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.

Two new bispecific T cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3ε binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3ε on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3 but not huNKG2D-OKT3 is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T cell cytokine production in comparison to B2-OKT3. No T cell pre-treatment was required for IFNγ production upon co-culture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T cell compartment was the primary source of IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density dependent production of IFNγ from both CD4+ and CD8+ T cells. There was two-fold more IFNγ produced per CD8+ T cell and five-fold greater percentage of CD8+ T cells producing IFNγ compared to CD4+ T cells. In addition, both BiTEs elicited significant anti-tumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust anti-tumor activity of these NKG2D ligand binding bispecific proteins and support their further development for clinical use.

Regimented activation of cyclin/CDK complexes is required for orderly cell cycle progression. Targeting this process has been an attractive therapeutic approach in neoplasia due to cell cycle deregulation and excessive proliferation. CDK inhibitors (notably flavopiridol and dinaciclib) have shown remarkable efficacy in CLL, attributed to their effect on CDK7/9 which is accompanied by a disruption of RNA polymerase-mediated transcription and subsequent deregulation of short-lived pro-survival proteins such as MCL1. Recently, endoplasmic reticulum (ER) stress has been implicated in CDK inhibition in CLL. A novel orally active kinase inhibitor P1446A (Piramal Healthcare Ltd) inhibits CDKs at nM concentrations and has demonstrated clinical activity in Phase I studies in solid tumors with minimal hematologic toxicity. Here we conducted a pre-clinical study of P1446A in CLL B-cells. Peripheral blood cells were obtained from patients with CLL and isolated using standard techniques. 83.3±3.3% of CLL cells underwent apoptosis upon 24-hour exposure to 1.5 µM P1446A, irrespective of chromosomal abnormalities [del(17p)/11q] or IGHV mutationalstatus. Apoptosis occurred as early as 4 hours after drug treatment and was accompanied by a dose-dependent inhibition of interphase CDKs (2/4/6) as evidenced by reduced phospho-Rb (T821/ S780). Apoptosis preceded reduction in RNA polymerase phosphorylation and thus occurred before transcriptional CDK7/9 were inhibited. Meanwhile, targeting CDK2 and CDK4/6 using specific inhibitors did not induce cell death in CLL. We supposed that P1446A could lead to ER stress induction in primary CLL cells, culminating in apoptosis. We noted rapid activation of JNK and p38 MAPK in CLL cells treated with P1446A in vitro. Phosphorylation of JNK and p38 MAPK occurred within 2 hours of treatment with P1446A, preceded onset of apoptosis as measured by PARP cleavage, and was followed by induction of activating transcription factors 2 and 3 (downstream targets of JNK/p38MAPK). Pharmacologic inhibitors of JNK/p38 conferred partial protection from P1446A-mediated apoptosis. We sought to identify the upstream regulators of JNK in this setting. Rapid phosphorylation of ASK1 at Thr845, a site required for its kinase activity, occurred in CLL cells treated with P1446A in a time and dose-dependent manner, concurrently with JNK activation. We observed formation of the trimeric complex between ASK1, IRE1, and TRAF2. Genetic knockdown of ASK1 led to a reduction in JNK phosphorylation and a partial rescue from apoptosis. P1446A did not induce unfolded protein response (UPR) beyond ASK1/JNK/p38 activation, consistent with previous reports on its limited functionality in CLL cells. By contrast, we found that treatment with P1446A led to a dramatic induction of NOXA mRNA and protein levels, in a JNK-dependent manner. In summary, we demonstrate that CDK inhibitor P1446A is a potent inducer of apoptosis in primary CLL cells in vitro. P1446A leads to partial activation of ER stress and the UPR in CLL cells manifested by ASK1-dependent signaling, leading to JNK/p38 MAPK activation and up-regulation of NOXA. This study provides rationale for additional investigations of P1446A in CLL. Disclosures Spurgeon: Bristol Meyers Squibb: Research Funding; Acerta Pharma: Research Funding; Gilead sciences: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria; Janssen: Research Funding. Srinivasa:Piramal Healthcare: Employment.

Introduction Natural killer (NK) cells are an important component of the innate immune system that play a key role in host immunity against cancer. NK cell recognition and activation is based on cell surface receptors recognizing specific ligands that are expressed on many types of tumor cells. Some of these receptors are capable of activating NK cell function while other receptors inhibit NK cell function. Therapeutic approaches to treat cancer have been developed based on preventing NK cell inhibition or using NK cell receptors and their ligands to activate NK cells or T cells to destroy tumor cells. Areas covered This article describes the various strategies for targeting NK cell receptors and NK cell receptor ligands using multivalent proteins to activate immunity against cancer. Expert opinion: NK cell receptors work in synergy to activate NK cell effector responses. Effective anti-cancer strategies will need to not only kill tumor cells but must also lead to the destruction of the tumor microenvironment. Immunotherapy based on NK cells and their receptors has the capacity to accomplish this through triggering lymphocyte cytotoxicity and cytokine production.

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-kappaB (NFκB) signaling in the CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that stromal-mediated NFκB pathway activity and CLL cell survival may be successfully abrogated in vitrousing MLN4924. MLN4924 is an investigational agent that inhibits the NEDD8-activating enzyme (NAE) and thereby prevents neddylation of Cullin-RING ubiquitin ligases (CRL), resulting in stabilization of their protein substrates, including inhibitor of NFκB. However, targeting NAE also slows degradation of other proteins and thus full biological consequences are tissue-dependent. In adherent solid tumor cell lines, MLN4924 induced expression of Cdt1, a DNA replication licensing factor, followed by DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B-cells has not been determined. Here we show that, under the growth conditions, targeting NAE induces DNA damage and cell cycle arrest in primary CLL B-cells. B-cells obtained from patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. CLL cells were incubated in the presence or absence of 25 ng/mL IL-21. MLN4924 was provided by Millennium Pharmaceuticals, Inc. CD40L-expressing stromal co-cultures rapidly induced anti-apoptotic BCL2 family proteins MCL1 and BCLX in peripheral blood CLL cells, leading to protection from spontaneous apoptosis and chemoresistance. Prolonged (72 h) co-cultures induced cell cycle progression in ~5% of the CLL cells. IL-21 further enhanced proliferation and sensitized CLL cells to MLN4924. Thus, treatment with 1 μM MLN4924 induced apoptosis in 44.6±5.4% of the CD40L-stimulated, and in 63.7±2.6% of the CD40L/IL-21-stimulated CLL cells at 24 h (p=0.00004). CD40-stimulated CLL cells treated with MLN4924 showed Cdt1 accumulation, DNA re-replication, activation of DNA damage response pathways as evidenced by accumulation of pRPA, pChk1, pChk2 and gH2AX which preceded PARP cleavage, and G2 arrest. DNA damage and G2 arrest were more prominent in IL-21-treated cells. Targeting NAE also resulted in early accumulation of the endogenous cyclin-dependent kinase inhibitors p21 and p27, both CRL targets. By contrast, retinoblastoma protein hypophosphorylation occurred late and did not precede apoptosis. siRNA-mediated knockdown of Cdt1, but not p21 or p27 prevented MLN4924-mediated DNA damage response and CLL cell apoptosis. Finally, MLN4924 did not lead to accumulation of Cdt1, p21 or p27 in CLL cells cultured off stroma. Since alkylating agents target primarily cycling cells, we further determined whether they would cooperate with MLN4924 to induce DNA damage in CLL. While either chlorambucil or bendamustine exhibited little toxicity against the CD40L-stimulated CLL cells, MLN4924 sensitized CLL cells to those agents. MLN4924 further augmented bendamustine toxicity in the presence of IL-21. MLN4924-mediated DNA damage response was augmented in the presence of bendamustine. Interestingly, this combination proved ineffective in the CLL samples with del(17p), suggesting that bendamustine effect depends on the intact p53 pathway. Here we characterize DNA damage response pathway in CLL, where it remains understudied. Targeting NAE in cycling CLL cells induces Cdt1 accumulation, DNA damage and cell cycle arrest. This data provide additional insights into the biological consequences of targeting NAE in CLL B-cells as well as preclinical rationale to study the potential clinical activity of MLN4924 in CLL, alone or in combination with bendamustine or chlorambucil. Disclosures Berger: Takeda: Employment.