GCB Diffuse Large B-cell Lymphoma Cell 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.

B-Cell Receptor Signaling Modulates Cholesterol Biosynthesis in Diffuse Large B-Cell Lymphoma

Cholesterol levels are associated with cancer progression and targeting cholesterol biosynthesis has been proposed as a therapeutic strategy in some neoplasms. It has been shown that inhibition of cholesterol synthesis in combination with chemotherapy resulted in encouraging clinical responses in patients with hematopoietic neoplasms including diffuse large B cell lymphoma (DLBCL) (Blood 2007; 109:2999-3006; Leuk. Lymphoma 2022; 63:1302-1313). Seminal work in DLBCL has shown that blocking the expression of SYK kinase, downstream of the B-cell receptor (BCR), impacted cholesterol biosynthesis (Cancer Cell 2013; 23:826-88). Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors in the endoplasmic reticulum, play a central role in lipid metabolism. Two genes, SREBF1, and SREBF2 codify three proteins (SREBP1a, SREBP1c, and SREBP2). SREBP1a and SREBP1c primarily regulate fatty acid metabolism, whereas SREBP2 is a master regulator of cholesterol biosynthesis. The underlying question of how BCR signaling is linked to the cholesterol biosynthesis encircling SREBP2 is not well understood. Using publicly available NCBI's Gene Expression Omnibus (GEO) DLBCL databases, we first analyzed the expression of genes directly regulated by SREBF2 involved in the biosynthesis of cholesterol, including ACAT1-2, HMGCS, HMGCR, MVK, PVMK, IDI1, SQLE, LSS, EBP, DHCR7, DHCR24, and NSDHL in DLBCL. We observed that SREBF2-mediated genes were upregulated in DLBCL as compared to non-neoplastic germinal center (GC) B-cells or low-grade B-cell lymphomas. These data were further confirmed by HTseq analysis of 66 DLBCL PDXs tumors established from DLBCL patients in our institution. Also, protein expression of nuclear SREBP2 was analyzed using immunohistochemistry in a DLBCL tissue microarray (n=84). Nuclear SREBP2 expression serves as an active form of transcriptional factor following cleavage. We confirmed strong nuclear positivity of SREBP2 in all non-GCB DLBCL; however, a subset of GCB DLBCL exhibited moderate nuclear SREBP2 positivity as compared to low-grade follicular lymphomas or reactive lymph node (control). These data suggest that SERBP2 is a key element in cholesterol biosynthesis, specifically upregulated, in non-GCB DLBCL. Next, in-vitro functional studies were performed to connect BCR signaling with SERBP2. We either genetically knock out the expression of IgH using CRISPR/Cas9 or pharmacologically inhibited Src kinase (downstream of the BCR signaling) with Dasatinib in non-GCB DLBCL cell lines. Both approaches reduce SREBP2 expression and its cholesterol-related downstream genes. In contrast, recovering IgH expression rescues the SREBP2-mediated cholesterol biosynthesis pathway establishing a direct role of BCR signaling in the regulation of the cholesterol pathway in non-GCB DLBCL through SREBP2. Mechanistically using Reverse Phase Protein Array (RPPA) proteomic analysis, we have identified cyclin-dependent kinase 1 (CDK1), a key player in cell cycle regulation, as a potential kinase involved in the phosphorylation of SREBP2 by activated BCR signaling. Our results suggest that CDK1 interacts with SREBP2 (as per colocalization studies), and that CDK1-mediated phosphorylation of SREBP2 preserves its stability to support enhanced cholesterol synthesis in non-GCB DLBCL cell lines. Although directly inhibiting of SREBPs is challenging, a promising approach is to inhibit SREBP translocation from the ER to the Golgi using PF-429242. Decreasing expression of SREBP2 at the genetic level or blocking the processing of SREBP2 with PF-429242 strongly reduces cell viability of DLBCL cell lines, with non-GCB DLBCL cell lines being more sensitive (IC50= 60-225nM) than the GCB DLBCL cells (IC50=200-330 nM). We also observed, in vitro, that co-treatments with PF-429242 and doxorubicin resulted in a synergistic response to doxorubicin in DLBCL cell lines of GCB, non-GCB and "double hit" suggesting that inhibiting SREBP2 could be a potential adjuvant therapeutic approach for DLBCL patients. In summary, cholesterol biosynthesis is tightly regulated by BCR signaling through SREBP2, specifically in non-GCB DLBCL. SREBP2 is a potential promising adjuvant therapeutic target for DLBCL as PF-429242 enhances responses to doxorubicin in cell lines expanding the therapeutic window of doxorubicin by increasing its efficacy.

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 A05: Olfactory receptors as a new therapeutic target in lymphomas

Abstract Background. Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are aggressive lymphomas in which up to 40% of patients are still in need of therapy. G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to endogenous and exogenous stimuli and are implicated in the pathophysiology of various diseases: CXCR3/4/5, S1PR1/2/3, or GPR34 are GPCRs already studied in lymphomas. The olfactory receptors (ORs) are also GPCRs, initially identified and characterized for their expression in olfactory cells but now shown to be expressed in various tissues. OR51E1 is known as prostate-specific G protein-coupled receptor 2 (PSGR2) due to its expression in prostate cancer, OR2B6 and OR2W3 are expressed in breast cancer, and OR51E1 in melanoma. Here, we studied OR13A1 in lymphoma cell lines and clinical specimens. Methods. RNA-Seq and real-time PCR for evaluating the expression of ORs in lymphoma cell lines. Gene expression data for clinical samples were obtained from public datasets. OR13A1 silencing was performed with both siRNAs (OCI-Ly1 and U2932) and doxycycline-inducible shRNAs (U2932 and VAL); viability was assessed after 3 days using MTT and even longer with live imaging. Computer modelling was performed using available ligands for the murine orthologue Olfr211. Results. Various ORs were expressed across a large panel of B cell lymphoma cell lines. In particular, OR13A1 was expressed in 35/47 cell lines and, mostly, derived from DLBCL and MCL. Among DLBCL, the expression was higher in cell lines derived from the germinal center (GC) B-cell type (GCB) rather than from activated B cell like (ABC) DLBCL (P value = 0.03). A similar pattern was observed in DLBCL patients: higher expression in GCB than ABC and in EZB than MCD (P < 0.001). OR13A1 also appeared higher in normal GC cells than in naïve and memory B cells. OR13A1 expression appears to identify GCB DLBCL (P value = 0.009) and MCL (P value = 0.006) patients with inferior outcome. Silencing of OR13A1 with siRNAs in GCB and ABC DLBCL cells (OCI-Ly1 and U2932) caused > 50% reduction in cell viability, and downregulation of pERK levels. Viability data were confirmed using a doxycycline-inducible shRNA system and an Incucyte live cell imaging instrument. Natural compounds reported as ligands for the murine orthologue Olfr211 or in silico identified to bind as agonists (ex: tyramine) increased cell line growth and compounds predicted to bind as antagonists (ex: cyclohexanone) decreased the cell viability in the presence but not in the absence of the GPCR. Conclusion. OR13A1 gene appeared more abundantly expressed in lymphomas compared to others ORs, in particular it was more expressed in MCL and in GCB DLBCL with a possible impact on clinical outcome. Genetic and chemical experiments showed that this GPCR has an essential function in lymphoma cells and represents a novel therapeutic target. Citation Format: Giulio Sartori, Luciano Cascione, Milos Matkovic, Hiro Matsunami, Andrea Cavalli, Francesco Bertoni. Olfactory receptors as a new therapeutic target in lymphomas [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A05.

Abstract 2655: The GPCR OR13A1 is essential for lymphoma cells

Abstract Background. G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to endogenous (amines, cations, lipids, peptides, glycoproteins) and exogenous (light, tastants, odorants) stimuli. Altered signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, such as CXCR3/4/5, S1PR1/2/3, GPR34 in lymphomas. Olfactory receptors (ORs) are GPCRs, initially identified and characterized for their expression in olfactory neurons but now recognized as expressed in different tissues. OR51E1 is known as prostate specific G protein coupled receptor 2 (PSGR2) due to its expression in prostate cancer, OR2B6 and OR2W3 are expressed in breast cancer, and OR51E1 in melanoma. OR13A1 has been included in the double-hit expression signature reported in diffuse large B cell lymphoma (DLBCL) (Ennishi et al 2019). Here, we studied OR13A1 in lymphoma cell lines and clinical specimens. Methods. RNA-Seq and real-time PCR for evaluating the expression of ORs in DLBCL cell lines. Gene expression data for clinical samples obtained from public dataset phs001444.v2.p1. OR13A1 silencing performed with both siRNAs (OCI-Ly1, U2932) and doxycycline-inducible shRNAs (U2932); viability assessed after 3 days using MTT and for longer with Incucyte live imaging. Computer modelling performed based on available ligands for the murine orthologue Olfr211 and similar known GPCRs structure. Results. Only one OR per cell line was usually expressed, as reported for olfactory cells. Using RNA-Seq and real-time PCR, OR13A1 gene appeared the most abundantly expressed in lymphomas cell lines compared to others ORs. Its expression was seen in 35/47 B cell lymphoma lines, with higher expression in germinal center B cell (GCB) DLBCL than activated B cell like (ABC) DLBCL (P 0.03). A similar pattern was observed in patients: higher expression in GCB than ABC and in EZB than MCD (P < 0.001). OR13A1 also appeared higher in normal GC cells than in naïve and memory B cells. Among GCB DLBCL patients, OR13A1 expression was associated with inferior outcome. Silencing of OR13A1 with siRNAs in GCB and ABC DLBCL cells caused > 50% reduction in cell viability, and pERK downregulation. Viability data were confirmed using a doxycycline-inducible shRNA system and live cell imaging instrument. Natural compounds reported as ligands for the murine orthologue Olfr211 or in silico identified to bind as agonists (ex: tyramine) increased cell line growth and compounds predicted to bind as antagonists (ex: cyclohexanone) decreased the cell viability in the presence but not in the absence of the GPCR. Conclusion. OR13A1 gene appeared more abundantly expressed in lymphomas compared to others ORs, higher in GCB than ABC DLBCL and with a possible impact on clinical outcome. Genetic and chemical experiments showed that this GPCR has an essential function in lymphoma cells and could represent a novel therapeutic target. Citation Format: Giulio Sartori, Luciano Cascione, Milos Matkovic, Hiroaki Matsunami, Andrea Cavalli, Francesco Bertoni. The GPCR OR13A1 is essential for lymphoma cells [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 2655.

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.

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.

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.

PRMT5 is upregulated by B-cell receptor signaling and forms a positive-feedback loop with PI3K/AKT in lymphoma cells.

PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. In diffuse large B cell lymphoma (DLBCL), the mechanisms of PRMT5 dysregulation and its role in lymphomagenesis remain largely unknown. Here we demonstrate that B cell receptor (BCR) signaling regulates PRMT5 expression in DLBCL cells. Immunohistochemical analysis reveals elevated levels of PRMT5 expression in DLBCL cases and in germinal center (GC) B cells when compared to naive B cells. PRMT5 can be induced in naive B cells by BCR stimulation. We discovered that BTK-NF-κB signaling induces PRMT5 transcription in activated B cell-like (ABC) DLBCL cells while BCR downstream PI3K-AKT-MYC signaling upregulates PRMT5 expression in both ABC and GCB DLBCL cells. PRMT5 inhibition inhibits the growth of DLBCL cells in vitro and patient derived xenografts. Genomic and biochemical analysis demonstrate that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a feedback regulatory mechanism to enhance cell survival and proliferation. Co-targeting PRMT5 and AKT by their specific inhibitors is lethal to DLBCL cell lines and primary cancer cells. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate PRMT5 and AKT inhibitors for DLBCL.

Combination of Enzastaurin and Ibrutinib Synergistically Induces Anti-Tumor Effects in Diffuse Large B Cell Lymphoma

Background:Diffuse large B cell lymphoma(DLBCL), the most common form of lymphoma, is characterized by a heterogeneous tumor entity. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, PI3K)inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers.Early studies demonstrated that PKCβ inhibitors alter phosphorylation level of BTK, which enhances activation of BTK signaling pathway.Here we aim to investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.Methods:In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole-transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effect of enzastaurin and ibrutinib was also evaluated in vivo.Results: Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of ABC (HBL-1,TMD8,SU-DHL-2)and GCB (SU-DHL-6, OCI-LY7)DLBCL cells lines. Exposure of these DLBCL cells to minimally toxic concentration of enzastaurin and ibrutinib resulted in more significant inhibitory activity than each agent alone. In addition, the combination of these two agents led to an enhanced apoptosis and induced G1 phase arrest compared with monotherapy, which were accompanied by marked regulation of apoptosis-related and cell cycle-related proteins. Combination treatment with enzastaurin and ibrutinib, to some extent, also functioned synergistically to inhibit cell migration and invasion compared with single agent. Moreover, combination of enzastaurin and ibrutinib seemed to be more effective in inhibition p-ERK, p-AKT and p-P38 in DLBCL cell lines. In addition, RNA-seq results showed that co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathways, and the pathway analysis results were consistent with the immunoblotting analysis. Furthermore, the mRNA expression analysis using real-time PCR further indicated that treatment with enzastaurin and ibrutinib significantly decreased the mRNA levels of NOTCH1 which has been recognized as an important oncogenic regulator of hematological malignancy. The combination effect of enzastaurin and ibrutinib in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment with enzastaurin and ibrutinib was also demonstrated using xenograft mice models.Conclusion:We investigated the combination of enzastaurin and ibrutinib in ABC and GCB DLBCL, demonstrating the co-treatment had synergistic anti-tumor effects in DLBCL. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be an new treatment strategy for DLBCL. DisclosuresNo relevant conflicts of interest to declare.

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.

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.

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.

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.

JMJD3 promotes survival of diffuse large B-cell lymphoma subtypes via distinct mechanisms.

JMJD3 (Jumonji domain containing-3), a histone H3 Lys27 (H3K27) demethylase, has been reported to be involved in the antigen-driven differentiation of germinal center B-cells. However, insight into the mechanism of JMJD3 in DLBCL (Diffuse large B-cell lymphoma) progression remains poorly understood. In this study, we investigated the subtype-specific JMJD3-dependent survival effects in DLBCL. Our data showed that in the ABC subtype, silencing-down of JMJD3 inhibited interferon regulatory factor 4 (IRF4) expression in a demethylase activity-dependent fashion. IRF4 reciprocally stimulated expression of JMJD3, forming a positive feedback loop that promoted survival in these cells. Accordingly, IRF4 expression was sufficient to rescue the pro-apoptotic effect of JMJD3 suppression in the ABC, but not in the GCB subtype. In contrast, ectopic overexpression of BCL-2 completely offset JMJD3-mediated survival in the GCB DLBCL cells. In vivo, treatment with siRNA to JMJD3 reduced tumor volume concordant with increased apoptosis in either subtype. This suggests it is a common target, though the distinctive signaling axes regulating DCBCL survival offer different strategic options for treating DLBCL subtypes.