Double-hit Diffuse Large B-cell Lymphoma Research Articles

Abstract 7276: Potent anti-proliferative activity of brincidofovir in Diffuse Large B-cell Lymphoma with overexpression of transcriptional repressor TLE1

Abstract Introduction: Brincidofovir (BCV) is a biologically-active acyclic nucleoside phosphonate and clinically-approved anti-viral agent. Recently, BCV has been demonstrated to display potent anti-proliferative and immunogenic properties in NK/T cell lymphoma (NKTCL). However, its effect on other lymphoma types remain unclear. Methods: We investigated BCV in an expanded cohort of B-cell lymphoma cell-lines (diffuse large B-cell lymphoma [DLBCL], n = 16; Burkitt lymphoma, n = 2; Hodgkin lymphoma, n = 1), in comparison with NKTCL cell lines (n = 11). Mouse xenograft models were deployed to assess in vivo effects. Whole transcriptomic sequencing (RNAseq) was performed to examine Epstein-Barr virus (EBV) status and explore potential mechanisms of action. Results: BCV inhibited viability in all B-cell lymphoma cell-lines in a dose-dependent manner (IC50 0.0798 to 8.414 µg/ml). Marked sensitivity (IC50 < 1 µg/ml) was demonstrated in nine cell-lines, including the MYC/BCL2-rearranged double-hit DLBCL cell-line OCI-LY18 (IC50 0.5 µg/ml). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice OCI-LY18 xenografts, compared with vehicle alone (p < 0.001, two-tailed t-test). RNAseq showed that most cell-lines (n = 16) were EBV negative. Interestingly, the transcriptional repressor TLE1 was amongst the top upregulated genes in the BCV-sensitive cell-lines (log2foldchange = 8.12, adjusted p = 0.0004). DLBCL patient samples from two independent datasets showed that TLE1-high tumors conferred worse overall survival (GSE11318, n = 200: HR 2.77, 95%CI 1.78 to 4.30, p < 0.0001; GSE10846, n = 414: HR 1.94, 95%CI 1.35 to 2.80, p = 0.0004). Gene set enrichment analysis showed that pathways involved in oxidative phosphorylation and oncogenic signaling were significantly upregulated in the BCV-resistant cell-lines compared to the rest. Notably, these findings are in stark contrast to NKTCL, in which TLE1 loss instead confers BCV sensitivity in vitro and is associated with poor survival outcomes in patients (n = 36; HR 3.44, 95% CI 1.23-10.49, p = 0.0301). Conclusions: Taken together, these results suggest that BCV may be a potential novel therapeutic agent for the treatment of B-cell lymphomas. Further work will be required to investigate the role of TLE1 and other molecular features as potential predictive biomarkers. Citation Format: Jing Yi Lee, Tun Kiat Ko, Elizabeth Chun Yong Lee, Bavani Kannan, Boon Yee Lim, Zhimei Li, Zexi Guo, Jui Wan Loh, Cedric Chuan-Young Ng, Kelila Xin Ye Chai, Nur Ayuni Binte Muhammad Taib, Kerry Lim, Dachuan Huang, Jing Quan Lim, Masatoshi Hazama, Koji Fukushima, Bin Tean Teh, Soon Thye Lim, Choon Kiat Ong, Jason Yongsheng Chan. Potent anti-proliferative activity of brincidofovir in Diffuse Large B-cell Lymphoma with overexpression of transcriptional repressor TLE1 [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 7276.

Abstract C076: Evidence of TCR and BCR clonal dynamics with enitociclib monotherapy in patients with MYC+ non-Hodgkin lymphoma (NHL)

Abstract Introduction: Enitociclib (VIP152) is a potent and selective CDK9 inhibitor with robust MYC downregulation (Frigault ASH 2021). We have previously reported safety, efficacy and MYC downregulation from 16 patients with B-cell lymphoma treated with enitociclib monotherapy, including 2 patients with double-hit diffuse large B-cell lymphoma (DH-DLBCL) who achieved complete metabolic remissions (CR) for 5.5+ and 4.0+ years (Diamond Clin Can Res 2022, Shadman ASH 2022). DH-DLBCL is characterized by MYC and BCL2/BCL6 rearrangements, and MYC+ NHL patients are known to be refractory to immune-oncology (IO) therapies. Herein, we evaluate the IO effect of enitociclib monotherapy in the blood of MYC+ NHL patients. Methods: Enitociclib is being evaluated in a phase 1 trial in patients with NHL receiving 30 mg i.v. once weekly (NCT02635672). From the previously reported 16-patient cohort (Shadman ASH 2022), 12 have evidence of MYC+ and had whole blood collections taken before and after dosing for at least 2 enitociclib doses (204 samples). RNA was purified from whole blood and bulk RNA seq performed by Illumina stranded total RNA prep with RiboZeroTM plus rRNA depletion and globin reduction RNA library preparation with strand-specific sequencing 50M paired reads (Discovery Life Science, AL, USA). TCR and BCR sequences were reconstructed using the TRUST4 algorithm (Song Nature Methods 2021). TCR beta chain (TRBC) sequences were annotated with antigen specificity data from VDJdb, McPAS-TCR and IEDB databases. Results: Demographics of the 12-patient cohort are described: 11 men; median age 68 (58-84) years; median prior therapy 2.5 (1-7); and includes DH-DLBCL (10; 2 CR, 8 clinical progression [PD]), triple hit-DLBCL (1 PD), and transformed follicular lymphoma (1 stable disease, currently on study). Simpson clonality of TRBC after 2 weeks of enitociclib compared with baseline correlates with response (CR, PD); however, the small sample size in each response category limits generalizability. Assuming BCR disease clones are identified by high frequency IGKC or lambda (IGLC), disease clonal switching occurs in 2 patients: DH-DLBCL (CR) from IGLC to IGKC and DH-DLBCL (PD) in reverse. In the CR patient, clearance of the top IGLC with enitociclib treatment is observed with simultaneous expansion of a high frequency TRBC. Although a viral specific TRBC is present in this patient, this top TRBC is unmapped to publicly known antigens suggesting the possibility of a cancer-specific TRBC. The second CR patient had low frequency TRBC. Tumor immune microenvironment deconvolution to describe dynamics of phenotypes will be presented to complement these findings. Conclusions: These data suggest that an IO mechanism of action may contribute to the durable 5.5 year CR observed in a DH-DLBCL patient treated with enitociclib monotherapy. Analysis in expanded datasets is required to support this preliminary observation including following active patients. Citation Format: Melanie M. Frigault, Joseph Birkett, Xin Huang, Raquel Izumi, Amy J. Johnson, Beatrix Stelte-Ludwig, Arushi Mithal, Ahmed Hamdy, Vanessa D. Jonsson. Evidence of TCR and BCR clonal dynamics with enitociclib monotherapy in patients with MYC+ non-Hodgkin lymphoma (NHL) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C076.

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 A07: 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

Abstract Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge as they 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, orally active compound with activity against DLBCL. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC50 of ~200 – 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma type. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% oral bioavailability and ~6 hours of serum half-life. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete response in all tumor-bearing animals and durable remission in 50% of animals with no tumor growth occurring for 2 weeks after dose cessation. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations demonstrated response in 100% of the lines with complete response in 6 of 9 PDX models tested. Dose scheduling studies indicate that 7 days of continuous dosing is sufficient to induce tumor regression and maintenance of a complete response. The therapeutic activity of BTM-3566 is related to a novel effect on mitochondrial proteostasis and a robust induction of the ATF4 ISR. Of the four eIF2α -kinases in the human genome we determined that HRI was uniquely required for BTM-3566 activity in DLBCL. HRI is activated by mitochondrial-related stress resulting in activation of the mitochondrial protease OMA1. Molecular analysis revealed that BTM-3566 activates OMA1, leading to induction of the ATF4 ISR pathway and apoptosis in DLBCL cell lines. Deletion of OMA1 reduces the ability of BTM-3566’s to induce apoptosis in DBLCL. Substrates of OMA1 include the dynamin OPA1 and DELE1, a protein recently shown to signal mitochondrial dysfunction through activation of HRI kinase and ATF4. Transfection of BJAB cells with a cleavage resistant OPA1 mutant has no effect on BTM-3566 induced apoptosis. In contrast, DELE1 KO suppresses BTM-3566 mediated apoptosis. BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Instead, BTM-3566 induces OMA1 activity through a novel mechanism regulated by the mitochondrial protein FAM210B. FAM210B expression is negatively correlated with response to BTM-3566, and overexpression of FAM210B blocks OMA1 activation and causes complete resistance to BTM-3566 induced apoptosis. Taken together, these data support a novel antitumor mechanism in DLBCL, where BTM3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An Investigational New Drug application for BTM3566 in B-cell malignancies will be submitted in Q2 2022 with initiation of first in human clinical trials planned for fall 2022. Citation Format: Adrian Schwarzer, Matheus Oliveira, Marc-Jens Kleppa, Andy Anantha, Alan Cooper, Mark Hannink, Todd Hembrough, Jedd Levine, Michael Luther, Michael Stocum, Linsey Stiles, Marc Liesa-Roig, Matthew Kostura. 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 [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 A07.

P1280: TARGETED INHIBITION OF DHODH IS SYNERGISTIC WITH BCL2 BLOCKADE IN LYMPHOMA WITH CONCURRENT MYC AND BCL2 REARRANGEMENT

Background: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, can be cured in approximately two-thirds of patients after initial therapy. The remaining one-third of patients have inferior survival outcomes due to relapse or refractoriness despite salvage chemotherapy with or without stem cell transplantation. The dual dysregulation of MYC and BCL2 is one of the most important pathogenic mechanisms. Thus, new therapies are needed to improve the prognosis of patients, and combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine and has been shown to be a potential therapeutic target for multiple diseases. Recent data have demonstrated that DHODH inhibitors can target MYC in some tumors. Aims: To develop and assess new therapeutics, we investigated the effects of the DHODH inhibitor brequinar in DHL lymphoma cell lines and the synthetic effects of brequinar and venetoclax. Methods: We first investigated the activity of DHODH inhibition in genetically defined double-hit DLBCL(DHL) cell lines. We then investigated whether c-MYC could be repressed by brequinar in DHL cells. Then we tried to uncover the synergistic mechanism of brequinar and venetoclax. To understand the genome-wide effects and target genes of brequinar and the combination with venetoclax, we performed an RNA-seq of SU-DHL4 cells treated with brequinar, venetoclax, or the combination of both agents versus DMSO-treated cells. At last, we confirmed the synergistic effect of DHODH inhibition and BCL2 blockade in vivo. Results: DHODH inhibition could repress DLBCL cell lines with MYC and BCL2 abnormalities. The protein level of c-MYC was upregulated 6 hours after brequinar treatment and then downregulated gradually with prolonged treatment time. The gene levels of c-MYC decreased over time. The synergistic mechanism of brequinar and venetoclax is the complementarity of different pathways. RNA-seq of SU-DHL4 cells treated with different agents further implied the possible synergistic mechanism of the two agents. Finally, in vivo study confirmed targeted inhibition of DHODH is synergistic with BCL2 blockade in lymphoma with concurrent MYC and BCL2 rearrangement Summary/Conclusion: Our study demonstrates that combined blockade of DHODH and BCL2 could synergistically inhibit DLBCL with concurrent MYC and BCL2 abnormalities through multiple pathways. Thus, it can be reasonably expected that a clinical-grade DHODH inhibitor combined with venetoclax might improve outcomes for patients with DHL or double-expression lymphoma and other cancers with high expression of MYC and BCL2 or MYC and MCL-1. Our findings provide new insights into the molecular basis of this effect and offer opportunities to design new targeted treatments for lymphoma.

Identification of CXCR4 Upregulation in Diffuse Large B-Cell Lymphoma Associated with Prognostic Significance and Clinicopathological Characteristics.

Background Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous malignant lymphoma with distinct characteristics. Patients with treatment failure after the standard immunochemotherapy have worse prognosis, which implies the necessity to uncover novel targets. The C-X-C chemokine receptor 4 (CXCR4) overexpression has been identified in several hematopoietic malignancies. However, the expression signatures and prognostic significance of CXCR4 in DLBCL associated with clinicopathological features remain unclear. Methods Gene expression profiles of DLBCL were obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Then, a meta-analysis with an integrated bioinformatic analysis was performed to assess the relationship between CXCR4 expression and clinicopathological features of DLBCL. Finally, experimental verification including immunohistochemical (IHC) staining and real-time quantitative PCR (qPCR) was carried out using patient samples. In vitro cell line viability tests were conducted using CXCR4 inhibitor WZ811. Results DLBCL patients with activated B-cell-like (ABC) subtype have higher expression level of CXCR4 with worse survival. Differential expressed genes in the CXCR4-upregulation group were enriched in canonical pathways associated with oncogenesis. DLBCL with CXCR4 upregulation had lower degree of CD8+ T cell infiltration. TIMER analysis demonstrated that the CXCR4 expression was positively correlated with the expression of CD5, MYC, NOTCH1, PDCD1, CD274, mTOR, FOXO1, and hnRNPA2B1 in DLBCL. IHC study in patient samples showed the positive correlation between CXCR4 and nongerminal center B-cell (non-GCB) subtype and mTOR expression. Meanwhile, quantitative polymerase chain reaction results revealed that high CXCR4 mRNA level was correlated to double-hit DLBCL. Finally, cell viability test showed that WZ811 exerted antiproliferation effect in DLBCL cell lines in a dose-dependent manner. Conclusion CXCR4 was upregulated in ABC-DLBCL associated with worse prognosis. Our analysis predicted CXCR4 as a potential target for DLBCL treatment, which may serve as an inhibitor both on BCR signaling and nuclear export warranting further investigation in clinical trials.

Abstract 2639: KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers

Abstract MYC is an early-response gene downstream of many receptor complexes, and a component that promotes several pro-growth signal transduction pathways. As a result, MYC expression is highly regulated, but once dysregulated in cancer, MYC promotes tumorigenesis via activation of cell growth, proliferation, and angiogenesis. CDK9 is both an upstream and downstream cofactor of MYC. CDK9 promotes MYC expression through its recruitment to super enhancers at the MYC amplicon and acts as a cofactor to drive gene expression programs. Previously, we profiled pan-cancer sensitivity to KB-0742, a potent, selective, and orally bioavailable small-molecule inhibitor of CDK9, and identified MYC-amplified cancers as being especially sensitive to KB-0742. To better understand the connection between MYC and KB-0742 sensitivity, 11 different tumor types were evaluated preclinically using immortalized cell lines, patient-derived cell lines (PDCs), patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). The 11 indications included head and neck, esophageal, gastric, liver, non-small cell lung cancer (NSCLC), pancreatic, ovarian, triple-negative breast cancer (TNBC), prostate, colon, and lymphoma. For 5 of the indications (head and neck, esophageal, gastric, liver, and colon), little activity was observed in cell lines and in vivo with weak correlations to MYC amplification. TNBC, ovarian cancer, and lymphoma showed good responses to KB-0742 treatment. For TNBC and ovarian, immortalized and patient-derived cell lines indicated lower IC50 values with increased MYC amplification or expression, but the results were not statistically significant. However, in vivo assessments using 15 PDX models showed good correlation of tumor growth inhibition (TGI) and MYC amplification/expression, with some tumor regressions observed in several ovarian models. Two PDO models of TNBC with different treatment histories were also treated with KB-0742 and compared to 4 standard-of-care (SOC) compounds. KB-0742 showed much greater activity in the models, with maximal inhibition rates of 100% and 89%. Cell-line screens of blood cancer-derived cell lines showed that lymphocyte-derived cell lines were the most sensitive to KB-0742 as compared to all other cancer types. We tested several PDX models for response to our compound. Treatment with KB-0742 resulted in TGIs of over 50% in several lymphoma models, including a TGI of 56% in 1 model of double-hit diffuse large B-cell lymphoma. These data support the continual development of KB-0742, which is currently being tested in a phase 1/2 clinical trial (NCT04718675). The dose-escalation phase is opened to relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommend phase 2 dose is determined, we plan to open dedicated expansion arms enrolling patients with MYC over-expression/amplification within the tumor types supported by these studies. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. KB-0742 is active in preclinical MYC high models of TNBC, ovarian, and DLBCL cancers [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 2639.

Double-drug loading upconversion nanoparticles for monitoring and therapy of a MYC/BCL6-positive double-hit diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a systemic hematological malignancy. Herein, through whole exome sequencing (WES), we found that DLBCL genome changes and expression characteristics are associated with various immune cells. Lenalidomide (Len) is a leading candidate for the immunomodulatory treatment of multiple myeloma in the clinic. Inspired by lenalidomide as an immunomodulatory drug for the treatment of multiple myeloma, we constructed a multifunctional nanoplatform with therapeutic and imaging properties for DLBCL by co-loading lenalidomide and dexamethasone (Dex) with upconversion nanoparticles using a GSH-sensitive linker (named as UCNPs-Len-Dex). In vitro cell experiments proved that the UCNPs-Len-Dex had good biocompatibility and obvious antitumor efficacy. UCNPs-Len-Dex also exhibited excellent anti-tumor efficacy and imaging properties in vivo. RNA sequencing showed that UCNPs-Len-Dex targeted and activated the E3 ligase of CRBN, resulting in IKZF1/3 degradation, which inhibited MYC/BCL6-positive DLBCL and maintained the stability of the immune microenvironment. Therefore, this study provided a new monitoring and therapeutic synergetic strategy for DLBCL.

Clinical Features and Immunophenotypes of Double-Hit Diffuse Large B-Cell Lymphoma.

Double-hit (DH) genetics induces a reduction in the complete remission (CR) and, consequently, in poor overall survival (OS) in diffuse large B-cell lymphoma (DLBCL) patients. Unfortunately, DH identification is time-consuming. Here, we retrospectively reviewed 92 newly diagnosed DLBCL patients, stratified them into the DH (n = 14) and non-DH groups (n = 78), and compared their clinical features and outcomes. The results revealed that the DH group had a higher percentage of bulky disease than the non-DH group (64.3% vs. 28.2%; p = 0.013). More patients in the DH group tested positive for double expresser (DE) (50.0% vs. 21.8%; p = 0.044). The three-year OS rates of patients with and without DH were 33.3% and 52.2%, respectively (p = 0.016). Importantly, advance stage and multiple comorbidities were correlated with a high mortality rate in multivariate analysis. Furthermore, by combining DE and the bulky disease, a specificity of 89.7% for DH prediction was achieved. In summary, DH genetics, not DE immunopositivity, could be a factor for an inferior OS in DLBCL. A combination of bulky disease and a positive DE immunophenotype could facilitate DH genetics prediction in newly diagnosed DLBCL patients.

Glofitamab (Glofit) in Combination with Polatuzumab Vedotin (Pola): Phase Ib/II Preliminary Data Support Manageable Safety and Encouraging Efficacy in Relapsed/Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL)

Glofitamab (Glofit) in Combination with Polatuzumab Vedotin (Pola): Phase Ib/II Preliminary Data Support Manageable Safety and Encouraging Efficacy in Relapsed/Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL)

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.

VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

Zanubrutinib, Lenalidomide Plus R-CHOP (ZR 2-CHOP) As the First-Line Treatment for Diffused Large B-Cell Lymphoma (DLBCL)

IntroductionTo evaluate the safety and efficacy of zanubrutinib, lenalidomide plus R-CHOP (ZR 2-CHOP) as the first-line treatment for DLBCL patients, we conducted this single-arm retrospective observational study.MethodsTreatment-naïve patients with DLBCL(including but not limited to double-hit, double expression) aged 18 to 75 years were enrolled.ZR 2-CHOP was given as follows, Oral zanubrutinib was given continuously (160 mg twice daily) from Day 0, lenalidomide 25 mg daily Day 1-7. Patients were administered intravenously rituximab (375 mg/m 2 Day 0), cyclophosphamide (750 mg/m 2 Day 1), doxorubicin (50 mg/m 2 Day 1), vincristine (1.4 mg/m 2 Day 1), and oral prednisone (100 mg Day 1-5). All patients were recommended to receive 6 cycles of ZR 2-CHOP (R-CHOP or R 2-CHOP were allowed in cycle 1-2 due to poor physical condition at treatment) and patients older than 70 years old were administered ZR 2-miniCHOP (Figure 1). CT or PET-CT scans were applied to mid-term efficacy and PET-CT scan was conducted after 6 cycles. ctDNA was dynamically detected before treatment, after 3 and 6 cycles to evaluate tumor mutational burden. The primary endpoint was complete response ratio (CRR) at mid-term and after 6 cycles. The secondary endpoint was overall response rate (ORR), ctDNA dynamics and adverse events (AE). AEs were graded based on CTCAE (version 5.0).Results12 treatment-naïve DLBCL patients diagnosed in Pukou CLL Center were enrolled in this cohort between July 2020 and June 2021. The median age was 56 years old and all patients had ECOG-PS ≤2. 1 patient (1/12) was diagnosed as double-hit DLBCL and 9 patients (9/12) as double-expression. 10 patients were non-GCB and 2 were GCB. 7 patients were classified as high-intermediate and high-risk group according to NCCN-IPI (Table 1). At data cutoff (1st July, 2021), the median follow-up was 8 months (3-12 months) with all patients have completed at least 3 cycles and mid-term assessment has been conducted. The ORR was 100.0%, with 10 patients achieved CR and 2 patients achieved PR (both two patients received R-CHOP regimen in cycle 1/cycle 1-2 due to poor physical condition at initial treatment, Figure 1). 10 patients have received 6 cycles, all of them achieved CR (Figure 2). ctDNA was dynamically detected in six patients. The median number of detectable ctDNA mutation among six patients was 8 (0-12) with two patients classified as MCD subtype and one patients as EZB subtype. All six patients showed undetectable ctDNA after 3 cycles. During end of treatment follow-up, one patient (triple-hit, EZB) who scheduled to receive auto-SCT underwent disease progression 4 months later and reemergence of ctDNA showed previous homologous clones. The most common hematological toxicity events were lymphocytes count decreased, neutrophil count decreased, thrombocytopenia and anemia, with 3-4 level occurrence rate was 66.7%, 25.0%, 25.0% and 16.7%. The most common non-hematological toxicity events were nausea, fatigue and anorexia. One patients discontinued oral zanubrutinib and lenalidomide in last two cycles due to drug rash.ConclusionZR 2-CHOP could attain high CR rate and ctDNA clearance in TN DLBCL, including patients with DEL and/or high-risk. The overall tolerability was manageable. ZR 2-CHOP could be one of the promising choice for TN DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:Zanubrutinib was used in the initial treatment of high-risk DLBCL.

Dose-adjusted EPOCH and rituximab for the treatment of double expressor and double-hit diffuse large B-cell lymphoma: impact of TP53 mutations on clinical outcome

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, including one-third of cases overexpressing MYC and BCL2 proteins (double expressor lymphoma, DEL) and 5-10% of patients with chromosomal rearrangements of MYC, BCL2 and/or BCL-6 (double/triple-hit lymphomas, DH/TH). TP53 mutations are detected in 20-25% of DEL. We report the efficacy of dose-adjusted EPOCH and rituximab (DA-EPOCH-R) in a series of 122 consecutive patients, including DEL (n=81, 66%), DEL-MYC (n=9, 7%), DEL-BCL2 (n=13, 11%), or high-grade lymphomas (DH/TH) (n=19, 16%). Central nervous system (CNS) prophylaxis included intravenous methotrexate (n=66), intrathecal chemotherapy (IT) (n=40) or no prophylaxis (n=16). Sixty-seven patients (55%) had highintermediate or high International Prognostic Index (IPI) and 30 (25%) had high CNS-IPI. The 2-year progression-free survival (PFS) and overall survival (OS) for the entire study population were 74% and 84%, respectively. There was a trend for inferior OS for DH/TH (2-year OS: 66%, P=0.058) as compared to all the others. The outcome was significantly better for the IPI 0-2 versus IPI 3-5 (OS: 98% vs. 72%, P=0.002). DA-EPOCH-R did not overcome the negative prognostic value of TP53 mutations: 2-year OS of 62% versus 88% (P=0.036) were observed for mutated as compared to wild-type cases, respectively. Systemic CNS prophylaxis conferred a better 2-year OS (94%) as compared to IT or no prophylaxis (76% and 65%, respectively; P=0.008). DA-EPOCH-R treatment resulted in a favorable outcome in patients with DEL and DEL with single rearrangement, whereas those with multiple genetic alterations such as DEL-DH/TH and TP53 mutated cases still have an inferior outcome.

Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer

Selective inhibition of exclusively transcription-regulating positive transcription elongation factor b/CDK9 is a promising new approach in cancer therapy. Starting from atuveciclib, the first selective CDK9 inhibitor to enter clinical development, lead optimization efforts aimed at identifying intravenously (iv) applicable CDK9 inhibitors with an improved therapeutic index led to the discovery of the highly potent and selective clinical candidate VIP152. The evaluation of various scaffold hops was instrumental in the identification of VIP152, which is characterized by the underexplored benzyl sulfoximine group. VIP152 exhibited the best preclinical overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats upon once weekly iv administration. VIP152 has entered clinical trials for the treatment of cancer with promising longterm, durable monotherapy activity in double-hit diffuse large B-cell lymphoma patients.

Abstract 1821: Integrative genomic analysis of diffuse large B cell lymphoma (DLBCL) identifies recurrent oncogenic alterations associated with distinct immune-related clusters

Abstract Introduction: Genetic alterations in tumor cells shape the cancer immune landscape and influence response to checkpoint blockade therapy (CBT). Despite recent advances in defining DLBCL genomics, the impact of genetic alterations on the DLBCL immune environment is not well-characterized. Here, we aim to define unique immune landscapes in DLBCL and determine the impact of lymphoma-cell intrinsic alterations on shaping immune environments in this disease, which may have implications for CBT response Methods: Gene set variation analysis (GSVA) was used to generate sample-wise enrichment scores for immune-related gene sets in ~500 DLBCLs. A cell-of-origin (COO) signature that classifies DLBCLs based on transcriptional similarity to activated B cells (ABC) or germinal center B cells (GCB) was included to control for COO-related genomic heterogeneity. Samples were hierarchically clustered. Paired exome sequencing was analyzed to identify mutations associated with each cluster. Cas9-mediated gene editing was used to modulate genes of interest in a murine lymphoma model (A20) Results: GSVA revealed 4 DLBCL immune clusters - ABC hot, ABC cold, GCB hot and GCB cold. Concordant with previous work, PD-L1 gene amplifications were highly enriched in ABC hot DLBCLs, while double-hit signature DLBCLs fell within the GCB cold cluster. Mutations and oncogenic pathways associated with each cluster were identified. ABC cold DLBCLs harbored recurrent inactivating mutations in TMEM30A and PRDM1, while mutations in KLHL6 and SPEN were associated with ABC hot DLBCLs. The GBC cold cluster was enriched for mutations in TP53 and FOXO1, and inactivating SOCS1 mutations were prevalent in GCB hot DLBCLs. MYC activation signatures characterized ABC and GCB cold clusters. A20 lymphomas with inducible c-Myc activation (A20MycER) contained fewer CD8+ T cells following tamoxifen versus control treatment, confirming our computational findings. Moreover, as SOCS1 mutations strongly associated with GCB hot DLBCLs and are prevalent in CBT-sensitive lymphomas, the extent to which Socs1-deficient lymphoma cells showed increased sensitivity to the critical T cell effector cytokine IFNγ was examined. Upon IFNγ exposure, A20Socs1-/- cells showed increased pStat1 levels, enhanced expression of PD-L1 and MHC-I, and strikingly diminished proliferation compared to A20WT cells, suggesting that enhanced JAK/STAT signaling via Socs1-/- increases IFNγ sensitivity, rendering lymphoma cells vulnerable to CBT Conclusion: Through computational analyses in a large genomic dataset, we have defined unique immune-related DLBCL clusters and associated recurrent genomic alterations. Through ongoing studies in murine models, we aim to elucidate mechanisms by which particular oncogenic alterations in lymphoma cells shape the DLBCL immune landscape and impact CBT response Citation Format: Sravya Tumuluru, James K. Godfrey, Jovian Yu, Alan Cooper, Michael Leukam, Sonali M. Smith, Xiufen Chen, Justin P. Kline. Integrative genomic analysis of diffuse large B cell lymphoma (DLBCL) identifies recurrent oncogenic alterations associated with distinct immune-related clusters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1821.

Combination of BR101801 and Venetoclax Demonstrates Synergistic Activity in DLBCL Cell Lines Harboring Double Hit and Double Expressor Alterations

Introduction: Double hit DLBCL with concurrent rearrangement of c-Myc and BCL-2 as well as double expressor DLBCL with concomitant overexpression of c-Myc and BCL-2 are characterized to have very poor outcomes in the following standard R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy as well as short survival in progressing clinical courses. In DLBCL patients, about 20% to 30% of patients have shown the dual c-Myc and BCL-2 aberrance. Thus, several new drugs are being developed to achieve a durable response in double hit and double expressor DLBCL. The phase I study in patients with RR DLBCL using Venetoclax monotherapy which is a BCL-2 inhibitor, showed a disappointing objective response [CR:4/34(12%), PR:2/34(6%)]. This result shows that a combined approach involving a c-Myc controlling agent and BCL-2 inhibitor may be required to overcome the poor response of RR DLBCL, especially double hit and double expressor DLBCL. BR101801 is a first-in-class, oral, dual target inhibitor of DNA-PK and PI3Kδ. The combined inhibition of dual targets tightly regulates the protein stability of c-Myc independent of genetic alterations such as translocation, amplification, and overexpression. The purpose of this study is to investigate the functional significance of BR101801 on the growth inhibition and c-Myc regulation in double hit and double expressor DLBCL cell lines.Methods: Westernblot of protein expression levels was analyzed with the human DLBCL cell lines that were treated with BR101801, PI3Kδ-specific inhibitors (Idelalisib, TGR-1202) and DNA-PK inhibitor (NU-7441) or DMSO for 24 hr. After treating SU-DHL-4 cell with cyclohexamide (CHX, 20 ug/ml), and BR101801 (0.5 μM) alone or in combination, the lysates was analyzed with the westernblot. Cells (5x103/well) were incubated with various concentrations of compounds and the cell viability was determined after 72 hr by the WST-8 assay. Cell lines were classified as c-Myc translocation (DOHH2, SU-DHL-6, SU-DHL-10, OCI-LY-18), c-Myc amplification (SU-DHL-4, OCI-LY-3), and c-Myc expressor (U2932). Cell lines additionally showed BCL-2 alteration such as translocation (DOHH2, SU-DHL-4, SU-DHL-6, OCI-LY-18) and amplification (OCI-LY-3, U2932).Results: BR101801 showed potent inhibitory activity on c-Myc protein stability whereas no activity was found in Idelalisib, TGR-1202, or NU7441 (Figure 1A; SU-SHL-4, 1B; SU-DHL-6). Using CHX to block de novo protein synthesis on SU-DHL-4 cells, the increased rate of c-Myc degradation by GSK3β regulation was observed (Figure 1C). SU-DHL-10 cell lines harboring c-Myc alterations exhibited no sensitivity to the PI3Kδ inhibitor such as Idelalisib (IC50≒4.4 μM) and TGR-1202 (IC50>10 μM) and DNA-PK inhibitor such as NU7441 (IC50≒1.1 μM). However, the combination of Idelalisib and NU7441 resulted in 200-fold (IC50≒ 0.02 uM) growth inhibition (Figure 1D), which was comparable to the effect of BR101801. SU-DHL-6 cells showed low sensitivity to Venetoclax alone with IC50≒0.16 μM. However, the IC50 value of Venetoclax at 0.1, 1, 10 nM of BR101801 was decreased to 0.12 uM, 0.003 uM and < 0.001 uM (11 pM), respectively in a dose-dependent manner (Figure 1E).Conclusions: In this study, the rationale of co-targeting of PI3Kδ and DNA-PK in c-Myc regulation and growth inhibition were demonstrated. The dual targets of DNA-PK and PI3Kδ were essential to control the protein stability of c-Myc in DLBCL cell lines harboring various c-Myc alterations. In conclusion, the potent effect of BR101801 with Venetoclax in DLBCL cells harboring alterations on both c-Myc and BCL-2 suggests a mechanistic rationale as a breakthrough treatment for RR DLBCL patients. To ensure translation to humans, the study on the efficacy of BR101801 in primary cells isolated from double hit and double expressor as well as RR DLBCL patients is currently conducted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Lenalidomide and Obinutuzumab with CHOP for Newly Diagnosed Diffuse Large B-Cell Lymphoma: Final Phase I/II Results

▪Introduction:Diffuse large B-cell Lymphoma (DLBCL) is the most common lymphoid cancer in the United States with 30K diagnoses each year. The standard therapy for all newly diagnosed patients is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP) which cures ~60% of patients. Lenalidomide (L) and obinutuzumab (O) have both shown promising efficacy and acceptable toxicity in DLBCL patients. Obinutuzumab, a humanized CD20 monoclonal antibody with increased pre-clinical affinity to the FcGRIIIa receptor in comparison to rituximab, may have increased antibody dependent cell-mediated cytotoxicity (ADCC). Lenalidomide, a potent immunomodulatory, is toxic to DLBCL via interferon signaling stimulation. As single agents, O and L achieve overall response rates (ORR) of 32% and 28%, respectively, in patients with relapsed DLBCL (Morschhauser, 2013, Hernandez-Ilizaliturri, 2011). The addition of L to RCHOP may overcome adverse outcomes of the non-germinal center (non-GCB) subtype of DLBCL. Randomized phase III clinical trials evaluating RCHOP +/- Len in non-GCB DLBCL are highly anticipated. The GOYA study found RCHOP was as effective as OCHOP in patients with unselected newly diagnosed DLBCL. We hypothesize the combination of OL-CHOP will result in enhanced NK cell-mediated cytotoxicity, down-regulation of interferon regulatory factor 4, and a complete response (CR) rate ≥ to OCHOP (57% in GOYA trial, Vitolo et al, ASH 2016).Methods: Newly diagnosed, CD20+ DLBCL patients were eligible if they have measurable disease, age ≥18 years, and adequate organ function. In the Phase Ib trial, Obinutuzumab was dosed at 1000mg IV days (d) 1, 8, and 15 during cycle 1, and d 1 on cycles 2-6. Lenalidomide dose was 15mg d 1 - 14 cycles 1 - 6 in the first cohort, with lower dose cohorts if toxicities occurred, using a 3+3 design. CHOP was dosed in standard fashion. Once the maximum tolerated dose (MTD) was identified, phase II was initiated with a planned total of at least 50 evaluable patients at the MTD. The primary objectives of this phase Ib/II trial were to determine the MTD and efficacy of LOCHOP. Correlative studies, including minimal disease assessment, cell of origin determination, and immune profiling are planned on blood and tumor samples.Results: Fifty three patients were enrolled, 6 in Phase Ib trial, 47 in Phase II with all evaluable for safety and 51 for efficacy. Two patients withdrew consent after 1 cycle of therapy. The median age was 62 years (26-83), with 42% males and a median IPI of 2 and NCCN IPI of 3. Five patients had double hit DLBCL, 5 patients had Double Expressor DLBCL, and 21 patients were non-GCB DLBCL. No dose limiting toxicities were identified in in Phase Ib. Toxicities encountered in PhIb include neutropenia (Grade 3: 50%, Grade 4 33%), thrombocytopenia (grade 3: 17%), and rash (grade 2: 17%), with similar findings in PhII. All six patients in PhIb achieved a complete response (CR). In the Phase II trial, 50 patients have end of therapy response assessment as of 8/2/17, with 45 with CR, 4 with partial response, 1 with progression. In the combined evaluable patients, the overall response rate is 98%, with CR of 90%. The CR rate was 90% in non-GCB and 92% in GCB. The median follow up 11 months and the median progression free and overall survival are not reached. There have been no deaths. Additional follow up and analyses will be presented at the meeting.Conclusions: The combination of Obinutuzumab, Lenalidomide, and CHOP is well tolerated, with no dose limiting toxicity encountered in the phase Ib trial, and efficacy is impressive in this single center single arm Phase Ib/II trial. Adverse events do not appear to be different than expected with standard RCHOP. Final results and correlative analyses will be presented at the ASH meeting [Display omitted] DisclosuresWestin:Apotex: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Nastoupil:TG Therapeutics: Honoraria, Research Funding; Gilead: Honoraria; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Celgene: Honoraria, Research Funding. Neelapu:Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Poseida Therapeutics, Inc: Research Funding; Cellectis Inc.: Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Karus: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lee:KITE PHARMA: Consultancy.

Salicylates Potentiate and Broaden CRM1 Inhibitor Anti-Tumor Activity Via S-Phase Arrest and Impaired DNA-Damage Repair

Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (selinexor), a first in class CRM1 inhibitor recently approved for multiple myeloma and diffuse large B-cell lymphoma (DLBCL), has been limited by substantial adverse effects (AEs). To address this clinical problem, we focused on identifying novel strategies to boost the potency, reduce toxicity, and broaden the applicability of CRM1 inhibitors to a wider range of malignancies. We discovered that salicylates could markedly enhance the anti-tumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. KPT-330 was chosen as the prototypical CRM1 inhibitor given its current FDA approval status and characterized pharmacokinetics; and choline salicylate (CS) was chosen as the prototypical salicylate given its favorable pharmacokinetics and reduced antiplatelet, renal, neurological and gastrointestinal AEs in humans compared to other salicylates. By using cell lines belonging to different hematologic malignancies and solid tumors, we demonstrated ex vivo that the combination of KPT-330 and CS (K+CS) could induce unique and significant antitumor effect at much lower dose of KPT-330 (at 25% of the dose used in the clinic), thereby potentially mitigating prohibitive clinical AEs (Figure 1a-d, and e-g). This significant synergetic antitumor effect observed with K+CS ex vivo was also validated in vivo by using an NSG mouse model of mantle cell lymphoma (Figure 1h). Moreover, the K+CS combination did not show this potent toxic effect on non-malignant cells in vivo and was safe without inducing toxicity to normal organs in NSG mice. Mechanistically, protein profiling through mass spectroscopy revealed that K+CS uniquely affects the cellular pathways of DNA damage repair, DNA synthesis and cell cycle progression. Studies involving immunoblotting, cell cycle analysis, immunofluorescence microscopy assessing nucleocytoplasmic molecular export and DNA damage, reporter assay to assess homologous recombination repair proficiency and immunohistochemistry showed that, compared to KPT-330 treatment alone, K+CS decreased the expression of CRM1, Rad51 and thymidylate synthase proteins in vitro and in vivo, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, and importantly a unique cell cycle arrest in S-phase, thus leading to cell apoptosis. These effects on cellular proteins and pathways were unique to K+CS treatment and were not observed with KPT-330 or CS single agent treatment. Pathway analyses through RNA sequencing also paralleled the findings of proteomic studies thus further validating the unique effect of K+CS treatment on the aforementioned cellular pathways. Importantly, K+CS treatment exerted unique and significant antitumor effect ex vivo on primary malignant cells obtained from patient's with high-risk hematologic malignancies such as double hit DLBCL, BTK and BCL2 inhibitor resistant mantle cell lymphoma, TP53 deleted/mutated chronic lymphocytic leukemia and high-risk multiple myeloma thus signifying its broader applicability as a treatment option for these aggressive hematologic malignancies where there is a dire need to find new treatment strategies (Figure 1i). In summary, we describe a unique all-oral drug combination with a novel constellation of mechanisms of action on cellular pathways that are exploited by cancer cells. K+CS represents a new class of therapy for multiple cancer types and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for therapy of blood cancers. Disclosures Witzig: Karyopharm Therapeutics: Research Funding; Acerta: Research Funding; Incyte: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Research Funding; MorphSys: Consultancy; Immune Design: Research Funding; Spectrum: Consultancy.

Analysis of primary central nervous system large B-cell lymphoma in the era of high-grade B-cell lymphoma: Detection of two cases with MYC and BCL6 rearrangements in a cohort of 12 cases

High-grade diffuse large B-cell lymphoma (HG-DLBCL) refers to DLBCL with MYC and BCL2 and/or BCL6 rearrangements (double-hit or triple-hit DLBCL) that exhibits poor prognosis. Double-expressor DLBCL (c-myc+/bcl-2+) has intermediate prognosis when compared to HG-DLBCL. Primary central nervous system lymphoma (PCNSL) has distinct pathophysiology (frequent non-germinal center-like subtype and double-expressor) and has worse prognosis than systemic DLBCL. By fluorescence in situ hybridization (FISH), 25–30% of PCNSLs harbor BCL6 abnormalities with rare alterations in MYC, BCL2, double-hit or triple-hit events. We describe the clinicopathologic features and status of MYC, BCL2 and BCL6 in 12 PCNSLs (7 women, 5 men; median age 63 years; range: 28–79). Six cases showed focal starry-sky pattern. Immunohistochemically, all (100%) were of non-germinal center-like subtype, and 8/10 (80%) cases were double-expressors. Ki-67 ranged from 70 to 100%. FISH was positive in 9/12 (75%) cases: 4 (33%) harbored a BCL6 rearrangement, 3 (25%) had a gain of BCL2, 2 (17%) cases each had a gain of BCL6 and gain of IGH, and gain of MYC and deletion of MYC were observed in 1 case each (8%). Two (16%) cases were MYC/BCL6 double-hit PCNSLs. No MYC/BCL2 or triple-hit cases were identified. Eleven (92%) patients received chemotherapy and one also received whole brain radiation. The median time of follow-up was 4.4 months (range, 0.3–40.3). Seven (58%) patients are alive, 4 (33%) have died, and 1 (8%) had no follow-up. Five alive patients are in remission, including one MYC/BCL6 double-hit PCNSL. Our results add two new cases of rare double-hit PCNSL to the literature.