SUMMARY Antibody blockade of the inhibitory CTLA-4 pathway has led to clinical benefit in a subset of patients with metastatic melanoma. Anti-CTLA-4 enhances T cell responses, including production of IFN-γ, which is a critical cytokine for host immune responses. However, the role of IFN-γ signaling in tumor cells in the setting of anti-CTLA-4 therapy remains unknown. Here we demonstrate that patients identified as non-responders to anti-CTLA-4 (ipilimumab) have tumors with genomic defects in IFN-γ pathway genes. Furthermore, mice bearing melanoma tumors with knockdown of IFN-γ receptor 1 (IFNGR1) have impaired tumor rejection upon anti-CTLA-4 therapy. These data highlight that loss of the IFN-γ signaling pathway is associated with primary resistance to anti-CTLA-4 therapy. Our findings demonstrate the importance of tumor genomic data, especially IFN-γ related genes, as prognostic information for patients selected to receive treatment with immune checkpoint therapy.

Enhancer of zeste homolog 2-mediated (EZH2-mediated) epigenetic regulation of T cell differentiation and Treg function has been described previously; however, the role of EZH2 in T cell-mediated antitumor immunity, especially in the context of immune checkpoint therapy, is not understood. Here, we showed that genetic depletion of EZH2 in Tregs (FoxP3creEZH2fl/fl mice) leads to robust antitumor immunity. In addition, pharmacological inhibition of EZH2 in human T cells using CPI-1205 elicited phenotypic and functional alterations of the Tregs and enhanced cytotoxic activity of Teffs. We observed that ipilimumab (anti-CTLA-4) increased EZH2 expression in peripheral T cells from treated patients. We hypothesized that inhibition of EZH2 expression in T cells would increase the effectiveness of anti-CTLA-4 therapy, which we tested in murine models. Collectively, our data demonstrated that modulating EZH2 expression in T cells can improve antitumor responses elicited by anti-CTLA-4 therapy, which provides a strong rationale for a combination trial of CPI-1205 plus ipilimumab.

Voltage-dependent calcium channels (CaV) open in response to changes in membrane potential, but their activity is modulated by Ca 2؉ binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca V1.2 ␣1 subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca 2؉ ⅐CaM in 2 distinct binding modes. The structure of the Ca V1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca 2؉ ⅐CaMs interacting with the pre-IQ regions and a canonical Ca V1-IQ-Ca 2؉ ⅐CaM complex. Native Ca V1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca 2؉ -dependent inactivation of heterologously expressed CaV1.2 channels.structure ͉ function ͉ voltage-gated calcium channel E xcitation-contraction coupling and other important cellular processes are controlled by the voltage-gated Ca 2ϩ channels (Ca V ). The ubiquitous Ca 2ϩ sensor and regulator molecule, calmodulin, is an essential component of Ca V regulation by Ca 2ϩ , and several regions of the cytoplasmic carboxyl terminus of the Ca V ␣1 subunit have been identified as critical molecular determinants for CaM's regulation of Ca V . Ca 2ϩ ⅐CaM bound to the IQ motif of the carboxyl terminus of the ␣ 1 subunit of L-type Ca 2ϩ channels is required for both a feed-forward regulation, Ca 2ϩ -dependent facilitation (CDF), and a feed-back regulation, Ca 2ϩ -dependent inactivation (CDI) (1, 2). CaM acts as the Ca 2ϩ sensor for CDI in Ca V 1.2, Ca V 2.1, Ca V 2.2 and Ca V 2.3, and CDF in Ca V 1.2 and Ca V 2.1. This duality of Ca V regulation by CaM suggests that there are either multiple binding sites or alternative interactions exist to regulate the channel based on different functional states of the channel.Regions upstream of the IQ motif, designated the pre-IQ region, have been implicated in Ca 2ϩ ⅐CaM regulation of the channel (3-5). Consistent with this, 2 different segments within the pre-IQ motif (1606-1627 and 1618-1652, identified as the A and C sequences, respectively) have been shown to bind CaM (4). Moreover, both the IQ motif and amino acids within the pre-IQ region (N1630-E1631) have been indicated to be critical for Ca V 1.2 CDI (6).Recent crystal structures of CaM in complex with the IQ motifs from Ca V 1.2 (7, 8), Ca V 2.2 and Ca V 2.3 (5) reveal few structural differences that could account for the differences in regulation of Ca V 1.2 and Ca V 2.2 by CaM. However, very little is known about the structure of the pre-IQ region or the molecular basis for its interactions with CaM.Here, we report the isolation and determination of the crystal structure at 2.1 Å resolution of a 77-residue (1609-1685) fragment of the carboxyl terminus of the ␣ 1 ...

Purpose MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated. Experimental design We performed RNA and exome sequencing on an exploratory set of TRCC (n=7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n=460). Results Using the TCGA dataset, we identified 7 TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP and KHDRBS2), which are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared to other RCC types, and showed activation of MITF, the transforming growth factor β1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared to ccRCC, with frequent mutations in chromatin remodeling genes (six of eight cases, three of which from the TCGA). In two cases, we identified mutations in INO80D, an ATP-dependent chromatin remodeling gene, previously shown to control the amplitude of the S phase. Knockdown of INO80D decreased cell proliferation in a novel cell line bearing LUC7L3-TFE3 translocation. Conclusions This genome-wide study defines the incidence of TRCC within a ccRCC-directed project and expands the genomic spectrum of TRCC by identifying novel MITF/TFE partners involved in RNA splicing and frequent mutations in chromatin remodeling genes.

Calmodulin activates the skeletal muscle Ca 2؉ release channel RYR1 at nM Ca 2؉ concentrations and inhibits the channel at M Ca 2؉ concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nM and M Ca 2؉ concentrations and are required for maximum inhibition of the channel at M Ca 2؉ concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nM and M Ca 2؉ concentrations, but destroyed its functional effects on RYR1 at nM Ca 2؉ . Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca 2؉ -calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca 2؉ binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the Nterminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.