Neural G0:a quiescent-like state found in neuroepithelial-derived cells and glioma

Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that 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 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas

Identification of Genetic Vulnerabilities in Glioblastoma and Other Cancers Using CRISPR-Cas9-based Functional Genomic Approaches

Thesis (Ph.D.)--University of Washington, 2020Glioblastoma (GBM) is the most common and aggressive form of brain cancer. It remains one of the deadliest cancers, with 90% of adult patients dying within two years of diagnosis with standard-of-care treatments. Although directly targeting oncogenic driver mutations is a popular strategy in developing new therapeutics, it has been met with limited success. Thus, we take the alternative, unbiased approach of using functional genomic screens to identify molecular vulnerabilities that arise in cancer cells due to the oncogenic state. In order to identify genes required for the proliferation and survival of GBM cells, the Paddison Lab previously performed lentiviral genome-scale pooled RNAi and CRISPR-Cas9 outgrowth screens in patient-derived GBM stem-like cells (GSCs) and human nontransformed neural stem cells (NSCs). Retesting screen hits using single CRISPR sgRNAs can prove challenging with lentivirus due to protracted windows of indel formation (editing over the course of ~5-12 days), resulting in phenotypically mixed populations. Here, to address this challenge, I optimized a method for direct nucleofection of ribonucleoprotein complexes (RNPs) composed of chemically synthesized 2'-O-methyl 3’phosphorothioate-modified sgRNA and purified Cas9 protein. With this technique, we can routinely achieve >90% indel formation as well as targeted genomic deletions in only 3 days, even in hyperdiploid cells (such as GSCs), with no need to create clonal lines for simple loss-of-function experiments. Additionally in this body of work, to further characterize candidate hits specific to GSCs (and not NSCs), we performed comprehensive pooled outgrowth retests of all putative screen hits that scored preferentially in GSC isolates in our whole-genome CRISPR-Cas9 screens. We then used these results to identify both GBM gene dependency groups and context-specific vulnerabilities, which was facilitated by incorporating published functional genomic screening data (DepMap database) into the analyses. By creating co-dependency networks, we identified GSC-specific gene vulnerability groups related to mitochondrial protein processing and turnover and membrane trafficking as well as metabolic enzymes and regulators. Furthermore, we also identified predicted context-specific vulnerabilities and further investigated the dsRNA-editing enzyme ADAR, which is required in cancer cells that have an interferon-stimulated gene expression signature, and the adapter protein EFR3A, which is required in cancer cells that have low expression of its paralog EFR3B. In addition, we investigated the particularly strongly GSC-specific screen hit FBXO42. We found this F-box protein to be essential in a subset of GSCs in vitro and in vivo but nonessential in NSCs in vitro, in addition to being required in subsets of cells of various other cancer types, suggesting the potential for both a large therapeutic window and broad applicability. Furthermore, we demonstrated that the ubiquitin ligase role of FBXO42 is responsible for the viability phenotype, but likely not through p53 as a previous study might suggest. In searching for possible interactors, we determined that FBXO42 and the gene CCDC6 (Coiled-Coil Domain Containing 6, a common translocation partner for receptor kinase oncogenes) are both necessary to promote viability in FBXO42 loss-sensitive cells, likely working together rather than redundantly. Lastly, we demonstrated that the GSC-specific viability loss is due to an extended metaphase arrest upon FBXO42 knockout due to prolonged spindle assembly checkpoint activation. Altogether, this body of work both improves upon existing CRISPR-Cas9 technologies and uses this technology as part of an effort to identify potential novel therapeutic opportunities in GBM and other cancers

A kinase-deficient NTRK2 splice variant predominates in glioma and amplifies several oncogenic signaling pathways.

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.We thank James Yan, Jenny Zhang, Deby Kumasaka, and Denis Adair for continued technical and administrative assistance and support throughout these experiments. Eero Castren at University of Helsinki provided pEF-BOS-TrkB plasmids used for RCAS-TrkB.T1 generation. We thank Francis S. Lee and Lino Tessarollo for providing breeding pairs of TrkB.T1-/- mice for antibody production. Luis Chiriboga at New York University School of Medicine provided helpful insight into histology protocols. We thank William A. Johnsen and Midori Clarke for assistance with antibody sequencing and protein purification. We thank the Tracy A. Goodpaster and Julie Randolph-Habecker at the Fred Hutchinson Experimental Histopathology Core for histology assistance during the antibody validation phase and Elizabeth Jensen at the Fred Hutchinson Genomics Core for help with all DNA sequencing. We thank Jeongwu Lee Do-Hyun Nam and Steven M. Pollard for providing cell isolates. Funding was provided by National Institutes of Health R01 CA195718 (E.C.H.), U54 CA193461 (E.C.H., F.S.), R01 CA100688 (E.C.H.), T32 CA965725 (S.S.P.), U54 DK106829 (S.S.P.), R21 CA223531 (S.S.P., E.C.H.), T32 CA080416 (P.H.), R01 CA190957 (P.J.P.; T.B.); Jacobs Foundation Research Fellowship (S.S.P.); American Cancer Society ACS-RSG-14-056-01 (P.J.P.); National Research Agency RTI2018-102035-B-I00 (M.S.); Seve Ballesteros Foundation (M.S.). Autopsy materials used in this study were obtained from the University of Washington Neuropathology Core, which is supported by the Alzheimer's Disease Research Center (AG05136), the Adult Changes in Thought Study (AG006781), and Morris K Udall Center of Excellence for Parkinson's Disease Research (NS062684)S

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

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent β-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML