Brenton R. Paolella

BET Bromodomain Inhibition of MYC-Amplified Medulloblastoma

Purpose: MYC-amplified medulloblastomas are highly lethal tumors. Bromodomain and extraterminal (BET) bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here, we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma. Experimental Design: We evaluated the effects of genetic and pharmacologic inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and genetically engineered mouse model (GEMM)-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed the in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice. Results: Treatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed downregulation of MYC expression and confirmed the inhibition of MYC-associated transcriptional targets. The exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged the survival of orthotopic xenograft models of MYC-amplified medulloblastoma (P < 0.001). Xenografts harvested from mice after five doses of JQ1 had reduced the expression of MYC mRNA and a reduced proliferative index. Conclusion: JQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma. Clin Cancer Res; 20(4); 912–25. ©2013 AACR.

MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

Id2 Is Required for Specification of Dopaminergic Neurons during Adult Olfactory Neurogenesis

Understanding the biology of adult neural stem cells has important implications for nervous system development and may contribute to our understanding of neurodegenerative disorders and their treatment. We have characterized the process of olfactory neurogenesis in adult mice lacking inhibitor of DNA binding 2−/− (Id2−/−). We found a diminished olfactory bulb containing reduced numbers of granular and periglomerular neurons with a distinct paucity of dopaminergic periglomerular neurons. While no deficiency of the stem cell compartment was detectable, migrating neuroblasts in Id2−/− mutant mice prematurely undergo astroglial differentiation within a disorganized rostral migratory stream. Further, when evaluated in vitro loss of Id2 results in decreased proliferation of neural progenitors and decreased expression of the Hes1 and Ascl1 (Mash1) transcription factors, known mediators of neuronal differentiation. These data support a novel role for sustained Id2 expression in migrating neural progenitors mediating olfactory dopaminergic neuronal differentiation in adult animals.

p53 directly represses Id2 to inhibit the proliferation of neural progenitor cells.

Neural progenitor cells (NPCs) have the capacity to proliferate and give rise to all major central nervous system cell types and represent a possible cell of origin in gliomagenesis. Deletion of the tumor suppressor gene Tp53 (p53) results in increased proliferation and self‐renewal of NPCs and is a common genetic mutation found in glioma. We have identified inhibitor of DNA binding 2 (Id2) as a novel target gene directly repressed by p53 to maintain normal NPC proliferation. p53(−/−) NPCs express elevated levels of Id2 and suppression of Id2 expression is sufficient to inhibit the increased proliferation and self‐renewal which results from p53 loss. Elevated expression of Id2 in wild‐type NPCs phenocopies the behavior of p53(−/−) NPCs by enhancing NPC proliferation and self‐renewal. Interestingly, p53 directly binds to a conserved site within the Id2 promoter to mediate these effects. Finally, we have identified elevated Id2 expression in glioma cell lines with mutated p53 and demonstrated that constitutive expression of Id2 plays a key role in the proliferation of glioma stem‐like cells. These findings indicate that Id2 functions as a proproliferative gene that antagonizes p53‐mediated cell cycle regulation in NPCs and may contribute to the malignant proliferation of glioma‐derived tumor stem cells. STEM CELLS 2011;1090–1101

Id2 mediates oligodendrocyte precursor cell maturation arrest and is tumorigenic in a PDGF- rich microenvironment

Maturation defects occurring in adult tissue progenitor cells have the potential to contribute to tumor development; however, there is little experimental evidence implicating this cellular mechanism in the pathogenesis of solid tumors. Inhibitor of DNA-binding 2 (Id2) is a transcription factor known to regulate the proliferation and differentiation of primitive stem and progenitor cells. Id2 is derepressed in adult tissue neural stem cells (NSC) lacking the tumor suppressor Tp53 and modulates their proliferation. Constitutive expression of Id2 in differentiating NSCs resulted in maturation-resistant oligodendroglial precursor cells (OPC), a cell population implicated in the initiation of glioma. Mechanistically, Id2 overexpression was associated with inhibition of the Notch effector Hey1, a bHLH transcription factor that we here characterize as a direct transcriptional repressor of the oligodendroglial lineage determinant Olig2. Orthotopic inoculation of NSCs with enhanced Id2 expression into brains of mice engineered to express platelet-derived growth factor in the central nervous system resulted in glioma. These data implicate a mechanism of altered NSC differentiation in glioma development and characterize a novel mouse model that reflects key characteristics of the recently described proneural subtype of glioblastoma multiforme. Such findings support the emerging concept that the cellular and molecular characteristics of tumor cells are linked to the transformation of distinct subsets of adult tissue progenitors.

Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency. DOI: http://dx.doi.org/10.7554/eLife.23268.001

Phosphorylation regulates Id2 degradation and mediates the proliferation of neural precursor cells.

Inhibitor of DNA binding proteins (Id1‐Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC‐like cells. We identified three phosphorylation sites near the N‐terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2‐/‐ NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N‐terminus had higher steady‐state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half‐life and reduced proteasome‐mediated degradation. Moreover, NPCs expressing constitutively de‐phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well‐characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell‐cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma‐derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321–1331

Behavioral abnormalities and Parkinson's-like histological changes resulting from Id2 inactivation in mice.

SUMMARY Characterizing dopaminergic neuronal development and function in novel genetic animal models might uncover strategies for researchers to develop disease-modifying treatments for neurologic disorders. Id2 is a transcription factor expressed in the developing central nervous system. Id2−/− mice have fewer dopaminergic neurons in the olfactory bulb and reduced olfactory discrimination, a pre-clinical marker of Parkinson’s disease. Here, we summarize behavioral, histological and in vitro molecular biological analyses to determine whether midbrain dopaminergic neurons are affected by Id2 loss. Id2−/− mice were hyperactive at 1 and 3 months of age, but by 6 months showed reduced activity. Id2−/− mice showed age-dependent histological alterations in dopaminergic neurons of the substantia nigra pars compacta (SNpC) associated with changes in locomotor activity. Reduced dopamine transporter (DAT) expression was observed at early ages in Id2−/− mice and DAT expression was dependent on Id2 expression in an in vitro dopaminergic differentiation model. Evidence of neurodegeneration, including activated caspase-3 and glial infiltration, were noted in the SNpC of older Id2−/− mice. These findings document a novel role for Id2 in the maintenance of midbrain dopamine neurons. The Id2−/− mouse should provide unique opportunities to study the progression of neurodegenerative disorders involving the dopamine system.

Abstract 4974: Pharmacogenomic interactions in glioblastoma cell line models

Glioblastoma (GBM) is the most common and malignant brain tumor. These tumors display a uniform and very short survival time even with treatment, but are highly heterogeneous at the histological and genomic level. To identify effective treatments and dependencies, we profiled the sensitivity of a panel of cancer cell lines to a small molecules and integrated this with systematic analysis of genetic and non-genetic determinants associated with chemical response. Methods. We profiled 381 drugs described in the Cancer Therapeutics Response Portal (CTRP) at 16 different duplicated concentrations across 78 GBM cell lines belonging to two different models: Patient-Derived GBM Cell Lines (PDGCL) and Long-Term GBM Cell Lines (LTGCL) previously included in the Cancer Cell Line Encyclopedia (CCLE). Cell lines were deeply characterized as to genotype and phenotype. As non-genomic determinants we considered: model, growth rate, behavior, stem cell and differentiation markers. Genomic determinants included mutations and somatic copy number alterations (SCNA) computed from whole exome sequencing. Each of these were integrated to determine oncogene and tumor suppressor gene pathway disruption (p53, RB and RTK signaling). At transcriptomic level we considered expression of 20.647 genes and patters described in GBMs (proneural, neural, classical, mesenchymal). Overall we correlated 10,859,643 pharmacogenomic features to discover associations with drug sensitivity. Summary. We developed a brain tumor living tissue bank as platform for preclinical pharmacogenomics analysis. Large-scale phenotypic characterization of GBM models showed increased cellular and molecular heterogeneity among PDGCLs compared to LTGCLs. PDGCLs better recapitulated patient GBM copy-number profiles. GBM cell lines exhibited all major driver mutations in human GBMs, except IDH1. PDGCLs and LTGCLs enriched for proneural and mesenchymal phenotypes, respectively. We identified NAMPT inhibitors as among the compounds with highest activity across cell lines. Integrative pharmacogenomic analyses showed MDM2/4 inhibitors were able to effectively suppress TP53 wild type GBM models, being CDKN1A (p21) expression a robust predictor of drug response in vitro and in vivo. Overall drug resistance across the screen in lines was highly associated with TP53 mutation, however a specific subset of TP53 mutant cell lines bearing simultaneous CDKN2A/B deletions were sensitive to CHK1/2 inhibitors, revealing a potential synthetic lethal interaction of clinical significance in these highly refractory cells. Analysis identified genetic alterations associated with vulnerabilities targeted by small molecules. About 85% of GBM patients display p53 pathway disruption, our results suggest independent pharmacological strategies for two genetic subtypes of GBM determined by TP53 and CDKN1A status. Our analyses provide molecular insights to drive targeted therapies in the new era of precision medicine. Citation Format: Ruben Ferrer-Luna, Shakti H. Ramkissoon, Karl H. Olausson, Lori A. Ramkissoon, Steven Schumacher, Rebecca Lamothe, Jaime H. Cheah, Kristine Pellton, Sam Haidar, Yun J. Kang, Brenton R. Paolella, Cecile Maire, Wenyu Song, Alice Meng, Ahmed Idbaih, Mikael L. Rinne, David A. Reardon, Patrick Y. Wen, Paul A. Clemons, Stuart L. Schreiber, Alykhan F. Shamji, Rameen Beroukhim, Keith L. Ligon. Pharmacogenomic interactions in glioblastoma cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4974. doi:10.1158/1538-7445.AM2017-4974

Abstract 2128: Phosphorylation of Id2 at the N-terminus modulates Id2 degradation and mediates cell cycle regulation in neural progenitor cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Glioblastoma is the most common and aggressive type of primary brain tumor in adults with more than 14,000 new cases diagnosed each year in the US. Although surgical resection, radiation, and cytotoxic therapies are available these current treatment methods are not curative and the median survival of patients remains at 12-15 months. Inhibitor of DNA binding proteins (Id1-Id4) are a family of genetically encoded dominant negative regulators of basic helix-loop-helix (bHLH) transcription factors. Id proteins are widely reported to inhibit differentiation and promote cell cycle transit in neural progenitor cells (NPCs) and have been implicated in the development of glioma. Our laboratory has observed a poor correlation between Id2 mRNA levels and Id2 protein levels in multiple cell types which led us to seek post-translational modifications that effected steady state Id2 protein levels and key Id2 mediated cellular functions. Using mass spectrometry we have identified three phosphorylation sites within the N-terminus of Id2 in proliferating NPCs. To interrogate the importance of Id2 N-terminal phosphorylation, Id2-/- NPCs were modified to express WT Id2 or various Id2 mutants expressing proteins that could not be phosphorylated at the N-terminus. We observed that NPCs expressing these mutants had higher steady state levels of Id2 than NPCs expressing WT Id2. It is known that WT Id2 is rapidly degraded by the proteasome. However, when proliferating NPCs were treated with cyclohexamide, phospho-ablated Id2 molecules exhibited a longer half-life than WT Id2 molecules indicating that loss of N-terminal phosphorylation results in resistance to proteasome-mediated degradation. Moreover, NPCs expressing this degradation-resistant, phospho-ablated Id2 protein proliferate more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Seeking to identify molecules whose inhibition might enhance Id2 phosphorylation, we evaluated the activity of multiple phosphatase inhibitors and identified phosphatases that could potentially function to stabilize pro-proliferative Id2 in NPCs. Calyculin A treatment caused a significant loss of Id2 protein suggesting that the PP1, PP2A, PP4 and/or PP6 phosphatases were likely regulators of Id2. To complement these pharmacologic studies we used a genetic approach to decrease PP2A expression and found that Id2 protein levels were decreased. Our findings indicate that inhibition of this phosphatase may provide a novel mechanism to decrease Id2 protein levels by causing rapid degradation of Id2. Citation Format: Jaclyn Sullivan, Matthew Havrda, Brenton Paolella, Arminja Kettenbach, Scott Gerber, Mark A. Israel. Phosphorylation of Id2 at the N-terminus modulates Id2 degradation and mediates cell cycle regulation in neural progenitor 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 2128. doi:10.1158/1538-7445.AM2015-2128