1 overlapped with those genes regulated by ETS1, the other transcription factor co-gained in DLBCL, suggesting a functional convergence within the ETS family. ASB2 was downregulated after FLI1 silencing and had FLI1 binding sites in both promoter region and distal enhancer regions. Furthermore, ASB2 is known to promote NF-kB activation in T-cell acute lymphoblastic leukemia and might be an essential gene in DLBCL cells according to a genetic screening. Consistently, ASB2 gene silencing was toxic in GCB DLBCL lines. We observed inhibition of NF-kB pathway by a strong protein downregulation of RELB, along with increased IκBα upon ASB2 and FLI1 silencing, although with no differences in NF-kB2 levels. Only FLI1 silencing caused downregulation of NF-kB1 and RELA protein levels, but no effect on these two proteins was observed upon ASB2 silencing. These results indicate that FLI1 regulates either the classic NF-kB pathway at transcriptional level or the alternative pathway, via ASB2, in GCB DLBCL. Conclusions: FLI1 is expressed at higher levels in GCB than ABC DLBCL and directly regulates a network of biologically crucial genes and processes in DLBCL, contributing to the regulation of NF-kB pathway in GCB DLBCL. ASB2, a subunit of a multimeric E3 ubiquitin ligase complex, is a novel FLI1 direct target, and its inhibition might represent a therapeutic approach for GCB DLBCL. Citation Format: Giulio Sartori, Sara Napoli, Luciano Cascione, Elaine Y.L. Chung, Valdemar Priebe, Alberto J. Arribas, Andrea Rinaldi, Michela Dall'Angelo, Mattia Forcato, Silvio Bicciato, Margot Thome, Francesco Bertoni. The FLI1 direct target ASB2 promotes NF-KB pathway activation in diffuse large B-cell lymphoma of the germinal center B-cell type [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-07.

Abstract 5215: Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma

Abstract The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Eμ-PIM and Eμ-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin, suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Citation Format: Patrizia Mondello, Chiara Tarantelli, Luciano Cascione, Alberto J. Arribas, Andrea Rinaldi, Anas Younes, Francesco Bertoni. Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5215.

Targeting PIN-1 Attenuates GCB DLBCL Cell Proliferation Through Inhibition of PI3K/AKT Signaling.

IntroductionDiffuse large B cell lymphoma (DLBCL) is a highly heterogeneous type of non-Hodgkin lymphoma with many molecular subtypes that can be distinguished by gene expression profiling (GEP). However, the pathogenesis of DLBCL is still unclear.Materials and MethodsThe expression levels of the prolyl isomerase PIN-1 and other related proteins were determined in 73 primary DLBCL patient samples and cell lines by Western blotting (WB) and immunohistochemical (IHC) staining. Cell cycle and apoptosis were evaluated by flow cytometry. Lymphoma cell viability was detected by CCK-8 proliferation assay.ResultsHigh levels of PIN-1 expression were detected in 55% of germinal center B cell (GCB) DLBCL patient samples, whereas such abnormal expression levels were found in only 11% of non-GCB DLBCL patient samples. PIN-1 expression was positively associated with activation of the oncogenic phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in both GCB DLBCL cell lines and primary patient samples. Depletion of PIN-1 was cytotoxic to GCB DLBCL model cell lines because it led to inhibition of the PI3K/AKT signaling pathway, revealing a GCB DLBCL subgroup that is dependent on this pathway. A PI3K inhibitor was selectively toxic to GCB DLBCL lines expressing high levels of PIN-1.ConclusionOur study used PIN-1 to identify a new subgroup of GCB DLBCL associated with the PI3K/AKT signaling pathway, and our findings reveal that inhibition of PI3K is a promising therapeutic approach for GCB DLBCL.

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.

Inhibition of PIM Kinases Targets Synthetic Vulnerabilities and Enhances Antigen Presentation in B-Cell Lymphoma

The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Em-PIM and Em-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. (Figure 1A)The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin (Figure 1B), suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. (Figure 1C) Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. (Figure 1D) Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. (Figure 1E) In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Disclosures Younes: Xynomics: Consultancy; Biopath: Consultancy; Genentech: Research Funding; AstraZeneca: Research Funding; Syndax: Research Funding; BMS: Research Funding; HCM: Consultancy; Celgene: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Takeda: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Pharmacyclics: Research Funding. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants.

SHP1 Deficiency Is Responsible for the Constitutive Activation of the BCR Pathway in GCB DLBCL

Functional and transcriptional profiling studies have identified a subset of diffuse large B cell lymphoma (DLBCL) tumors that rely on B cell receptor (BCR) signals for proliferation and survival. These BCR-dependent tumors have been identified among both the ABC and GCB DLBCL subtype. However, differences have been observed between the two subtypes in terms of the mechanism of BCR pathway activation and activity of downstream signaling molecules. BCR-dependent ABC DLBCL tumors are characterized by high basal NF-kB and PI3K/AKT activity and the presence of gain-of-function mutations in the CD79A or CD79B subunit of the BCR that are responsible for the chronic activation of the BCR pathway. In contrast, BCR-dependent GCB DLBCL tumors have low baseline NF-kB activity and lack CD79A/CD79B mutations, suggesting a different mechanism of BCR pathway activation. In this study, we investigated whether activation of the BCR pathway in GCB DLBCL is potentially caused by deficiency of the phosphatase SHP1, which is an important negative regulator of the BCR pathway and has been reported to be downregulated in approximately 40% of primary DLBCL tumors. For this purpose, we first correlated SHP1 expression with the presence of phosphorylated SYK and BLNK in the GCB DLBCL cell lines OCI-Ly1, OCI-Ly7, OCI-Ly18, SU-DHL-4, SU-DHL-6, WSU-NHL, SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage. Immunoblotting analysis revealed that SHP1 is expressed in SU-DHL-8, Toledo, BJAB, OCI-Ly19, OCI-Ly4, and Farage but not in the remaining 6 cell lines. Expression of SHP1 inversely correlated with expression of phosphorylated SYK (pSYK-Y352) and BLNK (pBLNK-Y84), suggesting that these two BCR-proximal signaling molecules are activated because of SHP1 deficiency. To further evaluate this possibility, we re-expressed SHP1 in OCI-Ly1 and SU-DHL-4 cells by transient transfection with a plasmid or a lentiviral SHP1 expression vector. Both approaches resulted in a substantial reduction of pSYK-Y352 and pBLNK-Y84 levels, confirming that SHP1 deficiency leads to activation of the BCR pathway.To determine whether activation of the BCR pathway contributes to tumor cell survival, we investigated the effects of the SYK inhibitor R406 (active substance of the drug fostamatinib) on the viability of the 6 SHP1-positive and 6 SHP1-negative GCB DLBCL cell lines. At concentrations up to 2 μM, R406 displayed only minimal toxicity against each of the SHP1-positive and SHP1-negative GCB DLBCL cell lines (<20% apoptotic cells), with the exception of WSU-NHL (60% apoptotic cells). However, analysis of the effects of R406 on the expression of BCL-2 family proteins showed downregulation of the antiapoptotic protein MCL-1 and upregulation of the proapoptotic proteins BIM and HRK in all of the SHP1-negative but in none of the SHP1-positive GCB DLBCL cell lines. Such changes were also observed following siRNA-mediated knockdown of SYK or overexpression of SHP1, further suggesting that they are on-target effects of R406. In addition, treatment with the BTK inhibitor ibrutinib or siRNA-mediated knockdown of BTK also resulted in downregulation of MCL-1 and upregulation of BIM in these cell lines. Similar changes were observed in 3 of the 5 investigated primary DLBCL samples following treatment with ibrutinib or fostamatinib. Importantly, downregulation of MCL-1 by RNA interference or upregulation of BIM by mRNA transfection resulted in substantially increased sensitivity of these cell lines to the BCL-2 inhibitor venetoclax, suggesting that SYK or BTK inhibitors could potentiate the cytotoxic effects of this drug. To further evaluate this possibility, we calculated the combination index of the R406 + venetoclax or ibrutinib + venetoclax drug combination in the 12 GCB DLBCL cell lines. Significant synergistic activity was seen in all five BCL-2-positive/SHP1-negative GCB DLBCL cell lines with the R406 + venetoclax combination and in four of them with the ibrutinib + venetoclax combination. In contrast, synergistic activity was detected in only one of the 6 SHP1-positive GCB DLBCL cell lines and was not seen in the BCL-2-negative/SHP1-negative cell line OCI-Ly7. Collectively, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-263: Preclinical efficacy of the ATR inhibitor AZD6738 in combination with the BTK inhibitor acalabrutinib in ABC-DLBCL models

Abstract Bruton tyrosine kinase (BTK) is an essential kinase in the B-cell receptor (BCR) signalling pathway. Acalabrutinib is a potent and highly selective irreversible BTK inhibitor that received accelerated approval by FDA for the treatment of Relapsed/Refractory Mantle Cell Lymphoma. It is proposed to be combined with the Ataxia telangiectasia and Rad3-related (ATR) kinase inhibitor AZD6738, in a Phase I/II proof of concept clinical trial to assess safety and efficacy in patients with B-cell malignancies. ATR is a key regulator of DNA replication, repair and cell cycle checkpoints. The Activated B-Cell (ABC) subtype of Diffuse Large B-Cell Lymphoma (DLBCL) is associated with chronic active BCR signalling, and given our previous identification of sensitivity to AZD6738 in ABC-DLBCL cell lines, we hypothesised that combination with acalabrutinib may have additional therapeutic benefit. Here we report preliminary data to support combination efficacy in models of ABC-type DLBCL. The growth inhibitory and cell kill effect of AZD6738 and acalabrutinib in combination was assessed in 3 ABC and 4 GCB DLBCL cell lines. Combination activity and cell kill was detected specifically in the ABC-type TMD8 cell line. Using flow cytometry, we show that the cell kill effects of the drug combination were dependent on dose and schedule. Single agent treatment with 5 nM acalabrutinib or 0.5 µM AZD6738 for 72 h, caused cell death in 12% and 17% of cells respectively. The equivalent doses given in combination for 72 h induced cell death in 39% of cells, indicative of a greater than additive effect. Cell killing was increased with longer co-exposure to the combination, shown by an increase in cell death from 18% at 24 h, to 30% at 48 h. Acalabrutinib is dosed on a BID schedule, thus we tested whether its activity in combination with AZD6738 was affected by pre- or post-addition of acalabrutinib. A 24 h pre-treatment of cells with acalabrutinib, followed by the combination of AZD6738 and acalabrutinib for 48 h resulted in 20% cell death, whereas 48 h combination treatment followed by 24 h acalabrutinib monotherapy caused a similar 27% increase in death, suggesting that combination efficacy is not dependent on sequence of drug administration. Furthermore, the mRNA expression of BTK target genes in ABC-DLBCL cell lines was modulated by acalabrutinib, however there was no additional modulation to this pathway by the combination with AZD6738, suggesting the two agents kill cells through different mechanisms of action. In the TMD8 xenograft mouse model, daily dosing of AZD6738 (25 mg/kg) with twice daily dosing of acalabrutinib (20 mg/kg) was well tolerated and resulted in complete and durable tumor regressions (8/8 tumors), whereas single agent treatments resulted in tumor growth delay. Collectively, our preliminary data support a rationale for combining BTK and ATR inhibitors for an effective and alternative treatment option in ABC-DLBCL. Citation Format: Lucy A. Young, Oona Delpuech, Brandon Willis, Alexandra Bussey, Zena Wilson, Michelle Dupont, Carlos Grajales, Andrew Bloecher, Todd Covey, Kate Wills, Alan Lau, Simon J. Hollingsworth. Preclinical efficacy of the ATR inhibitor AZD6738 in combination with the BTK inhibitor acalabrutinib in ABC-DLBCL models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-263.

XPO1 Inhibition By Selinexor Synergizes with BCR Inhibition, Blocks Tumor Growth and Prolongs Survival in a Bioluminescent Animal Model of Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is a very aggressive non-Hodgkin lymphoma localized in the cerebral parenchyma, eyes, leptomeninges or spinal cord in the absence of systemic involvement. Approximately 95% of PCNSL are classified as diffuse large B-cell lymphoma (DLBCL), being most of them more closely related to activated B-cell type (ABC-DLBCL). PCNSL is associated with poor prognosis, particularly because of the difficulty for drugs to cross the blood brain barrier. High dose methotrexate is the most effective treatment, but relapse is very common and salvage therapies are scarce. Therefore, the development of effective drugs with ability to penetrate the CNS is highly needed.Selinexor (KTP-330) is a Selective Inhibitor of Nuclear Export (SINE) compound that binds and inactivates XPO-1 protein. This inactivation induces anti-tumor effects by forcing nuclear retention and activation of tumor suppressor proteins. Selinexor has shown excellent brain penetration, exhibits promising results in pre-clinical models of glioblastoma and can inhibit both BCR and NF-kB signaling in malignant B-cells. Therefore, in order to provide a pre-clinical rationale for the use of selinexor for patients with PCNSL, we tested the role of XPO-1 inhibition in PCNSL using intracerebral xenograft murine models.We then determined the inhibitory concentration 50 (IC50) in 4 ABC and 5 GCB DLBCL cell lines. Cell survival and proliferation were tested 96 hours after incubation with selinexor. Analysis showed that the DLBCL cell lines have equivalent sensitivity to selinexor, regardless cell of origin (COO). In detail, survival assessed by AnnexinV-PI exclusion showed that mean ID50 for ABC cell lines was 4.98 µM +/- 3.6 and 6.3 µM +/- 3.8 for GCB (p=0.9). Proliferation assessed by MTS was also blocked by selinexor (mean ID50 for ABC-DLBCL was 1.35 µM +/- 0.7 vs. 16.16 µM +/- 11.17 for GCB-DLBCL (p=0.41)).Since SINE compounds have been shown to inhibit BCR signaling, we next tested the potential synergy between ibrutinib and selinexor. All three ABC-DLBCL cell lines tested were sensitive to ibrutinib and in two of them the combination with selinexor was strongly synergic. In contrast, none of the 3 GCB-DLBCL cell lines analyzed were sensitive to up to 100 µM single agent ibrutinib; interestingly, however, treatment with selinexor sensitized SUDHL4 cells to ibrutinib and the combination index values indicated strong synergism between the two drugs.Finally, we established an orthotopic xenograft model of PCNSL by stereotactic injection of the OCI-Ly10 (ABC) cell line stably transfected with firefly luciferase into the cerebral parenchyma of nude athymic mice. This allows the non-invasive longitudinal quantification of intracerebral tumor growth (bioluminescence detection, IVIS¨ Spectrum). Eleven days after the injection of cells all animals had developed detectable tumors confined to the CNS. Tumor size was measured and animals were randomly distributed into drug or vehicle experimental group (vehicle: n=8, mean radiance= 1.33á107 p/sec/cm2/sr ± 0.68á107; treatment: n=9, mean radiance=2.99á107 p/sec/cm2/sr ± 2.3á107). At this time point mice were treated with 5mg/kg of selinexor or vehicle via oral gavage three times a week. Treatment with selinexor significantly increased mice survival, with a median survival of 48 days in the treatment group compared to 34 days in the vehicle group (p< 0.0001; figure 1A). Mice in the treatment group showed a significantly slower increase in bioluminescence signal (two-way ANOVA: p< 0.0001; figure 1B). Specific time-point analysis showed that differences were significant as soon as 8 days after treatment. At final point, histopathological analysis showed diffuse infiltration in meninges and cerebral parenquima of highly proliferative CD20-positive B-cells (virtually 100% Ki-67-positive malignant B cells)In conclusion, selinexor inhibits proliferation and survival of DLBCL cell lines, regardless of the cell of origin and it can synergize with ibrutinib. Moreover, treatment of mice with CNS confined ABC-DLBCL with selinexor significantly reduces tumor growth and increases survival. Therefore, our results provide pre-clinical evidence for the development of selinexor as new therapeutic option for PCNSL or DLBCL with CNS involvement. [Display omitted] DisclosuresBosch:Hoffman La Roche: Consultancy, Honoraria, Research Funding; Millennium: Research Funding.

Primary Mediastinal Large B-Cell Lymphoma Exhibits Autonomous BCR Signaling and Responds to the Second Generation BTK Inhibitor Acalabrutinib

BackgroundPrimary mediastinal large B-cell lymphoma (PM LBCL) is putatively derived from thymic B cells. PM LBCL is clinically and morphologically distinct from diffuse large B-cell lymphoma (DLBCL), but shares certain phenotypic characteristics with nodular sclerosing classical Hodgkin's lymphoma. The expression and function of an intact B-cell receptor (BCR) in PM LBCL is controversial. In addition, the pathological mechanisms that drive lymphomagenesis in PM DLBCL are largely unknown. Here, we investigate the expression of functional BCR in PM LBCL and explore the hypothesis that autonomous signaling of the BCR as observed in CLL (Dühren-von Minden, Nature 2012) plays a key role in PM DLBCL pathogenesis.MethodsThe PM LBCL cell lines Farage, MedB-1, Karpas 1106P, and U2940 were tested in vitro by comparing calcium flux with and without inhibition of BCR signaling by blockade of Syk with the tyrosine kinase inhibitor R406.BCR transcripts were identified from archived fresh-frozen biopsies from two histologically confirmed PM LBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. To test for autonomous BCR signaling, murine triple KO (TKO) cells were transduced with functional BCR of PM LBCL (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon induction with tamoxifen as calcium flux by flow cytometry or as robust cellular proliferation.ResultsAll four PM-LBCL lines had higher calcium flux in the absence of R406, suggesting autonomous BCR signaling activity without antigenic stimulation or artificial BCR crosslinking. Functional BCR transcripts could be readily identified in both primary PM LBCL biopsies by ARTISAN PCR. In accordance with our hypothesis, TKO cells transduced with the BCR of both primary PM LBCL cases showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. To test whether this autonomous BCR signal would represent a druggable therapeutic target, the PM DLBCL cell lines were cultured in the presence of increasing doses of the second generation, highly BTK-specific kinase inhibitor acalabrutinib. The ABC DLBCL cell lines OCI-Ly10, TMD8, and U2932, and the GCB cell lines Karpas 422, OCI-Ly7, and OCI-Ly19 were tested in parallel as controls. Cell viability decreased in all cell lines with increasing concentrations of acalabrutinib (Figure). Consistent with their known dependency on active BCR signalling (Young, PNAS 2015; Koning, ASH 2016), ABC DLBCL cell lines were very sensitive to acalabrutinib. As suggested by the experimental evidence for autonomous BCR signalling, PM LBCL cell lines also responded to acalabrutinib, albeit to a lesser degree than ABC DLBCL. Growth inhibition of GCB DLBCL cell lines generally required excessively high doses of acalabrutinib, consistent with the notion that this DLBCL subtype depends on tonic BCR signaling only.ConclusionConsistent with our hypothesis, our data demonstrate expression of an intact BCR with autonomous signaling activity in all investigated cases of PM LBCL. These results point to an oncogenic role of structurally normal BCR in PM LBCL akin to CLL. Similarly to CLL and ABC DLBCL, PM DLBCL cell lines respond to BTK inhibitors, albeit less strongly than the ABC DLBCL cell lines tested. The results identify BCR signaling as a novel therapeutic target in PM LBCL and suggest the initiation of clinical trials with BTK inhibitors in PM LBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HCK Transcription Is Regulated By AP1, NF-Kb and STAT3 Transcription Factors in MYD88 Mutated WM and ABC-DLBCL Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family of tyrosine kinases (SFKs). HCK transcription is aberrantly upregulated in Waldenström’s Macroglobulinemia (WM) and Activated B-cell (ABC) subtype Diffuse Large B-cell Lymphoma (DLBCL) in response to activating mutations in MYD88 (Yang et al, Blood 2016). To clarify the mechanism responsible for the aberrant upregulation of HCK transcription inMYD88 mutated cells, we analyzed the promoter sequence of HCK using PROMO and identified consensus binding sites for transcription factors (AP1, NF-kB, STAT3, and IRF1) that are regulated by mutated MYD88 (Ngo et al, Nature 2011; Treon et al, NEJM 2012; Yang et al, Blood 2013; Juilland et al, Blood 2016; Yang et al, Blood 2016). We performed Chromatin Immuno-precipitation (ChIP) assays using ChIP grade antibodies to JunB, c-Jun, NF-kB-p65, STAT3 and IRF1 in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1, OCI-Ly3) cells that highly express HCK transcripts, as well as wild type MYD88 expressing GCB DLBCL (OCI-Ly7, OCI-Ly19) cells that show low HCK transcription. Following ChIP, a HCK promoter specific quantitative PCR assay was used to detect HCK promoter sequences. These studies showed that JunB, NF-kB-p65 and STAT3 bound more robustly to the HCK promoter in MYD88 mutated WM and ABC-DLBCL cells versus MYD88 wild type GCB DLBCL cell lines, while c-Jun bound more abundantly to the HCK promoter sequence in all DLBCL cell lines, regardless of MYD88 mutation status. In contrast c-Jun binding was low in MYD88 mutated WM cells. IRF1 binding to the HCK promoter was similar in all cell lines, regardless of the MYD88 mutation status. To further investigate HCK regulation, we developed an HCK promoter driven luciferase reporter vector (WT) with mutated AP-1 binding (AP1-mu-1~6), NF-kB binding (NF-kB-mu-1~5), and STAT3 binding (STAT3-mu) sites and investigated their impact on HCK promoter activity in MYD88 mutated BCWM.1 cells. We observed that mutation of AP1-mu-1,4,5,6; NF-kB-mu-1,4,5, as well as STAT3-mu binding sites greatly reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 transcription factors in HCK gene expression in MYD88 mutated cells. To further clarify the importance of these transcription factors in aberrant HCK gene expression in MYD88 mutated cells, we treated BCWM.1, MWCL-1, TMD-8 and HBL-1 cells with the AP-1 inhibitor SR 11302; NF-kB inhibitor QNZ; and the STAT3 inhibitor STA-21. Treatment of cells for 2 hours with SR 11302, QNZ, and STA-21 at sub-EC50 concentrations resulted in decreased HCK expression in MYD88 mutated all cell lines. Lastly, we investigated the contribution of BCR signaling to HCK transcription. BCWM.1, MWCL-1, TMD-8, and HBL-1 cells were treated with the Syk kinase inhibitor R406, and HCK transcription levels were then assessed. Differences in HCK expression were observed between MYD88 mutated WM and ABC DLBCL cells following R406, supporting a contributing role for BCR signaling in ABC DLBCL but not WM cells to HCK expression. Our data provide critical new insights into HCK regulation, and a framework for targeting pro-survival HCK signaling in WM and ABC DLBCL cells dependent on activating MYD88 mutations. DisclosuresCastillo:Biogen: Consultancy; Otsuka: Consultancy; Millennium: Research Funding; Janssen: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria. Treon:Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding.

A Novel Pan-PIM Kinase Inhibitor, SEL24-B489, Induces Apoptosis and Inhibits Proliferation of Diffuse Large B-Cell Lymphoma Cells through Inhibition of Protein Translation and Attenuation of Myc and NFkB Activity

Introduction. In 1/3of diffuse large B-cell lymphoma (DLBCL) patients current treatment options are ineffective, underscoring the need for more effective targeted therapies. Simultaneous genetic ablation of PIM kinases (PIM-1/2/3) or their pharmacological inhibition induce apoptosis in DLBCL cell lines, providing proof of concept that these proteins are promising targets in this disease. To better understand mechanisms of toxicity of PIM inhibition in DLBCL, we investigated biochemical and biological consequences of PIM inhibition using novel pan-PIM inhibitor (SEL24-B489) in a panel of DLBCL cell lines in in vivo xenograft model.Methods. Protein expression and phosphorylation status of PIM substrates was assessed by immunobloting. Proliferation and apoptosis were assessed by MTS assay and PI/AnnexinV staining. Gene expression profiling was performed on Illumina HT-12 v4 Chip. NFkB target genes expression was assessed by Real-Time Quantitative PCR (qPCR). For xenograft studies, 9-11 weeks old SCID/beige CB17 mice were subcutaneously injected with 5x106 U2932 cells suspended in 0.1 ml mixture of PBS with Matrigel. When tumor volumes reached ~100 mm3, mice were randomized into uniform groups and subjected to the compound administration (50 mg/kg daily, BID, per os). Tumor volume was monitored every second day.Results. The newly developed pan-PIM inhibitor (SEL24-B489) was toxic to DLBCL cell lines in low-micromolar or sub-micromolar concentrations (IC50: 0,29 to 1,17µM). To determine mechanisms underlying toxicity of PIM inhibition in DLBCL, we first assessed the activity of 4EBP1 and ribosomal protein S6, proteins involved in protein translation. PIM inhibitor rapidly and uniformly decreased 4EBP1 and S6 phosphorylation in all tested DLBCL cell lines (DHL4, DHL6, Ly1, Ly7, Karpas 422, Pfeiffer, Toledo, U2932, HBL-1). Since PIM kinases have been shown to increase stability and/or activity of NFκB-p65 (RelA) and Myc, we assessed the abundance/activity of these transcription factors upon PIM inhibitor treatment. PIM inhibition led to downregulation of Myc protein in GCB DLBCL cell lines. Since Myc and PIMs cooperate in augmenting transcription of active genes, we assessed global RNA levels upon PIM inhibitor treatment. The RNA abundance in SEL24-B489-treated cells decreased by 22-37%. The underlying mechanism of the decrease in cellular mRNA content involved global inhibition of PIM-dependent histone H3 serine 10 (H3S10) phosphorylation and decreased phoshorylation of RNA polymerase II (serine 2).To test whether PIM inhibition also attenuates specifically NFκB-dependent transcription in DLBCL cell lines, we assessed gene expression profiles of vehicle- or SEL24-B489-treated HBL-1, U2932 and DHL4 cells. Following PIM inhibition, we observed significant downregulation of multiple validated NFκB target genes in HBL-1 and U2932 cells, but not in DHL4 cells. We further confirmed decreased transcript abundance of 4 NFκB target genes (NFKBIA, CD40, TNFAIP 3 and MIR 155) by qPCR. Since NFκB target gene expression in ABC-DLBCL is essential for their survival and can be specifically blocked with the BTK inhibitor ibrutinib, we hypothesized that simultaneous targeting of PIMs and BTK would synergistically inhibit NFkB activity and cell survival. To test this hypothesis, we incubated the ABC-DLBCL cell lines with the ibrutinib, SEL24-B489, or their combination. We identified synergistic growth inhibitory effects of the drug combination in ABC-DLBCL cell lines, with combination index (CI) of 0.51 and 0.2 for HBL-1 and U2932 cells, respectively. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using U2932 cells. In contrast to progressive tumor growth in animals exposed to vehicle alone, we observed marked inhibition of tumor growth (>90%) in SEL24-B489-treated mice.Conclusions. In conclusion, a novel pan-PIM inhibitor SEL24-B489 induces apoptosis of DLBCL cell lines in low/sub-micromolar concentrations and exhibits activity in a xenograft model. The mechanisms of SEL24-B489 toxicity include blocking of protein translation, induction of Myc degradation, decrease of RNA transcription and attenuation of NFκB activity. Moreover, we show marked synergy between the PIM inhibitor and ibrutinib. Hence, these results provide new insights into mechanism of action of PIM inhibitors and rationale for targeting PIM activity in DLBCLs. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Golas:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Other: member of Selvita Scientific Advisory Board.

CC-122 Has Potent Anti-Lymphoma Activity through Destruction of the Aiolos and Ikaros Transcription Factors and Induction of Interferon Response Pathways

Background: IMiDs® Immunomodulatory agent lenalidomide (Len) is active in a range of hematologic cancers including diffuse large B-cell lymphoma (DLBCL). The molecular target of Len is cereblon (CRBN), a substrate receptor of the cullin 4 RING E3 ubiquitin ligase complex. In a CRBN-dependent manner, Len promotes ubiquitination of lymphoid transcription factors Ikaros and Aiolos leading to their subsequent degradation. This was shown to contribute to Len’s anti-Multiple Myeloma and T-cell activation properties. CC-122 is a non-phthalimide analog of thalidomide and has potent anti-proliferative, immunomodulatory and anti-angiogenic activities giving it a potentially broader range of activity than Len. CC-122, a first-in-class pleiotropic pathway modifier (PPM®), is currently in a Phase I clinical trial for DLBCL, multiple myeloma, and solid tumors (ClinicalTrials.gov NCT01421524) and showing encouraging clinical activity. The present study investigated the mechanism of action of CC-122 in pre-clinical models of DLBCL and in clinical samples from the ongoing Phase I study.Results: CC-122 inhibited proliferation (IC50=0.01-1.5μΜ) and induced apoptosis (6.5-12 fold) in 4 ABC and 3 GCB DLBCL cell lines in a dose-dependent manner. CC-122 induced rapid degradation of Aiolos and Ikaros in a CRBN-dependent manner; an effect blocked by MLN4924, a small molecule inhibitor of Cullin-RING E3 ligase complexes. In vivo anti-tumor activity of CC-122 was demonstrated in 2 xenograft models of human DLBCL, and was associated with significant reduction in Aiolos and Ikaros expression (94% and 69%, respectively, p<0.001) in tumor tissue within 6 hrs of administration. To identify proteins and signaling pathways affected by CC-122, tandem-mass tag proteomics was performed on DLBCL cells treated with vehicle or CC-122. While Aiolos and Ikaros levels decreased in a time- and dose-dependent manner, Interferon Stimulated Genes (ISGs) including IRF7, DDX58, and IFIT3 were notably increased (3, 5 and 12.5 fold, respectively). ISG induction with 24 hr CC-122 treatment was further confirmed by Western analysis. Surprisingly, the induction of ISGs occurred in the absence of increased interferon α, β, or γ levels, which was confirmed by qPCR and ELISA. Furthermore, shRNA targeting Aiolos led to inhibition of Aiolos protein expression (2-3 fold) and an increase in the mRNA and protein levels of IRF7, DDX58 and IFIT3 (10, 14 and 22 fold in mRNA expression, respectively). These data indicate that the increase in ISGs is a direct consequence of Aiolos degradation by CC-122 and bypasses the need for interferon production. Exploration of additional downstream functional gene candidates of CC-122 and shAiolos activity revealed a decrease in protein levels of Myc (up to 3 fold decrease with shAiolos), a key driver of DLBCL proliferation and survival associated with poor survival. Importantly, reduction of Aiolos expression and the consequent changes in the molecular pathways described led to inhibition of proliferation (2-4 fold versus control shRNA) and increased apoptosis (3-10 fold vs control shRNA), which paralleled CC-122 effects in DLBCL cells lines. Of note, CC-122 induction of ISGs was comparable in both ABC and GCB lines, whereas Len did not induce ISGs in GCB lines.The relationship between Aiolos and Ikaros protein expression and DLBCL cell of origin (COO) was further explored in primary DLBCL tissues. A tissue microarray containing 90 DLBCL patient cases immunohistochemically stained for Aiolos, Ikaros and COO markers (Hans) demonstrated Aiolos and Ikaros expression across GCB and non-GCB subtypes, suggesting that CC-122 may have broad-spectrum activity independent of COO. In fact, Aiolos and Ikaros were degraded in lymph node biopsies from both GCB and non-GCB DLBCL patients administered CC-122. The predictive value of Aiolos and Ikaros protein levels for clinical response will be assessed in the Phase I study.Conclusion: Aiolos degradation-dependent upregulation of ISGs represents a novel mechanism for the activity of CC-122. This mechanism bypasses the interferon-signaling pathway and occurs across cell of origin subtypes, suggesting this is a common mechanism accounting for the broad-spectrum activity of CC-122. Understanding of this mechanism has potential to greatly enhance the clinical development of CC-122 in DLBCL by guiding patient selection strategies and rational combination therapies. DisclosuresHagner:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Breider:Celgene Corp: Employment. Fontanillo:Celgene Corp: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Raymon:Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Barnes:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene Corporation: Employment, Equity Ownership. Daniel:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Pourdehnad:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership.

The Role Of Mir-155 In Controlling Key Growth and Survival Pathways In ABC-Subtype DLBCL

The incidence of diffuse large B-cell lymphoma (DLBCL) is rising, a situation that highlights the need for improved therapy with greater efficacy and fewer side effects. The fundamental clinical problem is that standard frontline combination chemotherapy for DLBCL (R-CHOP) achieves lasting remissions but is not curative. Development of a new, effective, low-toxicity frontline therapy against DLBCL that incorporates targeted agents is much needed. Constitutive activation of the NF-kB, NFAT, and STAT3 (NNS) pathways is a hallmark of DLBCL, particularly the activated B-cell–like (ABC) subtype. Although these pathways have been studied intensively recently, the precise mechanisms controlling these pathways in DLBCL is still uncertain. Elucidating the key mechanism(s) controlling these pathways should improve our understanding of the pathophysiology of DLBCL as well as the therapeutic strategy against this deadly disease. MicroRNAs (miRNA) are short 18-23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Aberrant expression of miRNAs has been implicated in numerous diseases, including cancers. MiRNA-155 (miR- 155) has shown to be overexpressed in aggressive hematologic malignancies, including DLBCL. The oncogenic nature of this miRNA was confirmed in Em-miR-155 transgenic mice, where the mice develop aggressive pre B cell neoplasms within 3-4 weeks. However, the role miR-155 in the pathophysiology of DLBCL is still unclear. We performed gene expression profiling (GEP) in 24 representative DLBCL cell lines (17 GCB- and 7 ABC-subtype). Our data show that miR-155 expression is significantly higher in ABC vs. GCB DLBCL cell lines (p-value<0.0001). We then analyzed the expression levels of miR-155 in primary DLBCL cells using Oncomine (https://www.oncomine.org), a publicly available cDNA cancer microarray database. Analysis of a representative data set (from Lenz G. et al) indicated that miR-155 levels were 3.12-fold higher in primary ABC-DLBCL (167 cases) than in GCB-DLBCL (183 cases). Since ABC-DLBCL subtype has been shown to have active B-cell receptor (BCR) signaling mediating NNS pathways and previous studies have shown that activation of mir-155 in normal and malignant B cells is through BCR stimulation, we examined whether miR-155 is involved in these pathways in DLBCL. Our results indicated that inhibition of miR-155 by transiently transfecting an anti-miR-155 into ABC-DLBCL cell lines harboring high expression of miR-155, suppresses NNS activation, leading to cell growth inhibition as wells as reducing DLBCL clonogenic activity. Next, we examined whether blocking the BCR pathway, using a novel small molecule Btk inhibitor, ibrutinib, can lead to miR-155 inhibition. Ibrutinib at low drug concentrations can effectively inhibit miR-155 expression, leading to NNS inhibition. To test the efficacy and specificity of ibrutinib in DLBCL, we screened ibrutinib in 24 DLBCL cell lines using proliferation assays. Our data showed that ibrutinib preferentially suppress proliferation of most ABC-DLBCL cell lines, whereas most GCB DLBCL cell lines were not highly responsive with drug concentrations less than 10 uM. Our result indicated that miR-155 plays a critical role in the BCR-mediated NNS growth survival pathways in ABC-DLBCL subtype and represents a potential target for therapy in personalized medicine. Disclosures:No relevant conflicts of interest to declare.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL

AbstractConstitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell–like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

MiR-181a Is a Master Regulator of the Nuclear Factor-κB Signaling Pathway in Diffuse Large B Cell Lymphoma

AbstractAbstract 417MicroRNAs (miRNAs) exhibit differential expression in cancer and can be used as prognostic biomarkers. MiR-181a expression is reported to be associated with survival and outcome in acute myeloid and chronic lymphocytic leukemia patients. We demonstrated that miR-181a levels are independently associated with improved survival of diffuse large B cell lymphoma (DLBCL) patients treated with R-CHOP (Rituximab, Cyclophosphamide, Adriamycin, Oncovin, Prednisolone). However, the mechanism underlying this observation and the function of miR-181a in DLBCL pathogenesis are unknown.MiR-181a was expressed at higher levels in centroblasts compared to naïve and memory B cells, and at significantly higher levels in GCB-like compared to ABC-like DLBCL cell lines (p=0.017). These observations suggested that miR-181a may differentially target critical signaling pathways in GCB and ABC DLBCL. NF-kB serves a critical role in ABC DLBCL survival. Utilizing 3 miRNA target prediction algorithms, multiple NF-κB signaling pathway transcripts harbored putative miR-181a binding sites. Consequently, we tested the effect of miR-181a on CARD11, IBKα, p105/p50, and C-Rel expression in DLBCL cell lines (HBL1, VAL). Compared with a scrambled miRNA control, miR-181a expression decreased protein and mRNA levels of these targets. To confirm the effect was direct, we fused the 3′-UTR sequences of CARD11, IBKα, p105 and C-Rel, each containing miR-181a putative binding sites, to a luciferase reporter gene. Co-transfecting miR-181a with the corresponding constructs, we demonstrated that all the constructs had significantly repressed luciferase activity compared with a non-targeting control. The effect was specific, since miR-181a did not affect luciferase activity of CARD11, IBKα, p105 and C-Rel reporter constructs with mutated binding sites. Using an NF-κB luciferase reporter assay, we next demonstrated that compared to a scrambled control, miR-181a significantly decreased NF-κB reporter activity in DLBCL cell lines (VAL, SUDHL6, OCILY7, OCILY19, HBL1, RCK8). MiR-181a also decreased NF-κB reporter activity induced by anti-IgM and TNFα stimulation. Concordantly, anti-miR-181a increased endogenous p105/p50 and C-Rel protein levels. Because ubiquitinated-IKKγ drives NF-κB signaling, we tested the effect of miR-181a in TNFα-stimulated 293T cells on ubiquitinated-IKKγ. MiR-181a decreased levels of ubiquitinated-IKKγ, corroborating the observed inhibitory effects on NF-κB signaling. We reasoned that NF-κB signaling repression should coincide with a decrease in endogenous transcription activity from NF-κB promoters. Indeed, miR-181a decreased mRNA expression levels of NF-κB target genes (BCL2, IRF4, IL-6, IKBa, FN1, PIM1, BLR1, CCL3, CFLAR, FCER2, TP53) as measured by qRT-PCR in miR-181a-transfected HBL1 cells. Because miR-181a directly targets p105/p50 and REL proteins, we postulated that this may be one of the main mechanisms of NF-κB signaling repression. Indeed, an electrophoresis mobility shift assay along with super-shifts analyses showed a decrease in the p105/p50 protein in HeLa nuclear extracts. To examine the biological significance of differential miR-181a expression between GCB- and ABC-like DLBCL and elucidate its potential role in DLBCL pathogenesis, we next assessed cell death (Annexin V, 7AAD) and cell proliferation (BrdU, 7AAD) in GCB (SUDHL4, OCILY7, OCILY19, VAL) and ABC (HBL1, OCILY10, RCK8, U2932) DLBCL cell lines transfected with GFP labeled precursor miR-181a. MiR-181a expression significantly increased cell death and apoptosis of ABC versus GCB DLBCL (p=0.006). This was associated with a more pronounced G1 phase growth arrest in the ABC DLBCL cells.Our studies demonstrate that miR-181a is a master regulator of canonical NF-kB signaling by regulating the expression of multiple components of this pathway, an effect that may underlie the distinct prognosis of DLBCL with different miR-181a expression levels. Furthermore, miR-181a down regulation may contribute to the pathogenesis of ABC DLBCL. Disclosures:No relevant conflicts of interest to declare.

Pharmacogenetic Approaches Identify ANTXR1 As a Potential Mediator of Response to Therapeutic NF-κB Inhibition in Diffuse Large B Cell Lymphoma

Abstract 234 Background:NF-κB is a family of transcription factors known to play an essential role in the development & survival of lymphocytes. In recent years, it has been clear that aberrant NF-κB activation is a hallmark of various lymphoid malignancies and appears to be associated with chemotherapy resistance and adverse prognosis. The canonical NF-κB pathway is frequently engaged in lymphoid malignancies wherein activated IKK phosphorylates IκB proteins inducing IκB polyubiquination and subsequent proteasomal proteolytic degradation; this allows for release and nuclear translocation of NF-κB dimers to activate target gene transcription.Gene Expression Profiling (GEP) has identified two distinct sub groups of Diffuse Large B cell Lymphoma (DLBCL). While the Activated B cell (ABC) type shows constitutive activation of NF-κB, the role for NF-κB activation in Germinal Center B Cell (GCB) DLBCL is currently unclear. Since NF-κB inhibition has been identified as a therapeutic possibility in DLBCLs, it is important to define the role of this pathway and its modulators. In this study, we sought to investigate key regulators of the NF-κB pathway that might mediate a therapeutic response to IKKβ inhibition of NF-κB in DLBCL including the GCB subtype. Through GEP and Exome Sequencing, we demonstrate that ANTXR1 is a key mediator of response to IKKβ inhibition in DLBCL. Methods/Results:We obtained a novel selective inhibitor of IKKβ, TLX-2001 that has been found to be safe in animal models. IC50 were obtained on 61 cell lines representing various lymphomas including DLBCL (N=25) using cell viability MTT assays. The drug showed efficacy in both ABC and GCB DLBCL cell lines at physiologically achievable concentrations. These results were unsurprising in ABC DLBCLs which are known to depend on NF-κB activation, but the lethality of this selective drug in GCB DLBCLs was unexpected. To better understand the role of individual genes in the response in GCB DLBCLs, gene expression profiling was performed on 61 cell lines using Human Gene 1.0 ST Array. We found that ANTXR1 expression significantly correlated with NF-κB resistance (p = 0.035).Additionally, we sequenced the exomes of DLBCL tumors (N=95) and matched normal tissue (N=34). 95 cases of DLBCLs consisted of 73 cases of primary human DLBCLs and 22 DLBCL cell lines. Whole exome sequencing was performed using the Agilent solution-based system of exon capture to sequence all protein coding exons in the CCDS database. We identified 465 recurrently somatically mutated genes in these DLBCL cases, and found that mutation status of ANTXR1 was associated with high sensitivity to IKKβ inhibition (p =0.015) of NF-κB. Cell lines with non-synonymous mutations in ANTXR1 had over 3-fold lower IC50 (mean= 2.39 μM) compared to cell lines with no mutation in ANTXR1 (mean IC50 = 8.72μM). Discussion/Conclusion:ANTXR1 is the docking receptor for bacillus anthracis toxin, and anti-tumor responses have been observed in mice injected with recombinant engineered anthrax toxin. It is also known as TEM8 and maps to chromosome 2p13.1. Increased levels of TEM8 (tumor endothelial marker 8) have been noted in various malignancies including melanoma. Our data suggest that pharmacogenetic approaches that combine gene expression profiling and whole exome sequencing are useful tools for identifying novel genes that modulate therapeutic responses in lymphoma. Disclosures:No relevant conflicts of interest to declare.