Lymphoma Cell Death Research Articles

Abstract LB081: Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma

Abstract Telomerase (TERT) is an enzyme critical for carcinogenesis expressed in about 90% of human cancers, including aggressive diffuse large B-cell lymphoma (DLBCL). Targeting TERT proven difficult due to on-target toxicities to non-malignant hematopoietic stem cells and potentially to activated immune effector cells. To overcome these challenges, we developed novel TLR9+ cell-selective CpG-oligodeoxynucleotides (ODNs) to deliver telomere-damaging nucleoside analogue, 6-thio-2’-deoxy-guanosine (6tdG), into target B cell lymphoma cells in vitro and in vivo. Human and mouse TLR9+/TERT+ lymphoma cells rapidly internalized CpG(6tdG)-oligonucleotides through active endocytosis. 6tdG molecules were released from the 3’ end of the oligonucleotide into cytosol and cell nucleus. TERT-mediated 6tdG incorporation into telomeres, led to DNA damage and lymphoma cell death. In vitro, CpG(6tdG)-oligonucleotides showed potency in targeting variety of human DLBCL cells in nanomolar range without detectable toxicity to non-malignant TERT— cells or human TERT+ hematopoietic stem cells and activated T cells. Importantly, the repeated systemic administration of CpG(6tdG)-oligonucleotides was well-tolerated by humanized hCD34/NOG mice, without detectable decrease in the percentage of hematopoietic stem cells, myeloid cells or T cells although the numbers of B cells were partly reduced as expected. Intravenously injected CpG(6tdG)-oligonucleotides, but not 6tdG nucleoside, had potent and dose-dependent antitumor effects against xenotransplanted models of human GC- or ABC-subtypes of DLBCL, such as OCI-Ly18 or OCI-Ly3, respectively. Furthermore, we assessed CpG(6tdG)-oligonucleotide activity against aggressive, syngeneic TERT+ Eμ-myc/15A lymphoma in immunocompetent mice. CpG(6tdG)-oligonucleotide, but not the CpG-oligonucleotide alone, 6tdG alone or co-injection of thereof, was effective in triggering regression of established 15A tumors with long-term survival of the majority of mice. The effect of CpG(6tdG)-oligonucleotide correlated with the activation of tumor antigen-specific T cell-mediated antitumor immune responses. In contrast, when tested in immunodeficient mice, CpG(6tdG)-oligonucleotide induced significant but transient regression of 15A lymphoma. These results suggest that the oligonucleotide elicits two-pronged activity triggering direct lymphoma cell cytotoxicity and also immune-mediated antitumor effects. Overall, this cell-selective strategy could provide an effective and safer strategy against aggressive TERT+ DLBCL cells with potential to engage and support T cell-based antitumor immunity. Citation Format: Chunsong Yu, Yong Liang, Piotr Swiderski, Larry Kwak, Marcin Kortylewski. Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB081.

JAK1/2 Inhibition Modulates Key Markers of Lymphomagenesis in 9p24.1 Amplified Lymphoma, Priming for Enhanced Antigenicity

Background Primary Mediastinal B-cell Lymphoma (PMBCL) and Hodgkin Lymphoma (HL) are frequently defined by chromosome 9p24.1 amplification, leading to upregulation of PDL-1/-2 and JAK2. JAK2, in addition to its known role in the JAK/STAT pathway, has also been shown to act as an epigenetic modifier by phosphorylation of H3Y41, enabling an euchromatin state and expression of MYC. When these epigenetic modifications are coupled with upregulation of PDL-1/-2, the 9p amplicon leads to immune escape, unchecked cell proliferation, and tumorigenesis. We hypothesize that if deranged histone modification and amplified expression of PD-1 ligands cooperate to drive cell growth in 9p amplified PMBCL and HL, then dual targeting with JAK inhibitor and immune checkpoint blockade will lead to altered gene expression and enhanced immunogenicity inducing synergistic cell death. The following abstract demonstrates preliminary data using ruxolintib monotherapy in cell lines, with goal of combining immunotherapy in mouse models. Methods In order to understand how ruxolitinib modulates key drivers of lymphomagenesis in 9p24.1 amplified lymphoma, human lymphoma cell lines with 9p amplification, (n=2): Karpas1106p (PMBCL) and L540 (HL)were compared to cell lines lacking 9p24.1 amplification, (n=2): Farage (PMBCL) and U2940 (PMBCL). All samples were treated for 3- and 6-day intervals. The IC50 concentrations were established for ruxolintib using Cell TiterGlo assay: Karpas1106p 5.72 µM, L540 22 µM, Farage 29.46 µM, and U2940 32.99 µM. All cells were treated for 48 hours with control or ruxolitnib and probed by western blot, flow cytometry, and RNA-sequencing. The expression of key determinants of survival including JAK/STAT, pro- and anti-apoptotic proteins (SOCS1 and MYC, respectively) and MHCII were measured by western blot. PD-1/-2 ligands were measured by flow cytometry. Transcriptomic changes were evaluated by RNA-seq and data was presented as Log 2 fold change compared to vehicle controls. Results Pharmacodynamic effects were most prominent in the 9p amplified Karpas1106p cell line demonstrating increased markers of immunogenicity by protein quantification: increased CIITA by 3.5-fold (SEM ± 1.15) and increased MHCII by 2.67-fold (SEM ± 0.33). Expression of tumor suppressor SOCS1 was increased by 2.67-fold (SEM ± 0.68) and expression of oncogene MYC was decreased by 6.06-fold (SEM ± 4.66). The 9p amplified L540 line similarly demonstrated increased CIITA by 3.22-fold (SEM ± 1.59) and decreased MYC by 2.69-fold (SEM ± 0.65), however no significant change in MHCII or SOCS1 was observed. Significant reduction of STAT3p and/or STAT6p was demonstrated across all cell lines (p < 0.01). Cell lines without 9p amplification, Farage and U2940, did not demonstrate consistent increase nor decrease in markers of immunogenicity or oncogenes, suggesting ruxolitinib preferentially modulates downstream markers of survival and immune recognition driven by the 9p amplicon. Flow cytometry demonstrates significantly reduced expression of PDL-1 (p = 0.01) and PDL-2 (p = 0.001) in 9p amplified cell lines (Karpas1106p and L540), and non-amplified cell lines (Farage and U2940), as shown in Figure 1. Down-regulation of PDL-1/-2 was more prominent in the 9p amplified cell lines compared to those without 9p amplification (p = 0.055 and p = 0.18, respectively), suggesting that ruxolitinib may prime for increased immunogenicity by reducing PDL-1/-2, especially in 9p amplified cell lines. RNA-seq of the 9p amplified Karpas1106p cell line demonstrated increased MHCII: HLA-DQ (1.12, p < 0.001), HLA-DO (0.70, p < 0.001), HLA DM (0.14, p = 0.001), and decreased PDL-1 (-1.04, p < 0.009), PDL-2 (-1.70, p < 0.001), C-MYC (-0.95, p < 0.001), and STAT1/3/4 (all p < 0.001). Gene-ontology analysis demonstrated increased lymphocyte differentiation (p < 0.001) and immune response activating pathways (p = 0.003) ( Fig. 2). Conclusion Altogether, data suggests that ruxolitinib enhances immunogenicity primarily in 9p amplified cell lines and supports the hypothesis that ruxolitinib may prime for lymphoma cell death when combined with immunotherapy. These findings provide a foundation for in vivo assessment in humanized mouse models treated with combination anti-PDL-1 and JAK1/2 inhibitor, targeting tumorigenesis driven by 9p amplification. *This work has been funded by the ASH RTAF grant.

Abstract A31: C-MYC Targeting by Degradation: Novel Dual c-MYC/GSPT1 Degrader GT19715 Induces TP53-independent Cell Death in MYC-amplified Acute Myeloid Leukemia and Lymphomas

Abstract The oncoprotein c-MYC, a major regulator of the epigenome and transcriptome, is dysregulated in 70% of all human cancers. MYC is highly expressed in Burkitt lymphoma and TP53 mutant and venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been successful and remains a major unmet clinical need. We developed the first cereblon E3 ligase modulators (CELMoDs) for c-MYC: GT19630 and GT19715 (salt form of GT19630). C-MYC was one of the top decreased proteins in chromatin-enriched proteomics, was pulled down by biotinylated GT19630 in in vitro affinity purification assay and was degraded with IC50 of 0.33 nM in MYC-amplified HL-60 cells. Proteasome inhibitor ixazomib completely blocked c-MYC reduction, suggesting a CRL4CRBN-dependent degradation. Blood cancer cell lines responded to GT19715 greater than other cancer cell lines such as lung, breast and brain tumors in a broad cell line panel, providing rationale to develop the degrader in hematologic malignancies. In agreement with other CELMoDs, proteomic analyses revealed degradation of translation termination factor GSPT1 (G1 to S phase transition proteins 1), an important factor in LSC survival Whereas a selective GSPT1 degrader CC-90009 reduced GSPT1 protein levels but not c-MYC, GT19715 reduced both c-MYC and GSPT1 and exerted a 20x higher cytoreduction than CC-90009 (IC50 of 1.8 nM vs 40.4 nM for GT19715 and CC-90009, respectively) in HL-60 cells. GT19630 degraded c-MYC and GSPT1 and inhibited tumor growth in a xenograft model with HL-60 cells. GT19715 eliminated circulating blasts and prolonged survival in the systemic Burkitt lymphoma model (Daudi). GT19715 significantly reduced human CD45+ AML blasts in peripheral blood, bone marrow (BM) and spleens compared to vehicle controls in vivo in a chemotherapy-resistant AML PDX model. MV4;11 venetoclax resistant (VR) cells demonstrated elevated protein levels of c-MYC and GSPT1 and GT19715 induced 4log10 cytoreduction in BM and prolonged survival of mice with MV4;11 VR cells. Baseline c-Myc protein levels associated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 (R2 = 0.86, P = 0.02). GT19715 induced comparable cell death in primary AML samples with wild-type or mutant TP53 (95.4% and 91.7% cytoreduction, P = 0.48 for wild-type and mutant TP53 samples at 64 nM of GT19715, respectively). CD34+ AML cells were more susceptible to GT19715 than CD34- AML cells, suggesting a greater efficacy in AML stem/progenitor than in more mature AML cells. Notably, single cell mass cytometry revealed that CD34+ AML cells had higher c-MYC protein levels than CD34+ normal BM cells and GT19715 reduced c-Myc levels in CD34+ AML but not normal BM cells. GT19715 induced greater cytoreduction in CD34+ AML than in normal BM cells, suggesting a therapeutic window. Conclusions: The novel dual c-MYC/GSPT1 degrader GT19715 exerts TP53 independent preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development. Citation Format: Yuki Nishida, Darah A Scruggs, Edward Ayoub, Lauren B Ostermann, Tallie Patsilevas, Vivian R Ruvolo, Po Yee Mak, Bing Z. Carter, Steffen Boettcher, Abhishek Maiti, Koji Sasaki, Qianxiang Zhou, Zhaohui Yang, Honghua Yan, Liandong Ma, Michael Andreeff. C-MYC Targeting by Degradation: Novel Dual c-MYC/GSPT1 Degrader GT19715 Induces TP53-independent Cell Death in MYC-amplified Acute Myeloid Leukemia and Lymphomas [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A31.

Synthetic lethality of drug-induced polyploidy and BCL-2 inhibition in lymphoma

Spontaneous whole genome duplication and the adaptive mutations that disrupt genome integrity checkpoints are infrequent events in B cell lymphomas. This suggests that lymphomas might be vulnerable to therapeutics that acutely trigger genomic instability and polyploidy. Here, we report a therapeutic combination of inhibitors of the Polo-like kinase 4 and BCL-2 that trigger genomic instability and cell death in aggressive lymphomas. The synthetic lethality is selective for tumor cells and spares vital organs. Mechanistically, inhibitors of Polo-like kinase 4 impair centrosome duplication and cause genomic instability. The elimination of polyploid cells largely depends on the pro-apoptotic BAX protein. Consequently, the combination of drugs that induce polyploidy with the BCL-2 inhibitor Venetoclax is highly synergistic and safe against xenograft and PDX models. We show that B cell lymphomas are ill-equipped for acute, therapy-induced polyploidy and that BCL-2 inhibition further enhances the removal of polyploid lymphoma cells.

Targeting positive cofactor 4 induces autophagic cell death in MYC-expressing diffuse large B-cell lymphoma

MYC-expressing diffuse large B-cell lymphoma (DLBCL) is one of the refractory lymphomas. Currently, the pathogenesis of MYC-expressing DLBCL is still unclear, and there is a lack of effective therapy. We characterized positive cofactor 4 (PC4) as an upstream regulator of c-Myc, and PC4 is overexpressed in DLBCL and is closely related to clinical staging, prognosis, and c-Myc expression. Furthermore, our in vivo and in vitro studies revealed that PC4 knockdown can induce autophagic cell death and enhance the therapeutic effect of doxorubicin in MYC-expressing DLBCL. Inhibition of c-Myc-mediated aerobic glycolysis and activation of the AMPK/mTOR signaling pathway are responsible for the autophagic cell death induced by PC4 knockdown in MYC-expressing DLBCL. Using dual-luciferase reporter assay and electrophoretic mobility shift assay assays, we also found that PC4 exerts its oncogenic functions by directly binding to c-Myc promoters. To sum up, our study provides novel insights into the functions and mechanisms of PC4 in MYC-expressing DLBCL and suggests that PC4 may be a promising therapeutic target for MYC-expressing DLBCL.

A Novel Peptide that Disrupts the Lck-IP3R Protein-Protein Interaction Induces Widespread Cell Death in Leukemia and Lymphoma.

There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.

Orelabrutinib and venetoclax synergistically induce cell death in double-hit lymphoma by interfering with the crosstalk between the PI3K/AKT and p38/MAPK signaling.

Double-hit lymphoma (DHL) is a rare and aggressive mature B-cell malignancy with concurrentMYCandBCL2rearrangements. When DHL becomes relapsed or refractory, it becomes resistant to the majority of therapeutic approaches and has subpar clinical results. Therefore, innovative therapeutics for this particular patient population are urgently needed. Orelabrutinib, a new oral BTK inhibitor, combined with the Bcl-2 inhibitor venetoclax, was used to confirm the antitumor effect of DHL. Cell counting kit-8 and Annexin V-FITC/PI assays were used to examine the interaction of this combined regimen on DHL cell lines and primary lymphoma cells. RNA sequencing, EdU incorporation assay, mitochondrial membrane potential assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of orelabrutinib with or without venetoclax against DHL cell lines. In this study, orelabrutinib combined with venetoclax synergistically induced DHL cell death, as evidenced by the inhibition of cell proliferation, the induct of cell cycle arrest, and the promotion of cell apoptosis via the mitochondrial pathway. Orelabrutinib treatment alters genome-wide gene expression in DHL cells. The combined regimen decreases the expression of BTK and Mcl-1, potentially interfering with the activity and crosstalk of PI3K/AKT signaling and p38/MAPK signaling. In addition, the combination of orelabrutinib and venetoclax shows cytotoxic activity in primary B-lymphoma cells. In summary, these findings reveal a novel therapy targeting BCR signaling and the Bcl-2 family for DHL patients with a poor prognosis.

C-MYC Targeting By Degradation: Novel Dual c-Myc/GSPT1 Degrader GT19715 Induces TP53-Independent Cell Death in Acute Myeloid Leukemia and Lymphomas

C-MYC Targeting By Degradation: Novel Dual c-Myc/GSPT1 Degrader GT19715 Induces TP53-Independent Cell Death in Acute Myeloid Leukemia and Lymphomas

Antitumor Activity of Simvastatin in Preclinical Models of Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive subtype of B-cell non-Hodgkin lymphoma that remains incurable with standard therapy. Statins are well-tolerated, inexpensive, and widely prescribed as cholesterol-lowering agents to treat hyperlipidemia and to prevent cardiovascular diseases through the blockage of the mevalonate metabolic pathway. These drugs have also shown promising anti-cancer activity through pleiotropic effects including the induction of lymphoma cell death. However, their potential use as anti-MCL agents has not been evaluated so far. The present study aimed to investigate the activity of simvastatin on MCL cells. We evaluated the cytotoxicity of simvastatin in MCL cell lines by CellTiter-Glo and lactate dehydrogenase (LDH) release assays. Cell proliferation and mitotic index were assessed by direct cell recounting and histone H3-pSer10 immunostaining. Apoptosis induction and reactive oxygen species (ROS) generation were evaluated by flow cytometry. Cell migration and invasion properties were determined by transwell assay. The antitumoral effect of simvastatin in vivo was evaluated in a chick embryo chorioallantoic membrane (CAM) MCL xenograft model. We show that treatment with simvastatin induced a 2 to 6-fold LDH release, inhibited more than 50% of cell proliferation, and enhanced the caspase-independent ROS-mediated death of MCL cells. The effective impairment of MCL cell survival was accompanied by the inhibition of AKT and mTOR phosphorylation. Moreover, simvastatin strongly decreased MCL cell migration and invasion ability, leading to a 55% tumor growth inhibition and a consistent diminution of bone marrow and spleen metastasis in vivo. Altogether, these data provide the first preclinical insight into the effect of simvastatin against MCL cells, suggesting that this agent might be considered for repurpose as a precise MCL therapy.

Ironomycin Induces Mantle Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction

Mantle-cell lymphoma (MCL) accounts for approximately 5 to 7% of all lymphomas, and in North America and Europe. MCL remains an aggressive and incurable cancer with existing therapies, presenting a significant unmet clinical need. Accumulating evidence reveals that abnormal iron metabolism plays an important role in tumorigenesis and in cancer progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify alterations of iron homeostasis in MCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of a cohort of patients with MCL (n=71) established the iron score (IS), a gene expression-based risk score enabling identification of patients with MCL with a poor outcome who might benefit from a suitable targeted therapy. We analyzed the therapeutic interest of ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Preclinical studies in Acute Myeloid Leukemia (Garciaz et al., 2022 Cancer Discovery) and Diffuse Large B-Cell Lymphoma (Devin et al., 2022 Can Res) have revealed the therapeutic interest of ironomycin in treating hematological malignancies. In a panel of 6 MCL cell lines, ironomycin inhibited MCL cell growth at nanomolar concentrations compared with typical iron chelators. Then, we investigated if ironomycin induces cell death through apoptosis. Flow cytometry analysis revealed that ironomycin treatment resulted in AnnV+/7AAD+ and AnnV+/7AAD- death profiles in MCL. This effect was partially inhibited by the pan-caspase inhibitor Q-VD-Oph. Ironomycin also induced caspases 8 and 9 cleavage, and the activation of caspases 3/7. Furthermore, we found that ironomycin induces ferroptosis with lipid peroxidation and the production of ROS monitored by the fluorescence probe CM-H2DCFDA. Ironomycin induced cell death is inhibited by the ferroptosis inhibitor, ferrostatin-1. The treatment did not induce ferritinophagy, as there was no significant change on the expression of LC3B-II, ferritin and the transferrin receptor (CD71).Taken together, these data indicate that ironomycin kills MCL cells by apoptosis mediated by caspase activation and ferroptosis. Ironomycin significantly decreased cell proliferation rate, with a reduced percentage of cells in S-phase together with Ki67 and Cyclin D1 downregulation. Western blot analysis showed an increase of Chk1 and Chk2 phosphorylation, two central factors of DNA damage response, and an increase of the phosphorylation of γ-H2AX, indicative of DNA damage. With major importance, we validated the therapeutic interest of ironomycin in primary MCL cells of patients (n=5). Ironomycin (20 nM) demonstrated a significant higher toxicity in MCL cells compared to normal cells from the microenvironment. Furthermore, ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment. We tested the therapeutic interest to combine ironomycin with conventional chemotherapy used in MCL. Interestingly, we identified a synergistic effect when ironomycin is combined with Ibrutinib BTK inhibitor. Furthermore, Significant synergistic effects were also observed by combining ironomycin with BH3 mimetics or Doxorubicin. Altogether, these data underline that MCL patients my benefit from targeting iron homeostasis using ironomycin alone or in combination with conventional MCL treatments.

Investigations of Redox Resistance in Aggressive Lymphomas with Cholesterol Modulating Lipid Nanoparticles

Introduction: High grade B cell lymphoma (HGBCL), characterized by C-MYC and BCL-2 rearrangements, are particularly aggressive with poor response to standard therapy and decreased overall survival (OS). We previously demonstrated that B-cell lymphomas, such as germinal center (GC) diffuse large B-cell lymphomas (DLBCL), are addicted to cholesterol uptake and that bioinspired gold core lipid nanoparticles (Au-LNPs) antagonize lipid uptake through the cell surface receptor called scavenger receptor type B-1 (SR-B1) and lead to cholesterol depletion in several lymphoma cell lines (Rink et al JBC 2020). Au-LNP treatment leads to lymphoma cell death by ferroptosis, through reduction of glutathione peroxidase (GPX4) and subsequent accumulation of oxidized lipids. The antioxidant Nrf2 pathway is known to resist oxidative stress and high levels of Nrf2 proteins predict poor response to chemotherapy in hematologic malignancies. We therefore hypothesized that 1) HGBCLs would be more resistant to Au-LNP and 2) antioxidant pathways might mediate that resistance. Methods: The GC DLBCL cell line (SUDHL-4) cells and HGBCL cell lines (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were used. Cells were treated with PBS (control) or Au-LNPs for 72h. Au-LNPs were synthesized as previously described (Rink JBC 2022). Cell viability was measured by MTS assay. Total RNA in RC and SUDHL-4 cells were extracted and used to construct cDNA libraries for RNA sequencing (RNAseq) by HiSeq 4000 with single-end 50bp reads and 20-25 million reads per sample in the NUSeq Core of Northwestern University. Metascape was used for functional analysis of identified differentially expressed genes (DEGs) and DEGs related to Nrf2 survival signaling, NQO1, FTH1, FTL, G6PD, TXNRD, NFE2L2, TXN, P4HB, and MGAT3 were confirmed in control and Au-LNP treated SUDHL-4, RC, OCI-ly1, OCI-ly8 and OCI-ly18 cells by RT-qPCR and Western blot analysis. Results: HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) were more resistant to treatment with Au-LNPs (IC50 = 31.42 nM, 27.22 nM, 58.24 nM, 26.06 nM, respectively) compared with SUDHL-4 (IC50 8.14 nM). RNAseq analysis identified 320 upregulated and 162 downregulated genes in SUDHL-4 cells and 346 upregulated and 182 downregulated genes in RC cells after treatment with Au-LNPs. Of these DEGs, 62 upregulated and 8 downregulated DEGs appeared in both cell lines after treatment. The top 5 upregulated common genes include ABHD4, DUSP5, JUN, RGS1 and SQSTM1. The top 5 downregulated common genes include ABCG1, DDN, GPX1, GPX4 and IKZF1. There were 284 upregulated and 174 downregulated DEGs only identified in Au-LNP treated RC cells. Among 284 upregulated DEGs the top 2 enriched clusters were Nrf2 survival signaling and nuclear receptors meta-pathways. Au-LNP treatment in RC cells showed log2 fold increase of NQO1 (4.6), FTH1 (1.28), FTL (2.1), and TXNRD1 (1.78), genes in the Nrf2 survival signaling pathway. In SUDHL-4 cells, only NQO1 and FTL1 showed significant differential expression with log2 fold increase of 0.7 and 0.45 after Au-LNP treatment. To confirm RNAseq results, we measured the expression of Nrf2 survival signaling genes, NQO1, FTH1, FTL, G6PD, TXNRD, NFE2L2, TXN, P4HB, and MGAT3 with or without Au-LNP in SUDHL4, RC, OCI-ly1, OCI-ly8, and OCI-ly18 cells by RT-qPCR. In SUDHL4 cells, compared with PBS control, Au-LNP treatment led to mild increase in expression of NQO1 (4.2 ± 0.6 fold). By contrast, in HGBCL cells, Au-LNPs increased NQO1 expression 19.9 ± 0.2 fold for RC cells, 9.0 ± 0.3 fold for OCI-ly1 cells and 36.5 ± 2.3 fold for OCI-ly8 cells compared with PBS control. Higher NQO1 and FTH1 protein expression in treated RC cells was also confirmed by western blot analysis. These data suggest that 1) HGBCL cell lines RC, OCI-ly1, OCI-ly8 and OCI-ly18 are more resistant to cell death by cholesterol depletion and that 2) this phenomenon is mediated by upregulation of genes in the Nrf2 pathway and NQO1, in particular. Conclusion: The redox related Nrf2 survival signaling pathway, especially the NQO1 gene, is upregulated after treatment with Au-LNPs. This was observed to a greater extent in the more resistant HGBCL cells (RC, OCI-ly1, OCI-ly8 and OCI-ly18) than the SUDHL4 cells. Strategies to target the Nrf2 pathway may reverse resistance to cholesterol depletion in lymphoma.

Consecutive Inhibition of Telomerase and Alternative Lengthening Pathway Promotes Hodgkin's Lymphoma Cell Death.

Telomere maintenance is key during cancer development. Malignant cells can either use telomerase or an alternative lengthening of telomere (ALT) pathway to maintain their telomere length. In Hodgkin’s Lymphoma (HL), the presence of telomerase activation is established. The activation of ALT has been reported recently. Our data confirm this notion describing co-localization of the phosphorylated form of telomeric repeat-binding factor 1 (pT371-TRF1) with ALT-associated promyelocytic leukemia bodies. Surprisingly, to our knowledge, there are no published studies targeting both telomere maintenance pathways in HL. Consequently, we investigated, for the first time, the effects of both telomerase and ALT inhibition on HL cell viability: We inhibited telomerase and/or ALT, given either individually, simultaneously, or consecutively. We report that the inhibition of telomerase using BIBR1532 followed by ALT inhibition, using trabectedin, caused a decrease of greater than 90% in cell viability in three patient-derived HL cell lines. Our results suggest that HL cells are most vulnerable to the consecutive inhibition of telomerase followed by ALT inhibition.

Constitutive Activation of p62/Sequestosome-1-Mediated Proteaphagy Regulates Proteolysis and Impairs Cell Death in Bortezomib-Resistant Mantle Cell Lymphoma.

Simple SummaryTo decipher the molecular mechanism underlying the resistance of a significant fraction of mantle cell lymphoma (MCL) patients to the first-in-class proteasome inhibitor bortezomib (BTZ), we have characterized the ubiquitin-related proteome (i.e., ubiquitome) of a set of MCL cell lines with different degrees of sensitivity to the drug by coupling a tandem ubiquitin-binding entity (TUBE) approach to mass spectrometry, followed by phenotypic and functional validations in both in vitro and in vivo models of MCL. We identified an enrichment of autophagy–lysosome system (ALS) components in BTZ-resistant cells, which was associated with constitutive intracellular inactivation of proteasome subunits by a process called proteaphagy. Blockade of this phenomenon by the pharmacological or genetic inactivation of the autophagy receptor p62/SQSTM1 reactivated normal proteasomal activity and restored the BTZ antitumor effect in in vitro and in vivo models of BTZ resistance.Protein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy–lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin–proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors.

Zinc Supplementation Enhances the Pro-Death Function of UPR in Lymphoma Cells Exposed to Radiation.

Simple SummaryIt is of fundamental importance to find strategies able to reduce the minimum doses of anticancer treatments, such as radiations, and concomitantly maintain efficient killing of cancer cells. The interconnection between ER stress and DNA repair may represent a promising approach to obtaining this goal. Here we found that pretreatment of lymphoma cells with Zinc chloride rendered these cells more sensitive to 2 Gy radiation. The exacerbation of ER stress and the activation pro-death function of UPR were among the underlying mechanisms leading to higher cytotoxicity of Zinc/radiation combination treatment. This evidence encourages the use of Zinc to reduce the doses of radiation in the treatment of lymphoma cells, allowing a high cytotoxicity to be obtained while minimizing the side effects.We have previously shown that Zinc supplementation triggered ER stress/UPR in cancer cells undergoing treatment by genotoxic agents, reactivated wtp53 in cancer cells harboring mutant p53 (mutp53) and potentiated the activity of wtp53 in those carrying wtp53. In this study, we used Zinc chloride alone or in combination with 2 Gy radiation to treat Primary Effusion Lymphoma (PEL) cells, an aggressive B-cell lymphoma associated with KSHV that harbors wt or partially functioning p53. We found that Zinc triggered a mild ER stress/UPR in these lymphoma cells and activated ERK1/2, molecule known to sustain cell survival in the course of UPR activation. In combination with radiations, Zinc triggered a stronger p53 activation that counteracted its mediated ERK1/2 phosphorylation, further upregulating the UPR molecule CHOP and promoting cell death. These data suggest that Zinc supplementation could be a promising strategy to reduce the doses of radiation and possibly of other DNA-damaging agents to obtain an efficient capacity to induce lymphoma cell death.

In Situ Vaccination with Combination of Class B and Class C Toll-like Receptor 9 Agonist CpG Immune Adjuvant Nanoparticles to Induce a Systemic Anti-Lymphoma Response

Introduction: Treatment of relapsed and refractory aggressive B cell lymphomas is challenging, especially after autologous stem cell transplant or chimeric antigen receptor T cell therapy. CpG oligodeoxynucleotides (CpGs) mimic bacterial DNA and bind to toll-like receptor 9 (TLR9). TLR9 is expressed in the endosomes of innate immune cells and B lymphocytes as well as B cell lymphomas. CpG binding to TLR9 in these cells leads to a pro-inflammatory response and induces apoptosis of lymphoma cells by altering NF-kB activation. However, clinical utility of CpGs is limited due to difficulties delivering the DNA directly to the innate immune cells and lymphoma cells, and these DNA strands are easily degraded in biological fluids. Therefore, we designed a nano-carrier to deliver two classes of CpGs to improve clinical efficacy of CpGs in treating lymphoma. Class B CpGs (B-CpGs) mainly stimulate B cells while class C CpGs (C-CpGs) act on both B cells and plasmacytoid dendritic cells. Though C-CpGs are the focus of current clinical trials, B-CpGs may have a special a role in the treatment of lymphoma due to their more potent, direct cytotoxic effect on the malignant lymphoma cells. Here, we evaluated the anti-lymphoma effect of the combination of B-CpG nanoparticles (BNP) and C-CpG nanoparticles (CNP) by optimizing lymphoma cell death and immune stimulation.Methods: BNP and CNP were synthesized by surface-functionalizing gold nanoparticles with modified B-CpGs and C-CpGs, followed by purification via centrifugation. We used a dual flank tumor murine lymphoma model to evaluate the local and systemic anti-lymphoma efficacy of the CpG NPs and screen for the most effective formulation. A20 lymphoma tumors were implanted on both flanks of Balb/c mice. When the tumors were 5-7mm in diameter, various treatments were injected intratumorally on days 1, 4, and 8 on the side with the larger tumor. Treatment groups include PBS, B-CpG, C-CpG, B+C-CpG, BNP, CNP, BNP+CNP. Free CpGs were injected at 50ug/ml in 50ul (2.5ug CpG per injection) and nanoparticles at 100nM (equivalent to 50ug/ml CpGs) in 50ul. Tumor growth were measured every other day. Event is defined as tumor volume > 3cm 3. Second, we used an advanced disease lymphoma model to evaluate intravenous delivery of combination NPs. We implanted A20 lymphoma cells on a flank tumor (on day -14), then we injected A20 cells intravenously to mimic advanced disease (on day -7). We treated the mice with intravenous (tail vein) injections of PBS, B+C-CpG, or BNP+CNP on days 1, 4, and 8.Results: We found that the combination of BNP and CNPs had the most significant reduction in total tumor volume (TTV) (treated + untreated tumor size) (393mm 3 on day 22). The PBS group demonstrated rapid tumor growth (TTV 3516mm 3). The experimental treatment groups, B-CpG (TTV 949mm 3), C-CpG (TTV 2230mm 3), B+C-CpGs (TTV 1680mm 3), BNP (TTV 1224mm 3), and CNP (1894mm 3) had similar TTV growth curves. Separating the treated tumor and untreated tumor growth curves, we found that B-CpG and BNP along with combination of BNP+CNP had the best tumor suppression on the treated side. This is not surprising as B-CpG sequences are more directly cytotoxic to lymphoma cells versus C-CpGs. Interestingly, combination nanoparticle treatment (BNP+CNP) was the only group that had significantly reduced tumor growth of the untreated tumor compared with all other conditions (p<0.03) where no statistical difference was measured when the PBS treatment group with the other groups. The BNP+CNP treatment group also had significantly prolonged event free survival compared with all other treatment groups. Furthermore, in the advanced disease model, when BNP and CNP were both injected intravenously, the flank tumor was notably smaller (149mm 3) compared with the other two groups (B+C-CpG 1310mm 3 or PBS 1555mm 3). Importantly, there were no visible lymphoma nodules on liver in the nanoparticle treatment group while there are obvious nodules for both the PBS and the free CpG groups.Conclusions: Overall, the combination of class B and class C CpG nanoparticles can generate a strong in situ anti-lymphoma vaccination response, presumably through the combination of by combining the superior cytotoxicity of BNPs with the enhanced immune stimulation effect of the CNPs. Further evaluation of the combined mechanism of action of this dual CpG nanoparticle approach and toxicity studies are in progress. DisclosuresThaxton: Zylem biosciences: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Gordon: Zylem Biosciences: Patents & Royalties: Patents, No royalties; Bristol Myers Squibb: Honoraria, Research Funding.

Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells.

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination.

Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Simple SummaryDiffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma with a high curability rate. However, 40% of patients will relapse or exhibit refractory disease, and compromised apoptotic pathways is among the prognosis-worsening factors. Therefore, drugging non-apoptotic modalities might be therapeutically promising. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells. Herein, we show that TQ selectively kills DLBCL cells, either cell lines or primary lymphoma cells bearing resistance features to standard treatment. Investigations show that, although TQ induced apoptotic markers, non-apoptotic death was the major mechanism responsible for TQ-induced cellular demise. We demonstrate critical and selective roles of cytosolic calcium and necroptosis in TQ-induced non-apoptotic cell death. Finally, TQ exhibits an improved selectivity profile over conventional chemotherapy. Collectively, this work provides new insights into the mode of action of TQ and points to the therapeutic relevance of non-apoptotic modalities as a fail-safe mechanism for pro-apoptotic DLBCL therapies.Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Sustained Reduction of Intracellular Calcium Is Important to Maximise GA101-Mediated Direct Cell Death in B-Cell Lymphomas

For over a decade, anti-CD20 monoclonal antibodies (mAbs) have successfully been used in the therapy of B-cell malignancies. Obinutuzumab (GA101), a type-II glycoengineered anti-CD20 mAb approved for treatment of Chronic Lymphocytic Leukaemia (CLL) and Follicular Lymphoma, was designed to have enhanced antibody-dependant cellular cytotoxicity compared with Rituximab. Pre-clinical data showed that in addition, GA101 also exhibited significantly enhanced direct cell death. The complete mechanism of increased direct cell death is still unknown.The biological role of CD20 remains unclear but it has been shown to act as part of an ion channel complex, a component of the store operated calcium (Ca2+) entry system. Numerous studies have investigated the immediate impact of anti-CD20 mAbs on intracellular Ca2+. Yet to date, none have assessed intracellular Ca2+ over a longer period.To investigate the effect of anti-CD20 mAbs on intracellular Ca2+, we initially used 4 B-cell lymphoma cell lines to assess Ca2+ flux immediately after treatment with either Rituximab or GA101. Cells were first stained with a fluorescent calcium indicator and baseline measurements recorded by flow cytometry. Cells were then treated with each antibody and fluorescence measurements were recorded for a further 5-10 minutes. Next, we conducted a time course experiment to visualise the kinetics of intracellular Ca2+ after 1, 2, 4, 8, and 24 hour treatments with either Rituximab or GA101; Trastuzumab was used as an isotype control. After treatment, samples were stained with a labelled calcium indicator before quantifying intracellular Ca2+ using imaging flow cytometry. We validated our findings in primary CLL samples using the same method. We conducted liquid chromatography tandem mass spectrometry based label-free phosphoproteomics on SUDHL4 cells in biological triplicates after 1 and 24 hour treatments with GA101 and Rituximab. Expression levels of >8500 phosphorylation sites were measured across treatments to establish changes in intracellular signalling and kinase activity.In line with published data, we observed an influx of Ca2+ when cells were treated with Rituximab but intracellular Ca2+ was not significantly altered for any of the extended treatments. In contrast, GA101 treatment did not induce an immediate influx of Ca2+,and a reduction in intracellular Ca2+ was observed after 1 hour treatment which was sustained across all extended treatment periods. This difference in kinetics was validated in primary CLL samples. Global phosphoproteomic analysis found that the activity of Polo-Like Kinase 2 (PLK2) was significantly increased following treatment with GA101. The increase in the activity of this kinase was specific to GA101 and was unchanged following treatment with Rituximab. Simultaneously there was a significant increase in the phosphorylation of Calumenin at Ser44, a known binding site of PLK2. Samples treated with GA101 for 24 hours exhibited a larger downregulation of pathways associated with calcium signalling compared to Rituximab-treated and control samples.Calumenin, a member of the CREC family, is able to bind 7 Ca2+ ions. It can be localised in the cytosol, in compartments of the secretory pathway, on the plasma membrane or in the extracellular space. We hypothesise that once phosphorylated, this protein bound with Ca2+ is secreted extracellularly, resulting in reduced intracellular Ca2+.In summary we report for the first time that intracellular Ca2+ is significantly decreased as a result of GA101 treatment in cell lines and primary samples. Using a global unbiased approach we have identified a significant increase in PLK2 activity and we observed a large increase in phosphorylation of Calumenin. Our data suggests that these proteins may contribute to the increased direct cell death observed as a result of GA101 treatment. We believe that expression and activity of these proteins are crucial in order to maximise GA101-mediated direct cell death. Ongoing studies are examining the functional roles of PLK2 and Calumenin in the reduction of intracellular Ca2+, comparing Ca2+ levels in response to antibody treatment in primary samples from different CLL subtypes, and assessing and comparing levels of apoptosis following antibody treatment to see whether there is a correlation with reduced intracellular Ca2+. DisclosuresGribben:Celgene: Honoraria; TG Therapeutics: Honoraria; Genentech/Roche: Honoraria; Karyopharm: Honoraria; Kite: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Janssen: Honoraria; Acerta: Honoraria.

Pre-Clinical Development of Targeted Therapies for Double Hit (DH) Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Gene expression profile classifies DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal B-cell lymphoma (PMBL). DH-DLBCL is a distinct subgroup, with a poor clinical outcome, harboring concurrent gene translocations involving the C-MYC and either/both BCL2 or BCL6 proto-oncogenes. DH-DLBCL has inferior response rates to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). In addition, pts with DH-DLBCL who are refractory to frontline chemoimmunotherapy fare dismally; a retrospective review at our institute yielded survival rates of zero and median OS of 6 months in these patients, highlighting the need for novel therapies. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, leading to rapid proliferation of neoplastic cells, and resistance to apoptotic stimuli. Upregulation of the anti-apoptotic protein Mcl-1 is implicated in chemotherapy resistance and poor clinical outcomes in ABC-DLBCL. Transcriptional regulation of MCL1 by c-MYC might further potentiate Mcl-1 induced resistance to chemotherapy in DH-DLBCL. Specific inhibitors targeting cell pro-survival proteins might provide a novel and alternative therapeutic approach in rituximab-chemotherapy resistant DH-DLBCL. We hypothesize that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 using readily available agents (Venetoclax and Proteasome inhibitors) is an effective strategy in inducing lymphoma cell death. Materials and Methods: We conducted pre-clinical in vitro and in vivo models targeting Bcl-2 with Venetoclax and Mcl-1 with either a c-Myc inhibitor (JQ-1) or Proteasome inhibitor (Carfilzomib) in DHL. Val, DOHH-2, and ROS-50 cells were exposed to Venetoclax (0-10 uM), JQ-1 (0-100 uM) and Carfilzomib (CFZ) (0-50 nM) for 24, 48 and 72 hours. IC50 drug concentrations were calculated for each agent. Subsequently, DHL cells were exposed to Venetoclax in combination with JQ-1 or CFZ for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) and changes in Bcl-2 family members and Caspase activation were determined by Western blotting. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including DHL were exposed ex vivo to Venetoclax +/- Carfilzomib. For in vivo experiments 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 ROS-50 cells via tail vein injection (IV). Subsequently, animals were treated with Venetoclax (100mg/kg/dose via gastric lavage on days 3-7 and 10-14); Carfilzomib (2mg/kg/dose IV days 3, 4, 10, 11), or both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro, Venetoclax, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both Venetoclax and CFZ and to a lesser degree with JQ-1. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Annexin/PI staining with the combination of Venetoclax and CFZ resulted in caspase dependent apoptosis. Western blotting confirmed changes in Bcl-2 family members favoring an apoptotic state as well as PARP cleavage following in vitro exposure to Venetoclax and CFZ. In vivo the combination of Venetoclax with Carfilzomib resulted in significant increased survival increase (P<0.05) when compared to single agent Venetoclax or CFZ. Conclusion: Our data suggests that Venetoclax exhibits strong synergistic activity with Carfilzomib in vitro and in vivo . Perhaps related to the synergy observed between Venetoclax and CFZ, is the effects of proteasome inhibition on Mcl-1 levels. Combination therapy targeting the proteasome inhibition and BCL-2 may provide an alternative therapeutic approach in DH-DLBCL. DisclosuresNo relevant conflicts of interest to declare.