Chad M. Toledo

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.

Causal Mechanistic Regulatory Network for Glioblastoma Deciphered Using Systems Genetics Network Analysis

We developed the transcription factor (TF)-target gene database and the Systems Genetics Network Analysis (SYGNAL) pipeline to decipher transcriptional regulatory networks from multi-omic and clinical patient data, and we applied these tools to 422 patients with glioblastoma multiforme (GBM). The resulting gbmSYGNAL network predicted 112 somatically mutated genes or pathways that act through 74 TFs and 37 microRNAs (miRNAs) (67 not previously associated with GBM) to dysregulate 237 distinct co-regulated gene modules associated with patient survival or oncogenic processes. The regulatory predictions were associated to cancer phenotypes using CRISPR-Cas9 and small RNA perturbation studies and also demonstrated GBM specificity. Two pairwise combinations (ETV6-NFKB1 and romidepsin-miR-486-3p) predicted by the gbmSYGNAL network had synergistic anti-proliferative effects. Finally, the network revealed that mutations in NF1 and PIK3CA modulate IRF1-mediated regulation of MHC class I antigen processing and presentation genes to increase tumor lymphocyte infiltration and worsen prognosis. Importantly, SYGNAL is widely applicable for integrating genomic and transcriptomic measurements from other human cohorts.

Molecular Pathways: Regulation and Targeting of Kinetochore–Microtubule Attachment in Cancer

Kinetochores are large protein structures assembled on centromeric DNA during mitosis that bind to microtubules of the mitotic spindle to orchestrate and power chromosome movements. Deregulation of kinetochore–microtubule (KT–MT) attachments has been implicated in driving chromosome instability and cancer evolution; however, the nature and source of KT–MT attachment defects in cancer cells remain largely unknown. Here, we highlight recent findings suggesting that oncogene-driven changes in kinetochore regulation occur in glioblastoma multiforme (GBM) and possibly other cancers exhibiting chromosome instability, giving rise to novel therapeutic opportunities. In particular, we consider the GLE2p-binding sequence domains of BubR1 and the newly discovered BuGZ, two kinetochore-associated proteins, as candidate therapeutic targets for GBM. Clin Cancer Res; 21(2); 233–9. ©2014 AACR.

Sensitivity to BUB1B Inhibition Defines an Alternative Classification of Glioblastoma

Glioblastoma multiforme (GBM) remains a mainly incurable disease in desperate need of more effective treatments. In this study, we develop evidence that the mitotic spindle checkpoint molecule BUB1B may offer a predictive marker for aggressiveness and effective drug response. A subset of GBM tumor isolates requires BUB1B to suppress lethal kinetochore-microtubule attachment defects. Using gene expression data from GBM stem-like cells, astrocytes, and neural progenitor cells that are sensitive or resistant to BUB1B inhibition, we created a computational framework to predict sensitivity to BUB1B inhibition. Applying this framework to tumor expression data from patients, we stratified tumors into BUB1B-sensitive (BUB1BS) or BUB1B-resistant (BUB1BR) subtypes. Through this effort, we found that BUB1BS patients have a significantly worse prognosis regardless of tumor development subtype (i.e., classical, mesenchymal, neural, proneural). Functional genomic profiling of BUB1BR versus BUB1BS isolates revealed a differential reliance of genes enriched in the BUB1BS classifier, including those involved in mitotic cell cycle, microtubule organization, and chromosome segregation. By comparing drug sensitivity profiles, we predicted BUB1BS cells to be more sensitive to type I and II topoisomerase inhibitors, Raf inhibitors, and other drugs, and experimentally validated some of these predictions. Taken together, the results show that our BUB1BR/S classification of GBM tumors can predict clinical course and sensitivity to drug treatment. Cancer Res; 77(20); 5518-29. ©2017 AACR.

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 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.

Abstract 1106: Genome-wide CRISPR-Cas9 screens reveal candidate therapeutic targets and tumor suppressor genes for human glioma

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. Our inability to develop new more effective therapies may arise from pre-clinical models that inadequately predict therapeutic window and the fact that many new GBM drugs are “hand-me-downs” from other cancers, not specifically developed for treating brain tumors. To identify patient-tailored drug targets for GBM, our group has performed a series of functional genetic screens in patient derived GBM stem-like cells (GSCs) and also non-transformed human neural stem cells (NSCs). GSCs retain tumor-initiating potential and tumor-specific genetic and epigenetic signatures, even during extended outgrowth in serum-free culture. NSCs represent non-transformed candidate cell of origin controls, which share similar gene expression signatures and identical in vitro growth conditions. Using these systems along with RNAi or CRISPR/Cas9 platforms, we have identified multiple molecular vulnerabilities specific to GSCs, which appear to be largely driven by oncogenic transformation, in processes ranging from kinetochore regulation to 3′ pre-mRNA splice site recognition. At this meeting, we present our latest findings from genome-wide CRISPR-Cas9 gene knockout screens in multiple GSC and NSC isolates. These include validation studies of patient tumor-lethal genes using gene knockout rather than gene knockdown technology. In addition, we will present validation studies of gene products growth limiting for NSC expansion/self-renewal from these screens, a subset of which are candidate tumor suppressors for glioma. Strengths and weaknesses of using gene editing technology and GSC isolates for identification of therapeutic targets will be discussed. Citation Format: Yu Ding, Chad Toledo, Pia Hoellerbauer, Ryan Basom, Emily Girad, Eunjee Lee, Philip Corrin, Qi Lin, Xiao-Nan Li, Do-Hyun Nam, Jeongwu Lee, Jun Zhu, Steven Pollard, Jeffery Delrow, Jim Olson, Patrick J. Paddison. Genome-wide CRISPR-Cas9 screens reveal candidate therapeutic targets and tumor suppressor genes for human glioma. [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 1106. doi:10.1158/1538-7445.AM2015-1106