Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Alterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)–like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243–induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.

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.

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.

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.