Systematic identification of cancer cell vulnerabilities to natural killer cell-mediated immune surveillance.

Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-γ signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 disruption induced an inflamed state in leukemic cells, including increased expression of lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 engagement. In AML patients, reduced DCAF15 expression was associated with improved survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory properties in the treatment of myeloid neoplasms.

Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation.

Acetylation of histone H3 at lysine 27 is a well-defined marker of enhancer activity. However, the functional impact of this modification at enhancers is poorly understood. Here, we use a chemical genetics approach to acutely block the function of the cAMP response element binding protein (CREB) binding protein (CBP)/P300 bromodomain in models of hematological malignancies and describe a consequent loss of H3K27Ac specifically from enhancers, despite the continued presence of CBP/P300 at chromatin. Using this approach to dissect the role of H3K27Ac at enhancers, we identify a critical role for this modification in the production of enhancer RNAs and transcription of enhancer-regulated gene networks.

Therapeutic Targeting of the CBP/p300 Bromodomain Blocks the Growth of Castration-Resistant Prostate Cancer.

Resistance invariably develops to antiandrogen therapies used to treat newly diagnosed prostate cancers, but effective treatments for castration-resistant disease remain elusive. Here, we report that the transcriptional coactivator CBP/p300 is required to maintain the growth of castration-resistant prostate cancer. To exploit this vulnerability, we developed a novel small-molecule inhibitor of the CBP/p300 bromodomain that blocks prostate cancer growth in vitro and in vivo Molecular dissection of the consequences of drug treatment revealed a critical role for CBP/p300 in histone acetylation required for the transcriptional activity of the androgen receptor and its target gene expression. Our findings offer a preclinical proof of concept for small-molecule therapies to target the CBP/p300 bromodomain as a strategy to treat castration-resistant prostate cancer. Cancer Res; 77(20); 5564-75. ©2017 AACR.

Trastuzumab uptake and its relation to efficacy in an animal model of HER2-positive breast cancer brain metastasis.

PURPOSE: The extent to which efficacy of the HER2 antibody Trastuzumab in brain metastases is limited by access of antibody to brain lesions remains a question of significant clinical importance. We investigated the uptake and distribution of trastuzumab in brain and mammary fat pad grafts of HER2-positive breast cancer to evaluate the relationship of these parameters to the anti-tumor activity of trastuzumab and trastuzumab emtansine (T-DM1). METHODS: Mouse transgenic breast tumor cells expressing human HER2 (Fo2-1282 or Fo5) were used to establish intracranial and orthotopic tumors. Tumor uptake and tissue distribution of systemically administered 89Zr-trastuzumab or muMAb 4D5 (murine parent of trastuzumab) were measured by PET and ELISA. Efficacy of muMAb 4D5, the PI3K/mTOR inhibitor GNE-317, and T-DM1 was also assessed. RESULTS: 89Zr-trastuzumab and muMAb 4D5 exhibited robust uptake into Fo2-1282 brain tumors, but not normal brains. Uptake into brain grafts was similar to mammary grafts. Despite this, muMAb 4D5 was less efficacious in brain grafts. Co-administration of muMAb 4D5 and GNE-317, a brain-penetrant PI3K/mTOR inhibitor, provided longer survival in mice with brain lesions than either agent alone. Moreover, T-DM1 increased survival in the Fo5 brain metastasis model. CONCLUSIONS: In models of HER2-positive breast cancer brain metastasis, trastuzumab efficacy does not appear to be limited by access to intracranial tumors. Anti-tumor activity improved with the addition of a brain-penetrant PI3K/mTOR inhibitor, suggesting that combining targeted therapies is a more effective strategy for treating HER2-positive breast cancer brain metastases. Survival was also extended in mice with Fo5 brain lesions treated with T-DM1.

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

Patients With Proneural Glioblastoma May Derive Overall Survival Benefit From the Addition of Bevacizumab to First-Line Radiotherapy and Temozolomide: Retrospective Analysis of the AVAglio Trial.

PURPOSE: The AVAglio (Avastin in Glioblastoma) and RTOG-0825 randomized, placebo-controlled phase III trials in newly diagnosed glioblastoma reported prolonged progression-free survival (PFS), but not overall survival (OS), with the addition of bevacizumab to radiotherapy plus temozolomide. To establish whether certain patient subgroups derived an OS benefit from the addition of bevacizumab to first-line standard-of-care therapy, AVAglio patients were retrospectively evaluated for molecular subtype, and bevacizumab efficacy was assessed for each patient subgroup. PATIENTS AND METHODS: A total of 349 pretreatment specimens (bevacizumab arm, n = 171; placebo arm, n = 178) from AVAglio patients (total, N = 921) were available for biomarker analysis. Samples were profiled for gene expression and isocitrate dehydrogenase 1 (IDH1) mutation status and classified into previously identified molecular subtypes. PFS and OS were assessed within each subtype. RESULTS: A multivariable analysis accounting for prognostic covariates revealed that bevacizumab conferred a significant OS advantage versus placebo for patients with proneural IDH1 wild-type tumors (17.1 v 12.8 months, respectively; hazard ratio, 0.43; 95% CI, 0.26 to 0.73; P = .002). This analysis also revealed an interaction between the proneural subtype biomarker and treatment arm (P = .023). The group of patients with mesenchymal and proneural tumors derived a PFS benefit from bevacizumab compared with placebo; however, this translated to an OS benefit in the proneural subset only. CONCLUSION: Retrospective analysis of AVAglio data suggests that patients with IDH1 wild-type proneural glioblastoma may derive an OS benefit from first-line bevacizumab treatment. The predictive value of the proneural subtype observed in AVAglio should be validated in an independent data set.

Hominoid-specific enzyme GLUD2 promotes growth of IDH1R132H glioma.

Somatic mutation of isocitrate dehydrogenase 1 (IDH1) is now recognized as the most common initiating event for secondary glioblastoma, a brain tumor type arising with high frequency in the frontal lobe. A puzzling feature of IDH1 mutation is the selective manifestation of glioma as the only neoplasm frequently associated with early postzygotic occurrence of this genomic alteration. We report here that IDH1(R132H) exhibits a growth-inhibitory effect that is abrogated in the presence of glutamate dehydrogenase 2 (GLUD2), a hominoid-specific enzyme purportedly optimized to facilitate glutamate turnover in human forebrain. Using murine glioma progenitor cells, we demonstrate that IDH1(R132H) exerts a growth-inhibitory effect that is paralleled by deficiency in metabolic flux from glucose and glutamine to lipids. Examining human gliomas, we find that glutamate dehydrogenase 1 (GLUD1) and GLUD2 are overexpressed in IDH1-mutant tumors and that orthotopic growth of an IDH1-mutant glioma line is inhibited by knockdown of GLUD1/2. Strikingly, introduction of GLUD2 into murine glioma progenitor cells reverses deleterious effects of IDH1 mutation on metabolic flux and tumor growth. Further, we report that glutamate, a substrate of GLUD2 and a neurotransmitter abundant in mammalian neocortex, can support growth of glioma progenitor cells irrespective of IDH1 mutation status. These findings suggest that specialization of human neocortex for high glutamate neurotransmitter flux creates a metabolic niche conducive to growth of IDH1 mutant tumors.

Targeting the PI3K pathway in the brain--efficacy of a PI3K inhibitor optimized to cross the blood-brain barrier.

PURPOSE: Glioblastoma (GBM), the most common primary brain tumor in adults, presents a high frequency of alteration in the PI3K pathway. Our objectives were to identify a dual PI3K/mTOR inhibitor optimized to cross the blood-brain barrier (BBB) and characterize its brain penetration, pathway modulation in the brain and efficacy in orthotopic xenograft models of GBM. EXPERIMENTAL DESIGN: Physicochemical properties of PI3K inhibitors were optimized using in silico tools, leading to the identification of GNE-317. This compound was tested in cells overexpressing P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP). Following administration to mice, GNE-317 plasma and brain concentrations were determined, and phosphorylated biomarkers (pAkt, p4EBP1, and pS6) were measured to assess PI3K pathway suppression in the brain. GNE-317 efficacy was evaluated in the U87, GS2, and GBM10 orthotopic models of GBM. RESULTS: GNE-317 was identified as having physicochemical properties predictive of low efflux by P-gp and BCRP. Studies in transfected MDCK cells showed that GNE-317 was not a substrate of either transporter. GNE-317 markedly inhibited the PI3K pathway in mouse brain, causing 40% to 90% suppression of the pAkt and pS6 signals up to 6-hour postdose. GNE-317 was efficacious in the U87, GS2, and GBM10 orthotopic models, achieving tumor growth inhibition of 90% and 50%, and survival benefit, respectively. CONCLUSIONS: These results indicated that specific optimization of PI3K inhibitors to cross the BBB led to potent suppression of the PI3K pathway in healthy brain. The efficacy of GNE-317 in 3 intracranial models of GBM suggested that this compound could be effective in the treatment of GBM.

Evidence for sequenced molecular evolution of IDH1 mutant glioblastoma from a distinct cell of origin.

PURPOSE: Mutation in isocitrate dehydrogenase 1 (IDH1) at R132 (IDH1(R132MUT)) is frequent in low-grade diffuse gliomas and, within glioblastoma (GBM), has been proposed as a marker for GBMs that arise by transformation from lower-grade gliomas, regardless of clinical history. To determine how GBMs arising with IDH1(R132MUT) differ from other GBMs, we undertook a comprehensive comparison of patients presenting clinically with primary GBM as a function of IDH1(R132) mutation status. PATIENTS AND METHODS: In all, 618 treatment-naive primary GBMs and 235 lower-grade diffuse gliomas were sequenced for IDH1(R132) and analyzed for demographic, radiographic, anatomic, histologic, genomic, epigenetic, and transcriptional characteristics. RESULTS: Investigation revealed a constellation of features that distinguishes IDH1(R132MUT) GBMs from other GBMs (including frontal location and lesser extent of contrast enhancement and necrosis), relates them to lower-grade IDH1(R132MUT) gliomas, and supports the concept that IDH1(R132MUT) gliomas arise from a neural precursor population that is spatially and temporally restricted in the brain. The observed patterns of DNA sequence, methylation, and copy number alterations support a model of ordered molecular evolution of IDH1(R132MUT) GBM in which the appearance of mutant IDH1 protein is an initial event, followed by production of p53 mutant protein, and finally by copy number alterations of PTEN and EGFR. CONCLUSION: Although histologically similar, GBMs arising with and without IDH1(R132MUT) appear to represent distinct disease entities that arise from separate cell types of origin as the result of largely nonoverlapping sets of molecular events. Optimal clinical management should account for the distinction between these GBM disease subtypes.

A distinct subset of glioma cell lines with stem cell-like properties reflects the transcriptional phenotype of glioblastomas and overexpresses CXCR4 as therapeutic target.

Glioblastomas contain stem-like cells that can be maintained in vitro using specific serum-free conditions. We investigated whether glioblastoma stem-like (GS) cell lines preserve the expression phenotype of human glioblastomas more closely than conventional glioma cell lines. Expression profiling revealed that a distinct subset of GS lines, which displayed a full stem-like phenotype (GSf), mirrored the expression signature of glioblastomas more closely than either other GS lines or cell lines grown in serum. GSf lines are highly tumorigenic and invasive in vivo, express CD133, grow spherically in vitro, are multipotent and display a Proneural gene expression signature, thus recapitulating key functional and transcriptional aspects of human glioblastomas. In contrast, GS lines with a restricted stem-like phenotype exhibited expression signatures more similar to conventional cell lines than to original patient tumors, suggesting that the transcriptional resemblance between GS lines and tumors is associated with different degrees of "stemness". Among markers overexpressed in patient tumors and GSf lines, we identified CXCR4 as a potential therapeutic target. GSf lines contained a minor population of CXCR4(hi) cells, a subfraction of which coexpressed CD133 and was expandable by hypoxia, whereas conventional cell lines contained only CXCR4(lo) cells. Convection-enhanced local treatment with AMD3100, a specific CXCR4 antagonist, inhibited the highly invasive growth of GS xenografts in vivo and cell migration in vitro. We thus demonstrate the utility of GSf lines in testing therapeutic agents and validate CXCR4 as a target to block the growth of invasive tumor-initiating glioma stem cells in vivo.

A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

Epigenome scans and cancer genome sequencing converge on WNK2, a kinase-independent suppressor of cell growth.

Human cancer genome and epigenome projects aim to identify new cancer genes and targets for therapy that have been overlooked by conventional approaches. Here we integrated large-scale genomics and epigenomics of 31 human infiltrative gliomas and identified low-frequency deletion and highly recurrent epigenetic silencing of WNK2, encoding a putative serine/threonine kinase. Prior cancer genome sequencing projects also identified point mutations in WNK1-4, suggesting that WNK family genes may have a role in cancers. We observed consistent gene silencing in tumors with dense aberrant methylation across 1.3 kb of the CpG island but more variable expression when the 5'-most region remained unmethylated. This primary tumor data fit well with WNK2 promoter analysis, which showed strong promoter activity in the 5'-most region, equivalent to the simian virus 40 promoter, but no activity in the 3' region. WT WNK2 exhibited autophosphorylation and protein kinase activity that was enhanced in cells exposed to hypertonic conditions, similar to WNK1. WNK2 inhibited up to 78% of colony formation by glioma cells but in an unexpectedly kinase-independent manner. The WNK2 silencing by epigenetic mechanisms was significantly associated (P < 0.01) with a known genetic signature of chemosensitive oligodendroglial tumors, 1p and 19q deletion, in two small but independent tumor sets. Taken together, the epigenetic silencing, occasional deletion and point mutation, and functional assessment suggest that aberrations of WNK2 may contribute to unregulated tumor cell growth. Thus, our integrated genetic and epigenetic approach might be useful to identify genes that are widely relevant to cancer, even when genetic alterations of the locus are infrequent.

Identification of IGF2 signaling through phosphoinositide-3-kinase regulatory subunit 3 as a growth-promoting axis in glioblastoma.

Amplification or overexpression of growth factor receptors is a frequent occurrence in malignant gliomas. Using both expression profiling and in situ hybridization, we identified insulin-like growth factor 2 (IGF2) as a marker for a subset of glioblastomas (GBMs) that lack amplification or overexpression of EGF receptor. Among 165 primary high-grade astrocytomas, 13% of grade IV tumors and 2% of grade III tumors expressed IGF2 mRNA levels >50-fold the sample population median. IGF2-overexpressing tumors frequently displayed PTEN loss, were highly proliferative, exhibited strong staining for phospho-Akt, and belonged to a subclass of GBMs characterized by poor survival. Using a serum-free culture system, we discovered that IGF2 can substitute for EGF to support the growth of GBM-derived neurospheres. The growth-promoting effects of IGF2 were mediated by the insulin-like growth factor receptor 1 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of phosphoinositide 3-kinase that shows genomic gains in some highly proliferative GBM cases. PIK3R3 knockdown inhibited IGF2-induced growth of GBM-derived neurospheres. The current results provide evidence that the IGF2-PIK3R3 signaling axis is involved in promoting the growth of a subclass of highly aggressive human GBMs that lack EGF receptor amplification. Our data underscore the importance of the phosphoinositide 3-kinase/Akt pathway for growth of high-grade gliomas and suggest that multiple molecular alterations that activate this signaling cascade may promote tumorigenesis. Further, these findings highlight the parallels between growth factors or receptors that are overexpressed in GBMs and those that support in vitro growth of tumor-derived stem-like cells.

Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis.

Previously undescribed prognostic subclasses of high-grade astrocytoma are identified and discovered to resemble stages in neurogenesis. One tumor class displaying neuronal lineage markers shows longer survival, while two tumor classes enriched for neural stem cell markers display equally short survival. Poor prognosis subclasses exhibit markers either of proliferation or of angiogenesis and mesenchyme. Upon recurrence, tumors frequently shift toward the mesenchymal subclass. Chromosomal locations of genes distinguishing tumor subclass parallel DNA copy number differences between subclasses. Functional relevance of tumor subtype molecular signatures is suggested by the ability of cell line signatures to predict neurosphere growth. A robust two-gene prognostic model utilizing PTEN and DLL3 expression suggests that Akt and Notch signaling are hallmarks of poor prognosis versus better prognosis gliomas, respectively.