Steven N. Quayle

PLAGL2 Regulates Wnt Signaling to Impede Differentiation in Neural Stem Cells and Gliomas

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state, which contributes to its plasticity and therapeutic resistance. Here, integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells.

Pattern of retinoblastoma pathway inactivation dictates response to CDK4/6 inhibition in GBM

Glioblastoma multiforme (GBM) is a fatal primary brain tumor harboring myriad genetic and epigenetic alterations. The recent multidimensional analysis of the GBM genome has provided a more complete view of the landscape of such alterations and their linked pathways. This effort has demonstrated that certain pathways are universally altered, but that the specific genetic events altered within each pathway can vary for each particular patients tumor. With this atlas of genetic and epigenetic events, it now becomes feasible to assess how the patterns of mutations in a pathway influence response to drugs that are targeting such pathways. This issue is particularly important for GBM because, in contrast to other tumor types, molecularly targeted therapies have failed to alter overall survival substantially. Here, we combined functional genetic screens and comprehensive genomic analyses to identify CDK6 as a GBM oncogene that is required for proliferation and viability in a subset of GBM cell lines and tumors. Using an available small molecule targeting cyclin-dependent kinases (CDKs) 4 and 6, we sought to determine if the specific pattern of retinoblastoma pathway inactivation dictated the response to CDK4/6 inhibitor therapy. We showed that codeletion of CDKN2A and CDKN2C serves as a strong predictor of sensitivity to a selective inhibitor of CDK4/6. Thus, genome-informed drug sensitivity studies identify a subset of GBMs likely to respond to CDK4/6 inhibition. More generally, these observations demonstrate that the integration of genomic, functional and pharmacologic data can be exploited to inform the development of targeted therapy directed against specific cancer pathways.

microRNA Regulatory Network Inference Identifies miR-34a as a Novel Regulator of TGF-β Signaling in Glioblastoma

UNLABELLED Leveraging The Cancer Genome Atlas (TCGA) multidimensional data in glioblastoma, we inferred the putative regulatory network between microRNA and mRNA using the Context Likelihood of Relatedness modeling algorithm. Interrogation of the network in context of defined molecular subtypes identified 8 microRNAs with a strong discriminatory potential between proneural and mesenchymal subtypes. Integrative in silico analyses, a functional genetic screen, and experimental validation identified miR-34a as a tumor suppressor in proneural subtype glioblastoma. Mechanistically, in addition to its direct regulation of platelet-derived growth factor receptor-alpha (PDGFRA), promoter enrichment analysis of context likelihood of relatedness-inferred mRNA nodes established miR-34a as a novel regulator of a SMAD4 transcriptional network. Clinically, miR-34a expression level is shown to be prognostic, where miR-34a low-expressing glioblastomas exhibited better overall survival. This work illustrates the potential of comprehensive multidimensional cancer genomic data combined with computational and experimental models in enabling mechanistic exploration of relationships among different genetic elements across the genome space in cancer. SIGNIFICANCE We illustrate here that network modeling of complex multidimensional cancer genomic data can generate a framework in which to explore the biology of cancers, leading to discovery of new pathogenetic insights as well as potential prognostic biomarkers. Specifically in glioblastoma, within the context of the global network, promoter enrichment analysis of network edges uncovered a novel regulation of TGF-β signaling via a Smad4 transcriptomic network by miR-34a.

Somatic Mutations of PIK3R1 Promote Gliomagenesis

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.

In Vitro and In Vivo Interactions between the HDAC6 Inhibitor Ricolinostat (ACY1215) and the Irreversible Proteasome Inhibitor Carfilzomib in Non-Hodgkin Lymphoma Cells

Interactions between the HDAC6 inhibitor ricolinostat (ACY1215) and the irreversible proteasome inhibitor carfilzomib were examined in non-Hodgkin lymphoma (NHL) models, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and double-hit lymphoma cells. Marked in vitro synergism was observed in multiple cell types associated with activation of cellular stress pathways (e.g., JNK1/2, ERK1/2, and p38) accompanied by increases in DNA damage (γH2A.X), G2–M arrest, and the pronounced induction of mitochondrial injury and apoptosis. Combination treatment with carfilzomib and ricolinostat increased reactive oxygen species (ROS), whereas the antioxidant TBAP attenuated DNA damage, JNK activation, and cell death. Similar interactions occurred in bortezomib-resistant and double-hit DLBCL, MCL, and primary DLBCL cells, but not in normal CD34+ cells. However, ricolinostat did not potentiate inhibition of chymotryptic activity by carfilzomib. shRNA knockdown of JNK1 (but not MEK1/2), or pharmacologic inhibition of p38, significantly reduced carfilzomib–ricolinostat lethality, indicating a functional contribution of these stress pathways to apoptosis. Combined exposure to carfilzomib and ricolinostat also markedly downregulated the cargo-loading protein HR23B. Moreover, HR23B knockdown significantly increased carfilzomib- and ricolinostat-mediated lethality, suggesting a role for this event in cell death. Finally, combined in vivo treatment with carfilzomib and ricolinostat was well tolerated and significantly suppressed tumor growth and increased survival in an MCL xenograft model. Collectively, these findings indicate that carfilzomib and ricolinostat interact synergistically in NHL cells through multiple stress-related mechanisms, and suggest that this strategy warrants further consideration in NHL. Mol Cancer Ther; 13(12); 2886–97. ©2014 AACR.

Synergistic Immunostimulatory Effects and Therapeutic Benefit of Combined Histone Deacetylase and Bromodomain Inhibition in Non–Small Cell Lung Cancer

Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein, we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T-cell activation and improved function of antigen-presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+FOXP3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell-mediated antitumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.Significance: Selective inhibition of HDACs and bromodomain proteins modulates tumor-associated immune cells in a manner that favors improved T-cell function and reduced inhibitory cellular mechanisms. These effects facilitated robust antitumor responses in tumor-bearing mice, demonstrating the therapeutic potential of combining these epigenetic modulators for the treatment of NSCLC. Cancer Discov; 7(8); 852-67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

Integrative functional genomics identifies RINT1 as a novel GBM oncogene

Large-scale cancer genomics efforts are identifying hundreds of somatic genomic alterations in glioblastoma (GBM). Distinguishing between active driver and neutral passenger alterations requires functional assessment of each gene; therefore, integrating biological weight of evidence with statistical significance for each genomic alteration will enable better prioritization for downstream studies. Here, we demonstrate the feasibility and potential of in vitro functional genomic screens to rapidly and systematically prioritize high-probability candidate genes for in vivo validation. Integration of low-complexity gain- and loss-of-function screens designed on the basis of genomic data identified 6 candidate GBM oncogenes, and RINT1 was validated as a novel GBM oncogene based on its ability to confer tumorigenicity to primary nontransformed murine astrocytes in vivo. Cancer genomics-guided low-complexity genomic screens can quickly provide a functional filter to prioritize high-value targets for further downstream mechanistic and translational studies.

Enhancement of pomalidomide anti-tumor response with ACY-241, a selective HDAC6 inhibitor

Thalidomide-based Immunomodulatory Drugs (IMiDs®), including lenalidomide and pomalidomide, are effective therapeutics for multiple myeloma. These agents have been approved with, or are under clinical development with, other targeted therapies including proteasome inhibitors, αCD38 monoclonal antibodies, as well as histone deacetylase (HDAC) inhibitors for combination therapy. HDAC inhibitors broadly targeting Class I and IIb HDACs have shown potent preclinical efficacy but have frequently demonstrated an undesirable safety profile in combination therapy approaches in clinical studies. Therefore, development of more selective HDAC inhibitors could provide enhanced efficacy with reduced side effects in combination with IMiDs® for the treatment of B-cell malignancies, including multiple myeloma. Here, the second generation selective HDAC6 inhibitor citarinostat (ACY-241), with a more favorable safety profile than non-selective pan-HDAC inhibitors, is shown to synergize with pomalidomide in in vitro assays through promoting greater apoptosis and cell cycle arrest. Furthermore, utilizing a multiple myeloma in vivo murine xenograft model, combination treatment with pomalidomide and ACY-241 leads to increased tumor growth inhibition. At the molecular level, combination treatment with ACY-241 and pomalidomide leads to greater suppression of the pro-survival factors survivin, Myc, and IRF4. The results presented here demonstrate synergy between pomalidomide and ACY-241 in both in vitro and in vivo preclinical models, providing further impetus for clinical development of ACY-241 for use in combination with IMiDs for patients with multiple myeloma and potentially other B-cell malignancies.

Abstract 5380: Selective HDAC inhibition by ricolinostat (ACY-1215) or ACY-241 synergizes with IMiD® immunomodulatory drugs in Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL) cells

Histone deacetylase (HDAC) enzymes are attractive therapeutic targets in oncology, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. Ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes in vitro and in vivo with bortezomib in models of MM (Santo, Blood, 2012) and with carfilzomib in models of lymphoma (Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in Phase I trials (Raje, Haematologica, 2014, Suppl 1). In addition to ongoing clinical studies with ricolinostat, we have identified ACY-241 as a next generation, orally available, selective inhibitor of HDAC6 for clinical evaluation. The IMiD® immunomodulatory class of drugs, including lenalidomide (len) and pomalidomide (pom), exhibit striking anti-myeloma properties in a variety of MM models, and have demonstrated significant clinical activity in MM patients. Beyond MM, len is also approved for the treatment of MCL patients. In support of our ongoing clinical development programs for ricolinostat and ACY-241, we show here that combinations with either len or pom leads to synergistic decreases in the viability of MM and MCL cells in vitro. Time course studies demonstrated accumulation of cell cycle arrest in cells after prolonged exposure to len or pom, as well as progressive induction of apoptosis in these cells. Notably, though, the addition of ricolinostat or ACY-241 to either len or pom resulted in synergistic increases in the percentage of MM and MCL cells undergoing apoptosis. At the molecular level, treatment with IMiD® immunomodulatory compounds reduced expression of the critical transcription factors MYC and IRF4, which were reduced even further by treatment with ricolinostat or ACY-241 in combination with either len or pom. The molecular mechanism underlying this effect is currently being explored, though retention of low level inhibition of HDAC1, 2, and 3 by these compounds may contribute to the enhanced effects on gene expression reported here in combination with len or pom. Finally, prolonged treatment with ACY-241 plus pomalidomide was well tolerated in vivo with no overt evidence of toxicity, and combination treatment resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241, and their preclinical activity in combination with IMiD® immunomodulatory drugs, these findings support clinical evaluation of these agents in MM and MCL patients. Citation Format: Steven N. Quayle, Ingrid Almeciga-Pinto, David Tamang, Min Yang, Simon S. Jones. Selective HDAC inhibition by ricolinostat (ACY-1215) or ACY-241 synergizes with IMiD® immunomodulatory drugs in Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5380. doi:10.1158/1538-7445.AM2015-5380

Selective Inhibitors of Histone Deacetylases 1 and 2 Synergize with Azacitidine in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and inhibitors of DNA methyltransferase or histone deacetylase (HDAC) enzymes have exhibited activity in preclinical AML models. Combination studies with HDAC inhibitors plus DNA methyltransferase inhibitors have potential beneficial clinical activity in AML, however the toxicity profiles of non-selective HDAC inhibitors in the combination setting limit their clinical utility. In this work, we describe the preclinical development of selective inhibitors of HDAC1 and HDAC2, which are hypothesized to have improved safety profiles, for combination therapy in AML. We demonstrate that selective inhibition of HDAC1 and HDAC2 is sufficient to achieve efficacy both as a single agent and in combination with azacitidine in preclinical models of AML, including established AML cell lines, primary leukemia cells from AML patient bone marrow samples and in vivo xenograft models of human AML. Gene expression profiling of AML cells treated with either an HDAC1/2 inhibitor, azacitidine, or the combination of both have identified a list of genes involved in transcription and cell cycle regulation as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML patients.