Philip Corrin

Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.

ABSTRACT Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFαR) is fused to the cytosolic domain of Drosophila Torso (αTor) or the mouse fibroblast growth factor receptor 1 (αFR). αTor homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFαR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of αTor to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The αFR chimeric receptor fails to rescue any aspect of the PDGFαR-null phenotype. Instead, αFR expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The αFR phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.

Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

Summary Factors that sustain self-renewal of mouse embryonic stem cells (ESCs) are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale small interfering RNA (siRNA) screen, we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ESC commitment. Tsc2 lies upstream of mammalian target of rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell-intrinsic rheostat for destabilizing ground-state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

We developed a versatile, high-throughput genetic screening strategy by coupling gene mutagenesis and expression profiling technologies. Using a retroviral gene-trap vector optimized for efficient mutagenesis and cloning, we randomly disrupted genes in mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that transcriptional target genes of platelet-derived growth factor (PDGF) can be efficiently and reliably identified in physiologically relevant cells and are immediately accessible to genetic studies to determine their in vivo roles and relative contributions to PDGF-regulated developmental processes. The same platform can be used to search for genes of specific biological relevance in a broad array of experimental settings, providing a fast track from gene identification to functional validation.

Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

Cancer-Specific Requirement for BUB1B/BUBR1 in Human Brain Tumor Isolates and Genetically Transformed Cells

UNLABELLED To identify new candidate therapeutic targets for glioblastoma multiforme, we combined functional genetics and glioblastoma network modeling to identify kinases required for the growth of patient-derived brain tumor-initiating cells (BTIC) but that are dispensable to proliferating human neural stem cells (NSC). This approach yielded BUB1B/BUBR1, a critical mitotic spindle checkpoint player, as the top-scoring glioblastoma lethal kinase. Knockdown of BUB1B inhibited expansion of BTIC isolates, both in vitro and in vivo, without affecting proliferation of NSCs or astrocytes. Mechanistic studies revealed that BUB1Bs GLE2p-binding sequence (GLEBS) domain activity is required to suppress lethal kinetochore-microtubule (KT-MT) attachment defects in glioblastoma isolates and genetically transformed cells with altered sister KT dynamics, which likely favor KT-MT instability. These results indicate that glioblastoma tumors have an added requirement for BUB1B to suppress lethal consequences of altered KT function and further suggest that sister KT measurements may predict cancer-specific sensitivity to BUB1B inhibition and perhaps other mitotic targets that affect KT-MT stability. SIGNIFICANCE Currently, no effective therapies are available for glioblastoma, the most frequent and aggressive brain tumor. Our results suggest that targeting the GLEBS domain activity of BUB1B may provide a therapeutic window for glioblastoma, as the GLEBS domain is nonessential in untransformed cells. Moreover, the results further suggest that sister KT distances at metaphase may predict sensitivity to anticancer therapeutics targeting KT function.

ZNF131 suppresses centrosome fragmentation in glioblastoma stem-like cells through regulation of HAUS5

Zinc finger domain genes comprise ∼3% of the human genome, yet many of their functions remain unknown. Here we investigated roles for the vertebrate-specific BTB domain zinc finger gene ZNF131 in the context of human brain tumors. We report that ZNF131 is broadly required for Glioblastoma stem-like cell (GSC) viability, but dispensable for neural progenitor cell (NPC) viability. Examination of gene expression changes after ZNF131 knockdown (kd) revealed that ZNF131 activity notably promotes expression of Joubert Syndrome ciliopathy genes, including KIF7, NPHP1, and TMEM237, as well as HAUS5, a component of Augmin/HAUS complex that facilitates microtubule nucleation along the mitotic spindle. Of these genes only kd of HAUS5 displayed GSC-specific viability loss. Critically, HAUS5 ectopic expression was sufficient to suppress viability defects of ZNF131 kd cells. Moreover, ZNF131 and HAUS5 kd phenocopied each other in GSCs, each causing: mitotic arrest, centrosome fragmentation, loss of Augmin/HAUS complex on the mitotic spindle, and loss of GSC self-renewal and tumor formation capacity. In control NPCs, we observed centrosome fragmentation and lethality only when HAUS5 kd was combined with kd of HAUS2 or HAUS4, demonstrating that the complex is essential in NPCs, but that GSCs have heightened requirement. Our results suggest that GSCs differentially rely on ZNF131-dependent expression of HAUS5 as well as the Augmin/HAUS complex activity to maintain the integrity of centrosome function and viability.

CRISPR-Cas9 Screens Reveal Genes Regulating a G0-like State in Human Neural Progenitors

In depth knowledge of the cellular states associated with normal and disease tissue homeostasis is critical for understanding disease etiology and uncovering therapeutic opportunities. Here, we used single cell RNA-seq to survey the cellular states of neuroepithelial-derived cells in cortical and neurogenic regions of developing and adult mammalian brain to compare with 38,474 cells obtained from 59 human gliomas, as well as pluripotent ESCs, endothelial cells, CD45+ immune cells, and non-CNS cancers. This analysis suggests that a significant portion of neuroepithelial-derived stem and progenitor cells and glioma cells that are not in G2/M or S phase exist in two states: G1 or Neural G0, defined by expression of certain neuro-developmental genes. In gliomas, higher overall Neural G0 gene expression is significantly associated with less aggressive gliomas, IDH1 mutation, and extended patient survival, while also anti-correlated with cell cycle gene expression. Knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down regulation of quiescence-associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 is a dynamic cellular state required for indolent cell cycles in neural-specified stem and progenitors poised for cell division. As a result, Neural G0 occupancy may be an important determinant of glioma tumor progression.

Abstract B14: Precision functional genomics for glioblastoma: Identifying molecular therapeutic targets using CRISPR-Cas9 and RNAi technologies in patient isolates

Glioblastoma (GBM) is the most aggressive and common form of adult brain cancer and is among the deadliest cancers, with a median survival of 15 months using standard-of-care therapies. Thus, improved treatments for GBM are desperately needed. To identify new GBM molecular therapeutic targets, our group has performed multiple functional genetic screens in patient-derived GBM stem-like cells (GSCs) and non-transformed human neural stem and progenitor cells (NPCs), which represent non-neoplastic controls. These screens, which have used both RNAi and CRISPR-Cas9 platforms, have led to the identification of several key molecular vulnerabilities in GSCs, including GBM-specific defects in: 39 splice site recognition, kinetochore function, and loss of redundancy between the kinase activities of PKMYT1 and WEE1. At this meeting we will present an overview of these studies, as well as our current efforts to: comprehensively retest all GBM-specific vulnerabilities scoring in these screens; address whether vulnerabilities arise from specific genetic alterations in patient samples (e.g. NF1 loss or PTEN loss); determine whether inhibition of specific molecular targets blocks tumor growth and/or maintenance; and demonstrate the mode of GBM-specific death for particular targets (e.g., cell cycle arrest, apoptosis, etc). In addition, we will highlight both strengths and limitations of applications of CRISPR-Cas9 technologies in patient samples. Collectively, our work illustrates the power of combining functional genetic technologies with the use of patient isolates to identify novel, patient-specific therapeutic strategies for GBM. Citation Format: Pia Hoellerbauer, Heather Feldman, Sonali Arora, Lucas Carter, Emily J. Girard, Philip Corrin, James M. Olson, Eric C. Holland, Patrick J. Paddison. Precision functional genomics for glioblastoma: Identifying molecular therapeutic targets using CRISPR-Cas9 and RNAi technologies in patient isolates [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B14.

Abstract 4370: Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells

Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults. There are currently no effective therapies for GBM. Even with standard of care treatments, such as surgery, radiation, and chemotherapy, ∼90% of adult patients die within 2 years of diagnosis. To identify therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 “knockout” (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality. From a biological standpoint, our results help re-discover PKMYT1 function in human cells. From a cancer standpoint, these results suggest that targeting PKMYT1 is a glioma-lethal gene and that tandem or sequential use of PKMYT1 and WEE1 inhibitors may illicit therapeutic responses in GBM. In addition to these results we will also present retest data for other candidate glioma-lethal genes and networks in multiple patient samples. Citation Format: Chad Toledo, Ding Yu, Pia Hoellerbauer, Ryan Davis, Ryan Basom, Emily Girard, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven Pollard, Jeffery Delrow, Bruce Clurman, James Olson, Patrick Paddison. Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4370.

Abstract C159: Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme

Precision oncology is currently based on the notion that genomic analysis of descriptive molecular signatures from patient tumors will lead to actionable therapeutic targets following the patient9s arrival into the clinic. However, this approach has failed to deliver new effective treatments for glioblastoma multiforme (GBM), which is the most common and aggressive form of brain cancer; and thus, ∼90% of adult GBM patients receiving standard of care therapies continue to die within 2 years of diagnosis. In addition, this approach has neglected the importance for using functional genetics in precision oncology. To identify new therapeutic targets for GBM, we applied functional genetics to perform lethal genome-wide CRISPR-Cas9 knockout screens in patient-derived GBM stem-like cells (GSCs) and also non-transformed human neural stem cells (NSCs), non-neoplastic tissue of origin controls. Here, we present our latest findings from these screens, which include multiple novel GBM-specific lethal genes that were validated by both in vitro and in vivo preclinical studies. Knockout of GBM-specific lethal genes, including the WEE1-like kinase, PKMYT1/Myt1, lead to lethality in GSCs, but not NSCs. Focused mechanistic studies revealed that PKMYT1 acts redundantly with WEE1 to phosphorylate CDK1-Y15 and to promote timely completion of mitosis in NSCs, but that this redundancy is lost in most GBM isolates and in NSCs harboring activated alleles of EGFR and AKT1, which are commonly altered signaling pathways in GBM. Moreover, PKMYT1 depletion in GSCs and genetically altered NSCs requiring PKMYT1 lead to cytokinesis failure and cell death during mitosis. In addition to lethal genes, genes promoting in vitro expansion of NSCs upon knockout were examined. For this category, we validated multiple genes that are candidate tumor suppressors and involved in: the negative regulation of Hippo signaling, TP53 signaling, epigenetic regulation, promoting neural development, and other cellular functions. Knockout of these genes caused shortened cell cycle transit times and drastic growth advantages in NSCs, and in the case of CREBBP knockout, caused precious entry into S-phase and deregulation of cell cycle gene expression. The identification of these potential tumor suppressors here reveal new genetic drivers in glioma/GBM, which contributes to a growing body of work that will help redefine GBM gene signatures. Furthermore, we found that the molecular signatures of pathways and genes commonly altered in GBM are not ideal GBM therapeutic targets since most of these targets failed to score as GBM-specific lethal hits in our knockout screens and likely are non-essential or essential in both the GSCs and NSCs. Nonetheless, these common GBM alterations give rise to cancer-specific vulnerabilities, which then lead to genes, such as PKMYT1, that are required to overcome functional impairments in cancer cells. Taken together, we demonstrated here that genomics and functional genetics are equally important for precision oncology, as the combination of both tools can identify genetically altered genes, cancer-specific therapeutic targets, and tumor suppressors. These results are part of a rapidly growing body of work by the science community that will one day allow oncologists to tailor therapies for each patient based upon his or her tumor genetic profile and characteristics. Citation Format: Chad M. Toledo, Yu Ding, Pia Hoellerbauer, Ryan J. Davis, Ryan Basom, Emily J. Girard, Eunjee Lee, Philip Corrin, Hamid Bolouri, Jerry Davison, Qing Zhang, Do-Hyun Nam, Jeongwu Lee, Steven M. Pollard, Jun Zhu, Jeffery J. Delrow, Bruce E. Clurman, James M. Olson, Patrick J. Paddison. Genome-wide CRISPR-Cas9 screens uncover therapeutic targets and tumor suppressor genes in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C159.