The BET (bromodomain and extra-terminal) proteins bind acetylated histones and recruit protein complexes to promote transcription elongation. In hematologic cancers, BET proteins have been shown to regulate expression of MYC and other genes that are important to disease pathology. Pharmacologic inhibition of BET protein binding has been shown to inhibit tumor growth in MYC-dependent cancers, such as multiple myeloma. In this study, we demonstrate that small cell lung cancer (SCLC) cells are exquisitely sensitive to growth inhibition by the BET inhibitor JQ1. JQ1 treatment has no impact on MYC protein expression, but results in downregulation of the lineage-specific transcription factor ASCL1. SCLC cells that are sensitive to JQ1 are also sensitive to ASCL1 depletion by RNAi. Chromatin immunoprecipitation studies confirmed the binding of the BET protein BRD4 to the ASCL1 enhancer, and the ability of JQ1 to disrupt the interaction. The importance of ASCL1 as a potential driver oncogene in SCLC is further underscored by the observation that ASCL1 is overexpressed in >50% of SCLC specimens, an extent greater than that observed for other putative oncogenes (MYC, MYCN, and SOX2) previously implicated in SCLC. Our studies have provided a mechanistic basis for the sensitivity of SCLC to BET inhibition and a rationale for the clinical development of BET inhibitors in this disease with high unmet medical need.

The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase casein kinase 2 (CK2) on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages. This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer. These findings demonstrate the modulatory effects of casein kinase 2 inhibitors on myeloid cell differentiation in the tumor microenvironment, which subsequently synergize with the antitumor effects of checkpoint inhibitor CTLA4. .

BackgroundThe role and sequencing of combination immuno-oncology (IO) therapy following progression on or after first-line IO therapy has not been well-established. The Fast Real-time Assessment of Combination Therapies in Immuno-ONcology (FRACTION) program is an open-label, phase 2 platform trial designed to evaluate multiple IO combinations in patients with advanced renal cell carcinoma (aRCC) who progressed during or after prior IO therapy. Here, we describe the results for patients treated with nivolumab plus ipilimumab. For enrollment in track 2 (reported here), patients with histologically confirmed clear cell aRCC, Karnofsky performance status ≥70%, and life expectancy ≥3 months who had previously progressed after IO (anti-programmed death 1 (PD-1), anti-programmed death-ligand 1 (PD-L1), or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4)) therapy were eligible. Previous treatment with anti-CTLA-4 therapy plus anti-PD-1/PD-L1 therapy precluded eligibility for enrollment in the nivolumab plus ipilimumab arm. Patients were treated with nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses, followed by nivolumab 480 mg every 4 weeks for up to 2 years or until progression, toxicity, or protocol-specified discontinuation. The primary outcome measures were objective response rate (ORR), duration of response (DOR), and progression-free survival (PFS) rate at 24 weeks. Secondary outcomes were safety and tolerability up to 2 years. Overall survival (OS) was a tertiary/exploratory endpoint. Overall, 46 patients were included with a median follow-up of 33.8 months. The ORR was 17.4% (95% CI, 7.8 to 31.4) with eight (17.4%) patients achieving partial response. Stable disease was achieved in 19 (41.3%) patients, while 14 (30.4%) had progressive disease. Median DOR (range) was 16.4 (2.1+ to 27.0+) months. The PFS rate at 24 weeks was 43.2%, and median OS was 23.8 (95% CI, 13.2 to not reached) months. Grade 3–4 immune-mediated adverse events were reported in seven (15.2%) patients. No treatment-related deaths were reported. Patients with aRCC treated with nivolumab plus ipilimumab may derive durable clinical benefit after progression on previous IO therapies, including heavily pretreated patients, with a manageable safety profile that was consistent with previously published safety outcomes. These outcomes contribute to the knowledge of optimal sequencing of IO therapies for patients with aRCC with high unmet needs.Trial registration numberNCT02996110.

Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control. Consistent with this finding, functional and RNA-sequencing analyses revealed greater changes in stimulated DGKζ-deficient T cells compared with DGKα-deficient T cells, which were enhanced relative to wildtype T cells. DGKζ also imparted greater regulation than DGKα in human T cells. Together, these data support targeting the ζ isoform of DGKs to therapeutically enhance T cell anti-tumor activity.

<p>Figure S1. Potency of I-BET762 in inhibition of proliferation of lung tumor cell lines. Lineage of lung cell lines is as indicated. Figure S2. Dose response curves of JQ1 in cell proliferation assays. Shown are examples of lung tumor cell lines with different sensitivity to JQ1. Figure S3. Induction of apoptosis in four SCLC cell lines was monitored by caspase 3 and 7 cleavage after 72 h treatment with JQ1. Data shown are fold changes over DMSO control. Figure S4. Probe sets induced by JQ1 in SCLC cell lines. Shown are microarray data for three probe sets that were induced by JQ1. Figure S5. Examples of probe sets that show dose-dependent changes, but were expressed only at background levels (RNF183) in all four cell lines, or were expressed at low levels in the sensitive cell lines (NR0B2). Figure S6. Dose response in expression of the three ASCL1 probe sets upon JQ1 treatment. Figure S7. (A) Enrichment of BRD4 binding at the ASCL1 gene enhancer compared to a gene desert on the same chromosome. Primers specific for ASCL1 enhancer or a gene desert on chromosome 12 were used to detect BRD4 binding. (B) JQ1 has no effect on non-specific binding of BRD4 to a gene desert in SCLC cell lines. Figure S8. (A) ASCL1 mRNA in SCLC and non-small-cell lung neuroendocrine (UMC-11 and NCI-H1155) cell lines. JQ1 sensitivity cut-off is arbitrarily defined as an IC50 of 0.5 µM. (B) ASCL1 protein abundance in SCLC cell lines that are sensitive or resistant to JQ1. (C) UMC-11 cells were treated with JQ1 for 24h and ASCL1 protein was analyzed by western blotting.</p>