Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.

CDK (cyclin-dependent kinase) inhibitors have shown remarkable activity in CLL, where its efficacy has been linked to inhibition of the transcriptional CDKs (7 and 9) and deregulation of RNA polymerase and short-lived pro-survival proteins such as MCL1. Furthermore, ER (endoplasmic reticulum) stress has been implicated in CDK inhibition in CLL. Here we conducted a pre-clinical study of a novel orally active kinase inhibitor P1446A in CLL B-cells. P1446A inhibited CDKs at nanomolar concentrations and induced rapid apoptosis of CLL cells in vitro, irrespective of chromosomal abnormalities or IGHV mutational status. Apoptosis preceded inactivation of RNA polymerase, and was accompanied by phosphorylation of stress kinases JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Pharmacologic inhibitors of JNK/p38 MAPK conferred protection from P1446A-mediated apoptosis. Treatment with P1446A led to a dramatic induction of NOXA in a JNK-dependent manner, and sensitized CLL cells to ABT-737, a BH3-mimetic. We observed concurrent activation of apoptosis stress-inducing kinase 1 (ASK1) and its interaction with inositol-requiring enzyme 1 (IRE1) and tumor necrosis factor receptor-associated factor 2 (TRAF2) in CLL cells treated with P1446A, providing insights into upstream regulation of JNK in this setting. Consistent with previous reports on limited functionality of ER stress mechanism in CLL cells, treatment with P1446A failed to induce an extensive unfolded protein response. This study provides rationale for additional investigations of P1446A in CLL.

While death receptor ligands (Fas and TRAIL) kill chemoresistant tumor cell lines, related therapies have limited clinical efficacy as single agents. Death receptor signaling is modulated by nuclear factor-κB (NFκB), a family of transcription factors which are constitutively active in B-cell malignancies. We and others have shown that pevonedistat, an investigational inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in B-cell neoplasia. Here we demonstrate that diffuse large B-cell lymphoma, particularly activated B-cell type, and primary chronic lymphocytic leukemia cells are re-sensitized to extrinsic apoptosis by pevonedistat. Pevonedistat enhanced caspase-8 processing following death receptor ligation, and downmodulated cFLIP, a NFκB-regulated protease-deficient caspase homolog. However, treatment with pevonedistat did not modulate death-inducing signaling complex in neoplastic B-cells, suggesting that they were sensitized to death ligands through the mitochondrial pathway. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies.

DLBCL accounts for >10,000 deaths in the U.S. annually. Despite efficacy of upfront chemo-immunotherapy, up to 50% of patients die within 5 years. While inhibitors of B-cell receptor signaling transformed the therapeutic paradigm in indolent lymphomas, they show poor activity in DLBCL, which thus represents an unmet clinical need. MYC oncogene, a transcription factor which regulates cell proliferation and growth, is a key driver in lymphomagenesis. Myc overexpression is associated with chemoresistance in NHL, while "double-hit" lymphomas characterized by MYC and BCL2 rearrangement are most aggressive. Myc proteins have extraordinarily short half-life. To regulate transcription, Myc recruits pTEFb, an elongation factor whose catalytic core comprises CDK9/cyclin T complexes. Hence, Myc expression and function may be susceptible to CDK9 inhibition. Non-specific inhibitors of multiple CDKs have shown promise in B-cell malignancies, where their pro-apoptotic effect has been attributed to a reduction in transcription and downmodulation of short lived pro-survival proteins, e.g. MCL1. However, such inhibitors lack a therapeutic window or a refined mechanism of action. Here we selectively targeted CDK9 in a pre-clinical study in DLBCL and demonstrate that this therapeutic approach disrupts Myc oncogenic function. Experiments were conducted in activated B-cell(ABC)-type (OCI-LY3, U2932, NuDUL-1) and germinal center(GC)-type (OCI-LY18/19, SUDHL4/6/10/16, VAL) DLBCL cell lines and in primary lymph node-derived B-cells from patients with refractory DLBCL. Tumor xenografts were established in NSG mice. We used AZ5576, a novel selective CDK9 inhibitor (Astra Zeneca), pan-CDK inhibitors (flavopiridol, dinaciclib) and genetic knockdown of CDK9. Treatment with AZ5576 promoted rapid apoptosis induction (within 2 hours) in DLBCL cell lines and primary tumor cells, as evident by PARP cleavage. Quantification of Annexin V+ cells after 24 hour drug exposure rendered an IC50 of 300-500 nM in DLBCL cell lines independent of cell of origin and 100 nM in primary DLBCL cells. Loss of RNA polymerase phosphorylation at Ser2 residue, a CDK9-dependent site, preceded apoptosis. Contrary to pan-CDK inhibitors, AZ-5576 did not inhibit CDKs 2, 4/6, and 7 (as measured by pRb and pRNApol [Ser5]), confirming its selective effect. CDK9 inhibition resulted in rapid (within 1 hour) and dose-dependent downregulation of Myc and MCL1 mRNA and protein in DLBCL cell lines and primary cells. Cell cycle profiling demonstrated that treatment with AZ-5576 led to S phase reduction, consistent with loss of Myc, while pan-CDK inhibitors downmodulated S/G2/M, reflecting inhibition of multiple CDKs. Cyclohexamide chase experiments demonstrated enhanced degradation of Myc in DLBCL cells treated with AZ-5576. Myc protein degradation is tightly regulated: while phosphorylation at Ser62 enhances Myc stability, MycT58 is primed for degradation. CDK9 inhibition decreased pMycS62 prior to apoptosis induction in DLBCL cell lines. Raji cells, which carry a stabilizing MycT58A mutation, were less sensitive to AZ-5576. This suggests that CDK9 may govern Myc protein turnover, thus regulating its expression through multiple mechanisms. Overexpression of Myc was associated with enhanced sensitivity to CDK9 inhibition. Treatment with 300 nM AZ-5576 for 24 h induced apoptosis of 35-70% of Myc-high cells (e.g., U-2932, VAL), compared with 10-20% of Myc-low cells (e.g., OCI-LY3, SU-DHL10). CDK9 inhibition did not disrupt Myc-CDK9 complex formation. Despite this, it abrogated Myc transcriptional activity in a functional reporter assay. Meanwhile, sensitivity to AZ-5576 did not correlate with expression of MCL1, BCL2 or BCLX. Our findings were confirmed in a mouse xenograft model. VAL cells were inoculated subcutaneously and treatment with AZ-5576 (30 mg/kg orally twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression (Figure), prolonged survival, decreased cell proliferation (Ki-67), enhanced apoptosis (Annexin V), and reduction in Myc total and p-S62 levels and MCL1. Thus, our data suggests that targeting CDK9 is a promising therapeutic strategy poised to disrupt Myc oncogenic activity in DLBCL and provides rationale for clinical development of AZ-5576. Figure 1 Figure 1. Disclosures Chen: Seattle Genetics: Consultancy. Drew:Astra Zeneca: Employment. Danilov:ImmunoGen: Consultancy; Pharmacyclics: Consultancy; Astra Zeneca: Research Funding; Prime Oncology: Honoraria; Dava Oncology: Honoraria; GIlead Sciences: Research Funding; Takeda: Research Funding.

Introduction: Novel therapies widely used in treatment of CLL and lymphoma, e.g. Bruton tyrosine kinase and phoshpoinotiside-3 kinase inhibitors, have complex immunomodulatory effects. Detailed understanding of the immune-modulatory effects of novel agents will help battle toxicities and inform the development of combination approaches in CLL and other lymphoid malignancies. Small ubiquitin-like modifier (SUMO) family proteins regulate target protein function by post-translational modification. Sumoylation regulates a variety of cellular processes, including nuclear import/export, transcriptional regulation, protein stability and cell cycle progression. While sumoylation has been shown to be deregulated in cancer and may contribute to carcinogenesis, it has also been implicated in T cell biology and function. Importantly, sumoylation may regulate NFκB signaling and PLCγ1-mediated NFAT activation, both indispensable for T-cell activation. Despite this, the role of sumoylation in broad aspects of T cell biology remain largely understudied. TAK-981 is a small molecule SAE inhibitor that forms an irreversible covalent adduct with SUMO molecules, thereby preventing transfer of SUMO from the E1 (SAE) to the E2 (Ubc9) enzyme, leading to a decrease in SUMO-conjugated proteins. Here, we investigated the immunomodulatory effects of TAK-981 in CLL ex vivo. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with TAK-981 for 1 hour and then subjected to concurrent T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for up to 7 days under Th1/2/17/Treg-polarizing conditions. Results: Protein sumoylation was induced within 15 minutes (SUMO1) and 24 hours (SUMO2/3) of TCR stimulation. Treatment with TAK-981 depleted nearly all polySUMO2/3-modified proteins but had less effect on SUMO1 conjugation in T cells at 24 hours. GSEA and Reactome pathway analysis of gene expression microarray data from TAK-981-treated CD4+ naïve TCR-stimulated T cells demonstrated minimal changes in NFκB- or NFAT-regulated genes. Significantly upregulated genes included those involved in transcriptional initiation/elongation (3 h), type I interferon response genes and PI3K/AKT signaling (24 h). Meanwhile, genes regulating cell cycle transition and DNA damage responses were downregulated. Activation (CD69, CD25, CD40L and HLA-DR) and survival of CD4+ andCD8+ T cell subpopulations was unimpeded by SAE inhibition within the first 24 hours of treatment, but was modestly reduced at 48 and 96 hours. T cell proliferation (CFSE, Edu) was reduced in a dose and time-dependent manner following exposure to TAK-981 (by 27.8% and 60.8% following treatment with 0.05 and 1.0 μM TAK-981 for 72 hours, respectively). Allogeneic T-cell cytotoxicity (using OCI-LY19 and OCI-LY3 lymphoma cells as target cells) was not disrupted by SAE inhibition. Remarkably, TCR-activated CD4+ naïve T cells treated with TAK-981 exhibited increased expression of CD38, a type I/II interferon response molecule [1]. Furthermore, sorted CD4+ naïve T cells showed enhanced IFNγ production, a type II IFN (as analyzed by flow cytometry), and increased TH1 differentiation in both TH1-polarized and non-polarized conditions. By contrast, differentiation of both TH17 and inducible regulatory T cells (iTregs) was reduced under the respective polarizing conditions. This was accompanied by diminished IL-2expression within the CD4+ T cell population. Conclusions: Our data suggest that targeting SAE may shift the T cell balance toward healthy immune cell subsets in CLL via induction of type I/II interferon response. TH1 polarization was accompanied by a reduction of immunosuppressive Treg phenotype. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies. Bürgler, S., et al., Chronic Lymphocytic Leukemia Cells Express CD38 in Response to Th1 Cell-Derived IFN-γ by a T-bet-Dependent Mechanism. The Journal of Immunology, 2015. 194(2): p. 827-835. Disclosures Huszar: Takeda Pharmaceuticals: Employment, Equity Ownership. Danilov:Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; Celgene: Consultancy; Aptose Biosciences: Research Funding; Seattle Genetics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Pharmacyclics: Consultancy; AstraZeneca: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Abbvie: Consultancy; Genentech: Consultancy, Research Funding; Bayer Oncology: Consultancy, Research Funding; Janssen: Consultancy; Gilead Sciences: Consultancy, Research Funding; TG Therapeutics: Consultancy; Curis: Consultancy.