Diffuse Large B-cell Lymphoma Lines Research Articles

Pharmacological Inhibitory Effect of Imetelstat, a Novel Human Telomerase Inhibitor in Diffuse Large B-Cell Lymphoma and Peripheral T-Cell Lymphoma

Telomerase activity is present in some non-Hodgkin lymphomas (NHL), including diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphomas (PTCL), and correlated with proliferation index, suggesting anti-telomerase drugs may be efficacious in some types of NHL (Ely et al. 2000). Imetelstat, a telomerase inhibitor that specifically targets the RNA template of human telomerase, is a potent, first-in-class, competitive inhibitor of telomerase enzymatic activity and has demonstrated clinical activity in myeloid malignancies (Steensma et al. 2021; Mascarenhas et al. 2021). No studies have been conducted to demonstrate the effects of imetelstat in DLBCL and PTCL. In our study, we characterized telomerase activity and telomere length, as well as the in-vitro effects of imetelstat on cell viability and apoptosis of a panel of DLBCL and PTCL cell lines. Six DLBCL cell lines, including germinal B cells (GCB: DOHH2, HT, SUDHL4), non-germinal B cells (HBL-1, TMD8), and one high-grade B cell lymphoma line RC with MYC and BCL2 gene rearrangements (double hit lymphomas; ), and five PTCL cell lines (Karpas 299, H9, MAC2A, SUDHL1, and SUPM2) were tested for imetelstat single agent activity as assessed by change of telomerase activity using ScienCell's Telomerase Activity Quantification qPCR Assay Kit, cell viability by Cell Titer-Glo Luminescent Cell Viability Assay and apoptosis by Annexin V/Propidium Iodide flow assay. Baseline telomerase activity and telomere length were measured for each cell line to correlate with imetelstat activity. In addition, PTCL cell lines were treated with Suberoylanilide hydroxamic acid (SAHA) and imetelstat to determine if the combination had an impact on cell viability over either of the single agents. All DLBCL and PTCL cell lines have higher baseline telomerase activity with Ct 20.6-23.2 cycles than the telomerase-positive cell lysate control with Ct 30.6 cycles used in the assay. The dose and time-dependent effects of imetelstat were examined through cell viability and apoptosis assays. Imetelstat sensitivity was higher in DLBCL than in PTCL cell lines used in this study. Preliminary experiments determined that IC50 3-17.5 μM imetelstat markedly reduced cell viability and induced apoptosis in DLBCL cell lines. Specifically, non-GCB DLBCL cell lines (IC50 = 3-4.1 μM) were more sensitive than the GCB-DLBCL (IC50 = 6-8.2 μM). While the level of effects in cell viability and apoptosis by imetelstat varied in the DLBCL cell lines, the double hit DLBCL cell line RC was the least sensitive to imetelstat (IC50 = 17.5 μM) after treatment for 8 days. In contrast, compared to DLBCL cell lines, imetelstat single-agent activity on cell viability was limited in PTCL cell lines, although a time and dose-dependent reduction of telomerase activity were noted. Additionally, the greater inhibitory effect in DLBCL than in PTCL cell lines by imetelstat treatment may be attributed to higher telomerase activity observed in DLBCL compared to PTCL cell lines. Furthermore, the PTCL cell lines had a ~7.3 fold longer telomere length than DLBCL cell lines, which was associated with less response to imetelstat than DLBCL lines. A combination of SAHA with imetelstat was more effective than a single agent alone, synergistic activity was observed in Karpas 299, while other PTCL lines had an additive effect. Our findings indicate that imetelstat was more sensitive in non-GCB subtypes than in GCB-DLBCL. While in PTCL cell lines, the activity of imetelstat was enhanced when combined with SAHA. In the lymphoma landscape, telomerase may be a therapeutic target for ABC-type DLBCL; the novel telomerase inhibitor Imetelstat may present one of the potential treatment options.

Abstract A02: Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981)

Abstract SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming. Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [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 A02.

Abstract A24: BTG1 mutations confer a fitness advantage and promote aggressive B cell lymphoma development by lowering the threshold for MYC induction

Abstract BTG1 somatic mutations are exclusively found in mature B cell malignancies, ~12% diffuse large B cell lymphoma (DLBCL) and are particularly enriched in the MCD/cluster 5 subtype of ABC-DLBCL, characterized by extranodal dissemination and poor clinical outcome. However, the mechanism of action and clinical relevance of BTG1 mutations remain unknown. We find that BTG1 mutations score among the top mutations with DLBCL driver potential, using a rigorous genomic and epigenomic covariates analysis. Most notably, BTG1 mutant patients presented with inferior clinical outcome (p=0.0011) in ABC-DLBCL cases from 5 cohorts and BTG1 mutation was independently associated with lower overall survival in a multivariable Cox regression analysis (p=0.0190) DLBCL originates from mature B cells having experienced the germinal center (GC) reaction. We therefore generated a conditional knockin mouse model to express the most frequent Btg1 Q36H in B cells. Btg1 Q36H GC B cells showed a dramatic fitness advantage in in vivo competitive assays. This effect was specific to the GC compartment and was dependent on T cells. RNAseq showed that Btg1Q36H GC B cells were markedly enriched for genes normally transiently induced upon positive selection by T cells, including MYC targets. The same signatures were enriched in BTG1 mutant DLBCL patients and isogenic BTG1Q36H vs BTG1WT human DLBCL cell lines. We observed a higher proportion of MYC-expressing cells in Btg1Q36H GCs without an increase in maximal MYC levels per cell, also confirmed in human DLBCL lines and primary human tonsillar B cells, suggesting a lowered threshold for MYC induction in mutant cells. Mechanistically, ~800 transcripts associated with BTG1WT, but not BTG1Q36H. These belong to the same gene sets that characterize positively selected GC B cell, including MYC itself. BTG1Q36H therefore showed reduced association with the MYC mRNA. We found that this associated with facilitated MYC protein synthesis. Polysome profiling also showed higher translation capacity in BTG1Q36H DLBCL cells. Crossing our Btg1 Q36H mice to the VavP-Bcl2 model showed that VavP-Bcl2+Btg1Q36H mice displayed shorter survival, earlier onset of lymphoma and dysplastic B cell infiltration into non lymphoid organs. Immunoglobulin genes sequencing showed that VavP-Bcl2+Btg1Q36H lymphoma B cells were highly clonal, extensively mutated and selected. Importantly, the lack of BTG1 deletions suggested that BTG1 missense mutations do not cause a full loss-of-function of the protein. Indeed, we observed that shRNA-mediated knockdown of BTG1 resulted in apoptosis. Collectively, BTG1 mutations contribute to the formation of aggressive lymphomas through an entirely novel mechanism, by lowering the threshold to MYC induction in response to T cell selection signals, conferring dramatic fitness. This effect appears to correspond to a partial loss of function disrupting a novel GC context-specific check point, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential. Citation Format: Coraline Mlynarczyk, Matt Teater, Juhee Pae, Ling Wang, Jonatan Ersching, Antonin Papin, Darko Barisic, Ersilia Barin, Kenneth B. Hoehn, Zhengming Chen, Diu T. T. Nguyen, Chiara Evans, Ashley S. Doane, Michael G. Kharas, David W. Scott, Gabriel Victora, Ari Melnick. BTG1 mutations confer a fitness advantage and promote aggressive B cell lymphoma development by lowering the threshold for MYC induction [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 A24.

Abstract CT210: A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, as single-agent in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM)

Abstract Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 demonstrated a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, DLBCL lines representing Double/Triple Hit Lymphoma (DHL/THL), Double Expressor Lymphoma (DEL), and MM cell lines harboring diverse mutational backgrounds. BMF-219 also exhibited high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and R/R MM. Methods: BF-MNN-101 (NCT05153330) is a prospective, open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of oral BMF-219 administered once daily in patients with R/R AL, DLBCL and MM who have received standard therapy. Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication following a modified Fibonacci sequence until 1 subject experiences either a ≥ Grade 2 adverse event or dose limiting toxicity (DLT). At that point, the current and all subsequent dose level(s) for the specific indication/cohort will follow a classical “3 + 3” design. Each treatment cycle will be 28 days until progression or intolerability occurs. Following conclusion of the parallel dose-escalations, expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Eligible patients include those with R/R AL, R/R DLBLC ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies who failed or are ineligible for any standard therapies. Patients must have ECOG ≤2, and adequate organ function with prior treatment-related toxicities resolved to ≤ Grade 2. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. The primary objective is to determine independently for each cohort/indication the optimal biological dose (OBD)/recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels in the escalation phase and during the expansion cohort portion of the study. The first patient in the study is anticipated to dose in Q1 of 2022. Citation Format: Farhad Ravandi-Kashani, Ashwin Kishtagari, Hetty Carraway, Emily Curran, Gary Schiller, Alex Cacovean, Bhagyashree Yadav, Thomas Butler, Jeffrey Lancet. A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, as single-agent in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM) [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 CT210.

Abstract 3932: Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is)

Abstract CC-99282 is a novel CELMoD® agent that was optimized for activity in non-Hodgkin lymphoma (NHL). CC-99282 potently degrades Ikaros and Aiolos, resulting in enhanced antiproliferative, apoptotic, and immune-stimulatory activity in diffuse large B-cell lymphoma (DLBCL) models, including those with acquired chemoresistance (Lopez-Girona A, et al. Hematol Oncol. 2021). In lymphocytes, Ikaros negatively regulates gene expression via histone modifications, including polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) (Oravecz A, et al. Nat Commun. 2015); in NHL, these epigenetic mechanisms are unclear. Using DLBCL models, we explored the relationship between CC-99282 activity and epigenetic status, and the mechanism of synergy of CC-99282 treatment and inhibition of EZH2, a PRC2 component. Baseline characteristics of human DLBCL lines and effects of CC-99282 ± tazemetostat (TAZ), a representative EZH2i, were evaluated in vitro and in vivo by chromatin immunoprecipitation sequencing, gene expression, flow cytometry, immunoblotting, enzyme fragment complementation assays, and/or CRISPR/Cas9 gene editing. Analysis of Ikaros/Aiolos degradation in > 20 DLBCL cell lines showed their loss is necessary, but not sufficient for CC-99282 efficacy. Evaluation of baseline histone marks showed that the sensitive cell lines exhibit aberrant, higher H3K27me3 coverage at promoter regions of expressed genes. This suggests a direct correlation between H3K27me3 status at these regions and CC-99282 sensitivity. In T cells, Oravecz et al. found that loss of Ikaros reduces H3K27me3 at specific chromatin sites due to its interaction with PRC2. We confirmed that these affected regions are enriched in genes upregulated upon CC-99282 treatment in DLBCL cell lines, suggesting a similar role for Ikaros in DLBCL. Pathway analysis following CC-99282 or TAZ treatment demonstrated high overlap between pathways altered by both agents. CC-99282 + TAZ combined demonstrated additive and/or synergistic antiproliferative and apoptotic effects in DLBCL cell lines. This combination did not alter Ikaros degradation or overall H3K27me3 status but increased downstream effects. Results were confirmed by CRISPR/Cas9 knockout competition assays with EZH2 and other PRC2 components. This effect was independent of cell of origin, EZH2 mutational status, or degree of CC-99282 sensitivity. Synergy was confirmed using the SU-DHL6 and DB xenograft models that are intrinsically resistant to EZH2is and CC-99282, respectively. Combination treatment yielded durable responses and tumor-free animals. Collectively, these data suggest that Ikaros could act as an epigenetic modulator through PRC2 recruitment and support the combination of CC-99282 with EZH2is in NHL (NCT03930953) to favor broad and durable clinical responses. Citation Format: Soraya Carrancio, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, Diana Jankeel, Adam Blattler, Preethi Janardhanan, Matthew Groza, Jim Leisten, Rama Krishna Narla, Antonia Lopez-Girona, Daniel W. Pierce. Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is) [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 3932.

COVALENT-101: A phase 1 study of BMF-219, a novel oral irreversible menin inhibitor, in patients with relapsed/refractory (R/R) acute leukemia (AL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM).

TPS7064 Background: Menin, a protein involved in transcriptional regulation, impacting cell cycle control, apoptosis, and DNA damage repair, plays a direct role in oncogenic signaling in multiple cancers. Inhibition of menin is therefore a novel approach to cancer treatment. Preclinical data of BMF-219, a highly selective, orally bioavailable, small-molecule irreversible inhibitor of menin, show sustained potent abrogation of menin-dependent oncogenic signaling in vitro and in vivo. BMF-219 exhibited a strong anti-proliferative effect on various menin-dependent acute myeloid leukemia (AML) cell lines, DLBCL lines representing Double/Triple Hit Lymphoma (DHL/THL) and Double Expressor Lymphoma (DEL), and MM cell lines with diverse mutational backgrounds. BMF-219 also showed high potency ex vivo in patient samples from MLL-rearranged and NPM1-mutant AML, THL and MYC-amplified DLBCL, and bone marrow mononuclear cells from treatment-naive and R/R MM. Methods: COVALENT-101 (NCT05153330) is an open-label, multi-cohort, non-randomized, multicenter Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of once daily oral BMF-219 in patients with R/R AL, DLBCL and MM who have received standard therapy. Utilizing an accelerated titration design, doses of BMF-219 will be escalated in single-subject cohorts independently for each indication until 1 subject experiences either a ≥ Grade 2 related-adverse event or dose limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 + 3” design. Treatment will continue in 28-day cycles until progression or intolerability. Expansion cohorts for each indication will enroll patients to obtain further safety and efficacy data. Patients with R/R AL, R/R DLBCL ≥ 2 but ≤ 5 therapies, and R/R MM who received ≥ 3 therapies and failed or are ineligible for any standard therapies are eligible. Patients must have ECOG PS ≤ 2, and adequate organ function. Key exclusion criteria include known CNS disease involvement, prior menin inhibitor therapy, and clinically significant cardiovascular disease. The primary objective is to determine independently for each cohort/indication the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-219 oral monotherapy. Key secondary objectives include further evaluation of safety and tolerability, characterization of the pharmacodynamics and pharmacokinetics of BMF-219, and assessment of its antitumor activity based on best overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), and time to progression (TTP) per disease-specific response criteria as assessed by the investigator. Food-effect studies will be performed in DLBCL and MM patients at certain dose levels. The enrollment commenced in January 2022. Clinical trial information: NCT05153330.

Btm-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma

Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or patients who fail CAR-T-cell therapy. r/r-DLBCLs are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class compound with activity against a variety of B-cell malignancies but with greatest effect in DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in DLBCL lines in vitro and complete tumor regression in vivo in DLBCL PDX mouse models harboring genetic alterations associated with poor prognosis.BTM-3566 is an oral small molecule based on a pyrazolothiazol-backbone, developed for treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC 50 of ~200 - 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma lines. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% of oral bioavailability and close to 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. BTM-3566 showed stability in human hepatocytes (IC < 5 ml/min*kg) as well and a favorable in vitro safety profile. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete regression in all tumor-bearing animals. Most importantly, no subsequent tumor growth occurred for 2 weeks after cessation of therapy, indicating that treatment with BTM-3566 resulted in a durable complete remission in this model of double-hit DLBCL. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1).Transcriptome and proteome analyses revealed that BTM-3566 strongly activated the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined, using CRISPR-Cas 9 gene depletion, that HRI was uniquely required for BTM-3566 eIF2a phosphorylation, induction of ATF4 ISR and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased ROS generation or blockage of mitochondrial ATP synthesis which result in an increase in mitochondrial proteostasis including activation of mitochondrial protease OMA1. We determined that BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Treatment with BTM-3566 induced OMA1-dependent OPA1 processing and mitochondrial fragmentation in as little as 30 minutes of treatment, in the absence of any reduction in mitochondrial oxygen consumption or membrane depolarization. This data indicates that BTM-3566 represents a new class of compounds that activate the mitochondrial protease OMA1.Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a mitochondrial membrane protein, negatively correlated with response to BTM-3566. Notably, overexpression of FAM210B completely prevents OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis in DLBCL cell line BJAB and the Burkitt lymphoma cell line Ramos. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation.In summary, we describe here a novel, highly potent activator of the mitochondrial ISR, which is well tolerated in mice and dogs, has favorable pharmacokinetics and induces robust DLBCL regression in-vivo. An IND application in B-cell malignancies will be completed by early Q1 2022 with initiation of first in human clinical trials the first half of 2022. [Display omitted] DisclosuresSchwarzer: Bantam Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anantha: Bantam Pharmaceutical: Consultancy, Current Employment, Current holder of stock options in a privately-held company. Cooper: Bantam Pharmaceutical: Consultancy. Hannink: Bantam Pharmaceutical: Research Funding. Hembrough: Bantam Pharmaceutical: Consultancy. Levine: Bantam Pharmaceutical: Consultancy, Current holder of individual stocks in a privately-held company. Luther: Bantam Pharmaceutical: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Stocum: Bantam Pharmaceutical: Current Employment, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Liesa-Roig: Bantam Pharmaceutical: Research Funding. Kostura: Bantam Pharmaceutical: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties: Named Inventor on patents with assignment to Bantam Pharmaceutical.

Repurposing NAD Salvage Inhibitors for GCB Diffuse Large-B Cell Lymphoma

Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of diseases in terms of cell of origin, genetics and clinical outcome. About 30% of all DLBCL patients represent an unmet clinical need as they either do not respond to the standard first line chemo-immunotherapy or recur after initial remission. DLBCL classifications based on the NMF (Non-Negative Matrix Factorization) and LymphGen algorithms have led to the identification of genetic subtypes based on the co-occurrence of specific lesions. Repositioning drugs that are approved or in active clinical development for other indications can be a powerful strategy to match these newly uncovered DLBCL subtypes to targeted therapies.We have previously shown that the screening of large panels of DLBCL cell lines representative of the genetics of the disease can facilitate the repositioning of approved drugs for biomarker-selected populations of DLBCL patients. Repositioned drugs can then be rapidly translated to the clinical use, as they have already been extensively characterized for their safety profile. As a proof of concept, we have demonstrated that Dasatinib, a Src/Abl inhibitor approved for B-cell Acute Lymphoblastic Leukemia and Chronic Myelogenous Leukemia, is highly effective in PTEN-positive DLBCLs, irrespective of their COO class (Scuoppo et al., PNAS 2019).Here we present the results of a new screening that was performed on a panel of eight cell lines (4 ABC- and 4 GCB-DLBCLs) to test the activity of 212 drugs, either approved or in advanced clinical development, for repositioning in DLBCL, followed by validation in a larger panel of 34 genetically characterized cell lines. Our results point to inhibitors of the Nicotinamide Phosphoribosyl Transferase (NAMPT) as potently active in 63% of GCB-DLBCLs. NAMPT catalyzes the conversion of Nicotinamide (NAM) to Beta-Nicotinamide Mononucleotide (Beta-NMN). This reaction is the rate-limiting step of the Nicotinamide Adenine Dinucleotide (NAD) salvage pathway, the major metabolic route of NAD regeneration in mammalian cells. We validated the on-target activity of NAMPT inhibitors by multiple genetic and pharmacological approaches. First, we found that the activities of three chemically distinct drugs (FK-866, STF-118804 and KPT-9274) were highly correlated across the 34 lines DLBCL panel. Second, we were able to abolish the activity of all three NAMPT inhibitors by supplementing cells with Beta-NMN. Third, we showed that transduction of the drug-resistant mutant NAMPT H191R offsets the activity of the drugs.Dose-response assays on the full DLBCL cell line panel confirmed ABC-DLBCL resistance and also highlighted the presence of sensitive (GCB-S) and resistant (GCB-R) GCB subsets that can be separated by a subnanomolar IC50 threshold. To generate biomarkers capable of predicting GCB-S patients, we examined the RNA-seq profiles and genetic make-ups of the DLBCL cell lines collection and observed that the GCB-S subset was associated to the LymphGen EZB subtype, characterized by the presence of EZH2 mutations and BCL2 translocations. Conversely, the GCB-R subtype was linked to a 5-gene expression signature for the Kynurenine De Novo pathway, an alternative route for NAD synthesis. Accordingly, expression of each of the five De Novo Kynurenine pathway genes induced resistance to NAMPT inhibitors. These results were validated in xenotransplants of luciferized DLBCL lines and Patient Derived Xenotransplant models (PDXs) that were transcriptionally classified for the status of the Kynurenine De Novo signature. Together, these data support the repositioning of NAMPT inhibitors as a therapeutically relevant strategy for EZB-type GCB-DLBCLs. DisclosuresPasqualucci: Astra Zeneca: Research Funding; Sanofi: Research Funding.

Repurposing Dasatinib for Ibrutinib-Resistant Diffuse Large B-Cell Lymphoma

Diffuse Large B-Cell Lymphoma (DLBCL) is the most frequent B-cell Non-Hodgkin Lymphoma (B-NHL). It derives from germinal-center (GC) B cells, and has been classified into GC B cell like (GCB) and activated B cell like (ABC) subtypes, which differ in their cell of origin, genetic alterations and responses to therapy, with ABC having inferior prognosis in response to the current immuno-chemotherapeutic regimen (R-CHOP). This standard first-line treatment is successful in ~70% of cases based on 5-year overall survival, but the remaining ~30% of patients remains incurable. While recently developed drugs, including the Bruton Tyrosine Kinase (BTK) inhibitor Ibrutinib and PI3K-delta inhibitors, are providing novel targeted strategies, their impact on DLBCL is still limited to a fraction of cases, mostly in the ABC subtype. In order to identify drugs that can be repositioned for selective efficacy against DLBCL subtypes (GCB- and ABC-DLBCL) compared to non-GC derived lymphomas, we screened a focused set including 2,160 FDA-approved drugs and other targeted drugs in advanced clinical testing on a panel of 9 cell lines consisting of 6 DLBCL (3 ABC and 3 GCB) and 3 Mantle Cell Lymphoma (MCL) lines. This screen identified the multi-kinase inhibitor Dasatinib as the most DLBCL-specific agent. Dose-response experiments on an extended panel of 32 DLBCL lines showed that Dasatinib has broader activity than Ibrutinib, since it is active against all ABC-DLBCL Ibrutinib-sensitive lines, as well as an additional set of 9 ABC- and GCB-DLBCL lines. Notably, Dasatinib overcomes Ibrutinib-resistance caused by BTK C481S mutation, as shown by the fact that Dasatinib is active against 3 Ibrutinib-sensitive ABC-DLBCL lines rendered Ibrutinib-resistant by transduction of BTK C481S mutant. In these cell lines, Dasatinib, analogous to Ibrutinib, is unable to suppress BTK-C481S auto-phosphorylation at Tyrosine 223, indicating that it may act independently of BTK. To understand which kinase or combination of kinases needs to be suppressed for Dasatinib activity, we employed Dasatinib-resistant, gatekeeper mutants of each BCR-associated Src-Family Kinase (LYN, FYN and BLK), which can all be targeted by Dasatinib, alone or in combination. Our results showed that FYN suppression is essential for Dasatinib activity in both DLBCL subtypes. Integrated genomic and bioinformatics approaches to explore genetic determinants of resistance to Dasatinib revealed PTEN disruption as the most significant predictor. PTEN is inactivated in the majority of resistant lines by both genetic and epigenetic mechanisms, resulting in the inability by Dasatinib to restrict PI3K-mediated AKT activation. These data were further validated by showing that 3 Dasatinib-sensitive DLBCL lines were made resistant by transduction of a constitutively active PIK3CA mutant (PIK3CA-H1047R). Notably, suppression of AKT activation by mTORC2 inhibitors can revert Dasatinib resistance, suggesting that combinatorial strategies involving Dasatinib and drugs targeting the PI3K/AKT/mTOR axis may be effective in PTEN-negative DLBCLs. The above results were validated in vivo using DLBCL xenograft mouse models. Overall, these results provide a preclinical framework for Dasatinib-based targeted therapies for DLBCL. DisclosuresNo relevant conflicts of interest to declare.

N-quinoline-benzenesulfonamide derivatives exert potent anti-lymphoma effect by targeting NF-κB.

SummaryWe previously identified the N-quinoline-benzenesulfonamide (NQBS) scaffold as a potent inhibitor of nuclear factor-κB (NF-κB) translocation. Now, we report the structure-activity relationship of compounds with the NQBS scaffold in models of diffuse large B-cell lymphoma (DLBCL). We identified CU-O42, CU-O47, and CU-O75 as NQBS analogs with the most potent cytotoxic activity in DLBCL lines. Their anti-lymphoma effect was mediated by NF-κB sequestration to the cytoplasm of DLBCL cells. Internal Coordinates Mechanics analysis suggested direct binding between CU-O75 and IκBα/p50/p65 which leads to the stabilization of the NF-κB trimer. A whole cellular thermal shift assay confirmed direct binding of the NQBS to IκBα, an inhibitory component of the IκBα/p50/p65 trimer. Lymphoma cell line sequencing revealed CU-O75 induced downregulation of NF-κB-dependent genes and DeMAND analysis identified IκBα as one of the top protein targets for CU-O75. CU-O42 was potent in inhibiting tumor growth in two mouse models of aggressive lymphomas.

Pharmaocogenomic Characterization of MCL-1 Inhibitor Response and Resistance in Aggressive B-Cell Lymphomas

Mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) are aggressive hematologic malignancies characterized by the accumulation of lymphoid cells defective in cell apoptosis biology and function. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. Notably, the anti-apoptotic myeloid cell leukemia 1 (MCL-1) protein is recurrently highly expressed in various kinds of non-Hodgkin's B-cell lymphomas and promotes the survival of lymphoma cells by counteracting pro-apoptotic protein activity. Collectively, these data support the hypothesis that MCL-1 plays a central role in B-cell lymphoma progression and drug resistance. Pharmacologically targeting MCL-1, therefore, represents an attractive strategy to combat these lymphomas. However, previous clinical and pre-clinical data suggest that treatment with single agent anti-BCL-2 family member therapy is associated with rapid acquisition of resistance. To this end, there is a great need to develop and apply selective small molecule MCL-1 inhibitors as part of a first-line therapy or upon emergence of tumor resistance characterized by upregulation of MCL-1 for lymphoma therapy. Here, we exploited the MCL-1 dependency in MCL and DLBCL by implementing pharmacogenomic and chemical proteomic approaches to investigate the molecular drug response and resistance mechanism to MCL-1 inhibitors. In anticipation of the evolution of MCL-1 inhibitor resistance, we modeled MCL-1 inhibitor resistance mechanisms by developing S63845 resistant lines with high doses of S63845 treatment for an extended period in MCL, DLBCL and MCL-derived lines. RNA sequencing and chemical proteomics on paired parental and resistant cells demonstrated that transcriptome and kinome reprograming linked to the MEK and ERK pathways contribute to MCL-1 inhibitor resistance via regulation of the BCL-2 family profile (BCL-2 and BIM), and as such, represent a novel targetable vulnerability in MCL-1 inhibitor resistant lymphoma. Additional analyses revealed synergistic activity of MCL-1 inhibitors (S63845, AZD5991) in combinations with inhibitors of MEK (Trametinib), ERK (SCH772984) and BCR (Ibrutinib) in MCL-1 inhibitor resistant MCL/DLBCL lines and primary samples. These results provide a strong rationale for further evaluation of MCL-1 inhibitor in combination with established therapy in the clinical setting and highlight a potential strategy for overcoming MCL-1 inhibitor resistance. Disclosures Shah: NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Kite/Gilead, Jazz, Incyte: Research Funding; Moffitt Cancer Center: Current Employment; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria. Shain:AbbVie: Research Funding; GlaxoSmithKline: Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive: Consultancy, Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Research Funding, Speakers Bureau.

Abstract PO-23: Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase

Abstract Somatic hypermutation (SHM) is a B cell-specific process that supports the affinity maturation of antibodies. The reaction targets the immunoglobulin loci (“on-target" activity) and is initiated by activation-induced cytidine deaminase (AID). AID is known to be able to mutate the genome more broadly (“off-target” activity) and hence was found to be linked to various types of B-cell malignancies. However, the mechanism by which AID is recruited and exerts its function is partially known. To unravel this puzzle, we have analyzed two types of diffuse large B-cell lymphoma (DLBCL) cell lines: germinal B cell like (GCB-DLBCL), in which AID is expressed and introduces mutations, and activated B cell like (ABC-DLBCL), where AID levels are typically increased but paradoxically, SHM is much reduced. To begin to accomplish this goal, we have taken advantage of a Diversification Activator (DIVAC)-GFP reporter assay to measure targeted SHM activity. We first confirmed that AID is expressed in ABC-DLBCL cell lines at levels up to fourfold higher than in GCB-DLBCL cell lines by RT-qPCR. In contrast to the high levels of AID seen in ABC-DLBCL cells, we have observed that SHM is reduced by as much as 10-fold in ABC versus GCB lines using the DIVAC–GFP assay. As this assay should provide a good surrogate for SHM activity at Ig loci, our data support the possibility that the Ig-locus-specific SHM targeting program is defective in ABC-DLBCL. To search for factors that might explain the differential activity of the SHM program in GCB versus ABC cells, we performed RNA-seq using cell lines from each group. Initial analysis revealed that expression of two cell surface markers, CD27 and CD38, is highly correlated with the high mutation phenotype. Flow cytometry analysis for these two markers along with the pan B cell marker CD20 revealed various subpopulations in some DLBCL lines. Isolation, characterization, and measurement of SHM activity using the DIVAC-GFP assay of these subpopulations further strengthened the correlation between CD27/CD28 expression and SHM activity. These analyses also uncovered SHM-permissive subpopulations in ABC lines and SHM-resistant subpopulations in GCB lines. This observation explains residual SHM activity in ABC lines and argues for heterogeneity in the activity of the SHM program in DLBCL cell lines and perhaps within tumor populations in patients. Future studies will include deletion and introduction of specific factors from GCB or ABC lines to validate phenotype and identify regulators of the SHM program. Finally, sequence-analysis will be done to quantify mutations with the broad goal of understanding how AID targeting and mis-targeting are regulated. Citation Format: Huseyin Saribasak, Ravi Dinesh, Lizhen Wu, David G. Schatz. Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase [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-23.

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.

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.

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma.

It has been suggested that stimulation of B-cell receptors (BCR) by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCR in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of three of five activated B-cell type DLBCL cell lines and two of 11 primary DLBCL cases. Compared to controls, Ars2 was hypophosphorylated exclusively in cases and cell lines with Ars2-specific BCR. In a validation cohort, hypophosphorylated Ars2 was found in eight of 31 activated B-cell type DLBCL, but in only one of 20 germinal center B-cell like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA’ toxin conjugate induced killing of cell lines with Ars2-reactive BCR. Ars2 appears to play a role in a subgroup of activated B-cell-type DLBCL. Moreover, transformed DLBCL lines with Ars2-reactive BCR still showed growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

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

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

An OCT2 / OCA-B / MEF2B Ternary Complex Controls the Activity and Architecture of an Essential Locus Control Region for Normal and Malignant Germinal Center B-Cells

Melnick: Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding.

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

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

JNJ-64457107, a CD40 Agonist, Induces Cell Death in BCL6hiIRF4neg GCB Subtype of DLBCL

CD40 is a member of the TNF receptor family that is expressed in multiple tumor types and normal cells including antigen presenting cells (APC), endothelium and platelets. CD40-CD40L signaling is one of the pivotal mechanisms essential for robust antigen presentation and adaptive immunity. JNJ-64457107 (JNJ-107) is an anti-CD40 IgG1 agonistic antibody that is currently in a Phase 1 clinical trial (NCT02829099). JNJ-107/ADC-1013 induced significant anti-tumor activity and immunological memory (Mangsbo 2015). It was also reported that a CD40 agonist, SGN-40, induced direct cytotoxicity in diffuse large B cell lymphoma (DLBCL) cell lines (Burlington 2011), and clinical responses have been noted in this indication (Advani 2009). However, not all DLBCL patients and DLBCL cell lines respond to SGN-40. Thus, a patient selection strategy in B cell malignancies may be beneficial. Here, we investigated the mechanism of action and potential biomarkers of response to JNJ-107 in DLBCL.Here, we show that JNJ-107 induced dose-dependent APC activation measured by upregulation of co-stimulatory molecules CD86, CD54, HLA-DR and CD23 on peripheral B cells in ex vivo whole blood and in vitro generated dendritic cells by flow cytometry. The highest APC activation was at 0.1-5 µg/ml of JNJ-107. We also found that JNJ-107 induced cell death of CD40 positive DLBCL cell lines representing both, germinal center B-cell (GCB) and activated B-cell (ABC) subtypes at similar doses. Cell death was measured by the Cell Titer-Glo assay and confirmed by staining with PI/AnnexinV. JNJ-107 at 1 µg/ml induced cell death (in 30-90% of cells) in 11/20 DLBCL cell lines (called “sensitive”) after 4 days of treatment, with 2 GCB lines (BJAB and SU-DHL4) showing >90% cell death. No DLBCL cell line proliferation was observed in response to JNJ-107.CD40 signals through the NF-kB pathway in DLBCL. We investigated, if basal expression of NF-kB signaling components predicts responses to JNJ-107. First, we assessed activation of the NF-kB pathway in DLBCL lines by Western blot before and after treatment with JNJ-107. We used p65 and p52 as markers of canonical and non-canonical NF-kB activation, respectively. We found that JNJ-107 predominantly activated non-canonical NF-kB signaling in DLBCL cell lines by inducing p100 cleavage and release of p52 (Figure 1). A lower rate of p52 induction was observed in JNJ-107 “sensitive” lines that had constitutive expression of p52 (Pfeiffer, OCI-Ly7). Thus, we did not observe strong correlation between p52 induction and cell death in response to JNJ-107.BCL6 and IRF4 are markers of cell of origin in DLBCL. BCL6 and IRF4 are constitutively expressed in GCB and ABC subtypes, respectively. Interestingly, “sensitive” GCB lines had higher BCL6 expression than resistant lines. BCL6 protein was rapidly degraded (within 16h) in “sensitive” GCB DLBCL lines stimulated with JNJ-107. BCL6 is a transcriptional repressor that is regulated by many factors including IRF4. IRF4 is induced downstream of CD40, and directly represses BCL6 expression by binding to its promoter (Saito 2007). Consistent with constitutive activation of NF-kB, ABC DLBCL lines constitutively expressed IRF4. Surprisingly, several GCB lines also constitutively expressed IRF4 and were unable to further upregulate IRF4 in response to JNJ-107. The latter lines showed a lower degree of cell death in response to JNJ-107 compared to lines negative for IRF4 expression (except Pfeiffer).In summary, APC activation in whole blood, direct cytotoxicity and non-canonical NF-kB activation in DLBCL cell lines were seen with CD40 agonist, JNJ-107. GCB cell lines with high BCL6 expression and negative for IRF4 (BCL6hiIRF4neg) were more sensitive to JNJ-107 induced cell death. Based on these findings, GCB DLBCL patients with functional CD40 signaling in the tumor cells (BCL6hiIRF4neg phenotype) may show enhanced anti-tumor responses.Figure 1. JNJ-107 potently induces non-canonical NF-kB signaling in DLBCL cell lines. DLBCL lines were treated with 0.1 µg/ml JNJ-107 for 15 min, then 5 µg/ml cross-linker, IgG F(ab')2 for 16h. p65 and p52 fold induction was determined by Western blot of whole lysates. Representative data of 2 independent experiments are shown. [Display omitted] DisclosuresRusbuldt:Janssen R&D: Employment. Smith:Janssen R&D: Employment. Balasubramanian:Janssen Research & Development: Employment, Equity Ownership. Borzillo:Janssen R&D: Employment. Alvarez Arias:Janssen R&D: Employment.

Development of First-in-Class Histone Acetyltransferase (HAT) Activators for Precision Targeting of Epigenetic Derangements in Lymphoma

Monoalleleic inactivating mutations in histone acetyltransferase (HAT) enzymes promote lymphomagenesis in germinal center derived B-cell lymphomas, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), occurring in about 40% of patients. The intact wild-type allele offers an opportunity to leverage the normal enzyme to overcome the pathogenic impact of the mutated allele. We hypothesize that if inactivating mutations in HATs are critical to FL and DLBCL lymphomagenesis, then drugs capable of inducing enhanced function of the wild-type HAT allele product should be cytotoxic in cells harboring HAT mutations.We designed and synthesized a library of new chemical entities with HAT activating properties (N=70). The cytotoxic effects of the compounds (N=29) were evaluated via medium-throughput screening in 4 DLBCL cell lines. IC50 ranged from 3.6 to 43.2 µM. Focusing on 6 analogue compounds, which share the same Nphenylbenzamide scaffold, we evaluated cytotoxicity across an expanded panel of 11 DLBCL cell lines. The median IC50 of 6 analogues tested was lower in the EP300 mutated cell lines (median 9.6 µM, range 5 - 11 µM) compared to the wildtype lines (median, 17 µM, range 15 - 24 µM). YF2 was chosen as the lead compound because it was the most selective of the analogues in inducing cytotoxicity in cell lines harboring EP300 mutations compared to wildtype (IC50 5 µM and 19 µM respectively, p<0.0005).To determine YF2's functional effect on activating p300 in a cell free assay, p300-mediated histone and p53 acetylation was measured by combining recombinant p300, substrate and acetyl-CoA. YF2 increased p300-mediated histone H3 lysine27 acetylation (EC50=38.64 nM) and H3 lysine18 acetylation (EC50=1.656 nM). YF2 also induced acetylation of p53 by 5-fold in a dose dependent manner. In cellular assays, YF2 induces acetylation of histone (H3K27 2-fold and H3K14 1.6-fold) after exposure in the SUDHL-6 cell line (EP300 mutated) as measured by mass spectrometry and confirmed by immunoblot of histone extracts.To assess the pharmacokinetics (PK) and preliminary in vivo efficacy of YF2, SUDHL-6 (EP300-mutated) xenograft bearing mice were treated once daily i.p. for 6 days with YF2 doses of 40mg/kg or 60mg/kg. Serum and tumor samples were collected at sequential time points. The Cmax of YF2 60mg/kg was 2424 ng/mL (5.1 µM) whereas Cmax of 40mg/kg was 2091.96 ng/mL (4.5 µM) which is consistent with the cellular IC50. Both concentrations of YF2, 40mg/kg and 60mg/kg, accumulated in tumor with Cmax 9536.71 and 9858.15 ng/g, respectively. YF2 is rapidly absorbed in the serum (Tmax 0.25 h) and sustained in the tumor (Tmax 4h). Significant effects on tumor size were observed in 13 of 19 mice demonstrating decreased tumor volume following only 6 days of YF2 treatment. Mice treated with YF2 40mg/kg induced H3K27 acetylation in tumor specimens as determined by mass spectrometry.YF2 40mg/kg was well tolerated in SCID/beige mice for 30 days without significant weight loss, while 60mg/kg YF2 led to 20% weight loss during 6-days of treatment. Additionally, YF2 demonstrated cytotoxic effects in 2 primary patient lymphoma samples but were non-cytotoxic to peripheral blood mononuclear cells from healthy donors.Furthermore, we hypothesized that if DLBCL is sensitive to an enhanced acetylation state, then combined targeting of epigenetic machinery with HAT activators and HDAC inhibitors may induce profound epigenetic modification leading to synergistic induction of programmed cell death. The concentration : effect relationship of YF2 and the pan-HDAC inhibitor, romidepsin, was evaluated over time across a panel of lymphoma cell lines (N=7). Synergy was calculated by Excess over Bliss (EOB>10 connotes synergy). Combination of YF2 and romidepsin demonstrated strong synergism in DLBCL lines (EOB = 48). The combination led to enhanced histone acetylation compared to either single agents. The combination is safe in mice and murine xenograft studies of the combination are underway.In summary, YF2 induces HAT-mediated acetylation of histone and p53. It demonstrates selective cytotoxic effects in EP300-mutated DLBCL cell lines, and is both well tolerated and effective in xenograft mouse models of lymphoma suggesting potential clinical application and precision medicine opportunities for patients harboring this mutation. [Display omitted] DisclosuresO'Connor:Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.