Abstract: Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency. Experimental Design: We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition. Results: Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice. Conclusions: Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.

Abstract: Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Abstract: Therapeutic resistance and intolerance of Bruton tyrosine kinase (BTK) inhibitors is an emerging need in CLL. SYK is integral to the activation of BTK and the B-cell receptor (BCR) signaling cascade and is overexpressed in CLL. We have shown that BAFF-mediated SYK activation triggered BCR signaling and rendered protection of CLL cells from spontaneous apoptosis in vitro. Single agent small molecule SYK inhibitor entospletinib was efficacious in treatment of patients with R/R CLL. Here we report final results of a single arm, open label, investigator-initiated phase 1/2 clinical trial which evaluated safety and efficacy of entospletinib in combination with obinutuzumab, a glycoengineered monoclonal anti-CD20 antibody, in patients with R/R CLL (NCT03010358). Patients enrolled in the Phase 1 dose-escalation portion of the trial included adults with CLL or non-Hodgkin lymphoma (Phase 1 part only) after ≥1 prior therapy. Patients were enrolled at 2 dose levels to receive entospletinib 200 or 400 mg twice-daily orally according to 3+3 design. The primary endpoint for the phase 1 portion of the study was to determine the RP2D of the combination. All patients received single agent entospletinib as part of a 7-day run-in. Thereafter, patients received entospletinib on days 1-28 of each 28-day cycle continuously, and obinutuzumab intravenously in standard doses for 6 cycles. Once the RP2D was determined, a phase 2 study enrolled patients with R/R CLL only, where complete response (CR) was the primary endpoint. A total of 24 patients (22 CLL, 2 follicular lymphoma) were enrolled. Twelve patients were enrolled in the phase 1 part of the study. The phase 2 part of the study included 17 patients with CLL. Of 6 patients who received entospletinib 200 mg on the Phase 1 part of the study, one patient experienced a DLT (grade 3 asymptomatic AST/ALT abnormalities) attributed to entospletinib. No DLTs were observed among the six patients who received entospletinib 400 mg. Thus, entospletinib 400 mg twice-daily was determined to be the RP2D in combination with obinutuzumab. Efficacy of entospletinib+obinutuzumab was analyzed in the 21 patients with CLL, of which 17 received entospletinib at RP2D (400 mg twice daily). Patients with CLL had a median age of 66 years. Thirteen patients (62%) had TP53 aberration (n=9), complex karyotype (n=6), or NOTCH1 or SF3B1 mutation. The median number of prior therapies was two (range, 1-6). Seven patients had received prior ibrutinib (4 patients discontinued due to intolerance and 2 due to progression). Median follow-up was 31 months. Among the 21 efficacy-evaluable participants with CLL, the ORR was 67% (95%CI, 43-85%). Three patients (14%, 95%CI 3-36%) achieved a CR, and 11 patients (53%) had a partial response (PR). patients with confirmed CR had undetectable MRD in the bone marrow. Median event-free survival was 27.5 months (95%CI: 16 months-NR), treatment duration - 31 months (95%CI: 27-40; Figure). Thirteen patients with high-risk CLL had an ORR of 54% (5 PRs and 2 CRs). Among the eight patients who had previously received kinase inhibitors, ORR was 62.5% (all PRs). Treatment-related adverse events were reported in 96% of patients (Table). Grade 3 or higher AEs occurred in 65%. Neutropenia (43.5%; including 4 patients [17%] who had transient grade 4 neutropenia attributed to obinutuzumab) was the most common grade ≥3 hematologic toxicity. The median onset of neutropenia was 7 days after the first obinutuzumab infusion, median duration was 28 days. Growth factor support was not required and grade ≥3 infection occurred in only 1 patient. Only one patient on study discontinued therapy due to adverse events (recurrent AST/ALT abnormalities which resolved upon cessation of entospletinib). Pharmacodynamic analysis demonstrated that treatment with entospletinib led to rapid downmodulation of pSTAT3 and the anti-apoptotic protein MCL1 in CLL cells. Furthermore, six months of combination therapy was accompanied by a reduction in IFNγ secretion in CD4 + T-cells and a reversal of exhausted phenotype, as evidenced by downregulation of PD-1. Thus, the combination of entospletinib and obinutuzumab shows an acceptable safety profile. Efficacy of this combination (EFS 27.5 months in predominantly high-risk population ) compares favorably with chlorambucil/obinutuzumab in R/R CLL (13 months), thus warranting continued exploration of the regimen. Figure 1 Figure 1. Disclosures Danilov: Genentech: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding. Spurgeon: Bristol Myers Squibb: Other: Institution: Research Grant/Funding; BeiGene: Other: Institution: Research Grant/Funding; AstraZeneca: Other: Institution: Research Grant/Funding; Acerta Pharma: Other: Institution: Research Grant/Funding; Pharmacyclics: Consultancy; Janssen: Consultancy, Other: Institution: Research Grant/Funding; Genentech: Consultancy, Other: Institution: Research Grant/Funding; Karyopharm: Consultancy; Velos Bio: Consultancy, Other: Institution: Research Grant/Funding; Gilead Sciences: Other: Institution: Research Grant/Funding; Ionis: Other: Institution: Research Grant/Funding; Merck & Co., Inc.: Other: Institution: Research Grant/Funding; Fred Hutchinson Cancer Research Center: Other: Data Safety Monitoring Board. Kittai: Abbvie: Consultancy; Bristol-Meyers Squibb: Consultancy; Janssen: Consultancy.

Abstract: Deregulation of the MYC transcription factor is a key driver in lymphomagenesis. MYC induces global changes in gene expression that contribute to cell growth, proliferation, and oncogenesis by stimulating the activity of RNA polymerases. A key feature in its ability to stimulate RNA Pol II activity is recruitment of pTEFb, an elongation factor whose catalytic core comprises CDK9/cyclin T complexes. Hence, MYC expression and function may be susceptible to CDK9 inhibition. We conducted a pre-clinical assessment of AZ5576, a selective CDK9 inhibitor, in diffuse large B-cell lymphoma (DLBCL). The in vitro and in vivo effects of AZ5576 on apoptosis, cell cycle, Mcl-1, and MYC expression were assessed by flow cytometry, immunoblotting, qPCR and RNA-Seq. We demonstrate that, in addition to depleting Mcl-1, targeting CDK9 disrupts MYC oncogenic function. Treatment with AZ5576 inhibited growth of DLBCL cell lines in vitro and in vivo, independent of cell-of-origin. CDK9 inhibition downregulated Mcl-1 and MYC mRNA transcript and protein in a dose-dependent manner. MYC-expressing cell lines demonstrated enhanced susceptibility to AZ5576. CDK9 inhibition promoted turnover of MYC protein, and decreased MYC phosphorylation at the stabilizing Ser62 residue and downregulated MYC transcriptional targets in DLBCL cells, a finding confirmed in a functional reporter assay, suggesting that CDK9 may govern MYC protein turnover, thus regulating its expression through multiple mechanisms. Our data suggest that targeting CDK9 is poised to disrupt MYC oncogenic activity in DLBCL and provide rationale for clinical development of selective CDK9 inhibitors.

Abstract: Novel targeted agents used in therapy of lymphoid malignancies are recognized to have complex immune-mediated effects. Sumoylation, a posttranslational modification of target proteins by small ubiquitin-like modifiers (SUMO), regulates a variety of cellular processes indispensable in immune cell activation. Despite this, the role of sumoylation in T-cell biology in context of cancer is not known. TAK-981 (subasumstat) is a small-molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein. Using T cells derived from patients with chronic lymphocytic leukemia (CLL), we demonstrate that targeting SAE activates type I IFN response. This is accompanied by largely intact T-cell activation in response to T-cell receptor engagement, with increased expression of CD69 and CD38. Furthermore, TAK-981 decreases regulatory T cell (Treg) differentiation and enhances secretion of IFNγ by CD4+ and CD8+ T cells. These findings were recapitulated in mouse models, suggesting an evolutionarily conserved mechanism of T-cell activation regulated by SUMO modification. Relevant to the consideration of TAK-981 as an effective agent for immunotherapy in hematologic malignancies, we demonstrate that the downstream impact of TAK-981 administration is enhancement of the cytotoxic function of CD8+ T cells, thus uncovering immune implications of targeting sumoylation in lymphoid neoplasia.

Abstract: Introduction: Immune checkpoint inhibitors have limited single agent activity in B-cell non-Hodgkin lymphoma (NHL). Hence, it is important to develop strategies which will thwart immune evasion in this disease. Neddylation is a sequential enzyme-based process which ultimately regulates protein turnover. In the initial step, NAE activates NEDD8 in an ATP-dependent reaction in which a high-energy thioester bond is formed between NEDD8 and the catalytic cysteine of NAE. Active NEDD8 is then transferred to the NEDD8-specific E2 conjugating enzyme (UBE2M) and is conjugated to cullin proteins which are part of the Cullin-RING E3 ubiquitin ligases (CRL). Pevonedistat (pevo) forms a covalent adduct with NEDD8, thereby inhibiting NAE and thus reduces CRL activity and diminishes ubiquitination and proteasomal degradation of CRL substrates (IκB, HIF-1α, etc). We have recently reported that neddylation regulates T cell activation and polarization (Best et al, Leukemia 2021). Here we investigate how pharmacologic targeting of neddylation modulates anti-tumor immunity using NHL models. Methods: Peripheral blood mononuclear cells were isolated from patients with NHL and T cells were purified using Dynabeads. A20 cells were transplanted into flanks of syngeneic BALB/c mice. When tumors reached 100 mm 3, mice were randomized into groups and treated with pevo 60 mg/kg subcutaneously daily for 10 days or vehicle control. Once moribund, mice were sacrificed, tumors were processed into single-cell suspension and analyzed by flow cytometry. Pevo was provided by Takeda Development Center Americas Inc. (Cambridge, MA). Results: Primary patient-derived CD3/28-stimulated CD3 + T cells exhibited upregulation of TNFα and IFNγ in vitro in the presence of pevo. Concurrently, we observed increased expression of PD-1 and CTLA-4. Pre-treatment of T cells with pevo enhanced killing of NHL cell lines (JeKo-1, Mino, Maver-1 and VAL) in allogeneic cytotoxicity assays. Expectedly, treatment with pevo resulted in increased expression of HIF-1α in TCR-stimulated T cells. shRNA-mediated knockdown of HIF-1α abrogated the pevo effect, suggesting that NAE inhibition modulates T cell function in HIF-1α-dependent manner. While A20 cells showed resistance in vitro, treatment with pevo delayed lymphoma progression in A20 mice in vivo (Fig 1A). This was accompanied by an increase of tumor-infiltrating lymphocytes (TILs; Fig 1B). CD8 + TILs from pevo-treated mice exhibited activated phenotype as manifested by increased secretion of IFNγ (Fig 1C). Meanwhile, expression of the exhaustion molecules CTLA-4 and PD-1 by CD4/CD8 + TILs remained unchanged. To further investigate the role of T-cell immunity in this setting, we employed 1) CD8 depletion by pre-treatment with 12.5 mg/kg anti-CD8 antibody (IV); or 2) CRISPR/Cas9-mediated knockout of β2-microglobulin (MHC class I protein) in A20 cells. Either approach led to a partial decrease of pevo efficacy in vivo compared with respective controls. To exclude tumor-intrinsic effect of NAE inhibition, we knocked down UBE2M in A20 cells. Loss of UBE2M had no effect on growth of control tumors, or pevo therapeutic effect, implying that the anti-tumor efficacy of NAEi was T cell-mediated in this model. Since pevo modulates PD-1 on human T cells, we explored its effect on PD-L1 expression. Treatment with pevo upregulated PD-L1 expression in A20 cells in a MYC-dependent manner. Hence, we explored a combination of pevo and αPD-1 blockade in A20 model. Combination treatment significantly increased the CD4 + and CD8 + TILs. A decrease in tumor growth was significantly more pronounced than with either drug alone (Fig 1A). The combination benefit was fully reversed by loss of B2M, again highlighting the importance of immune mechanism . We observed expansion of IL-2, IL-4 and IL-17-secreting CD4+ TILs following the combined treatment, compared with either drug alone. In addition, CD4+ and CD8+ TILs derived from these mice secreted high levels of IFNγ (Fig. 1C). Conclusions: NAE inhibition enhanced T cell-mediated cytotoxicity in vitro. Treatment with pevo promoted activation of TILs and restricted tumor growth in an A20 mouse lymphoma model. Pevo-treated tumors were sensitized to αPD-1 . Thus, targeting NAE enhances anti-tumor immunity. Our data provide a strong rationale for future studies of pevo in combination with immune checkpoint inhibitors in lymphoma and other tumors. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding. Berger: Takeda Development Center Americas, Inc.: Current Employment. Danilov: Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Takeda Oncology: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria.

Abstract: Neddylation is a sequential enzyme-based process which regulates the function of E3 Cullin-RING ligase (CRL) and thus degradation of substrate proteins. Here we show that CD8 + T cells are a direct target for therapeutically relevant anti-lymphoma activity of pevonedistat, a Nedd8-activating enzyme (NAE) inhibitor. Pevonedistat-treated patient-derived CD8 + T cells upregulated TNFα and IFNγ and exhibited enhanced cytotoxicity. Pevonedistat induced CD8 + T-cell inflamed microenvironment and delayed tumor progression in A20 syngeneic lymphoma model. This anti-tumor effect lessened when CD8 + T cells lost the ability to engage tumors through MHC class I interactions, achieved either through CD8 + T-cell depletion or genetic knockout of B2M . Meanwhile, loss of UBE2M in tumor did not alter efficacy of pevonedistat. Concurrent blockade of NAE and PD-1 led to enhanced tumor immune infiltration, T-cell activation and chemokine expression and synergistically restricted tumor growth. shRNA-mediated knockdown of HIF-1α, a CRL substrate, abrogated the in vitro effects of pevonedistat, suggesting that NAE inhibition modulates T-cell function in HIF-1α-dependent manner. scRNA-Seq-based clinical analyses in lymphoma patients receiving pevonedistat therapy demonstrated upregulation of interferon response signatures in immune cells. Thus, targeting NAE enhances the inflammatory T-cell state, providing rationale for checkpoint blockade-based combination therapy.