Chemical genomics reveals targetable programs of human cancers rooted in pluripotency.

Overlapping principles of embryonic and tumor biology have been described, with recent multi-omics campaigns uncovering shared molecular profiles between human pluripotent stem cells (hPSCs) and adult tumors. Here, using a chemical genomic approach, we provide biological evidence that early germ layer fate decisions of hPSCs reveal targets of human cancers. Single-cell deconstruction of hPSCs-defined subsets that share transcriptional patterns with transformed adult tissues. Chemical screening using a unique germ layer specification assay for hPSCs identified drugs that enriched for compounds that selectively suppressed the growth of patient-derived tumors corresponding exclusively to their germ layer origin. Transcriptional response of hPSCs to germ layer inducing drugs could be used to identify targets capable of regulating hPSC specification as well as inhibiting adult tumors. Our study demonstrates properties of adult tumors converge with hPSCs drug induced differentiation in a germ layer specific manner, thereby expanding our understanding of cancer stemness and pluripotency.

PRMT5 inhibition disrupts splicing and stemness in glioblastoma.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.

Brain Tumor Stem Cell Dependence on Glutaminase Reveals a Metabolic Vulnerability through the Amino Acid Deprivation Response Pathway.

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. SIGNIFICANCE: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.

Isolation and Culture of Glioblastoma Brain Tumor Stem Cells.

Cancer stem cells (CSCs) have been identified in glioblastoma (GBM) and are proposed to be the main actors of post-treatment recurrence contributing to the very dismal prognosis of this devastating disease. Consequently, this important population of cells needs to be further studied to uncover potential vulnerabilities, identify novel therapeutic targets, and develop drugs that can be translated to the clinic. One obstacle preventing progress in understanding the biology of GBM and the development of novel therapies has arguably been the absence of biologically relevant in vitro models representative of the CSC population in GBM. Adherent and non-adherent serum-free culture methods, initially developed for culturing neural stem cells, have been adapted to identify, isolate, maintain, and expand brain tumor stem cells (BTSCs) from GBM. In this chapter, we describe a method to isolate and culture these BTSCs from fresh GBM patient samples.

Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells.

Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance.

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma.

Cellular senescence is a tumor suppressor mechanism where cells enter a permanent growth arrest following cellular stress. Oncogene-induced senescence (OIS) is induced in non-malignant cells following the expression of an oncogene or inactivation of a tumor suppressor. Previously, we have shown that protein kinase C iota (PKCι) depletion induces cellular senescence in glioblastoma cells in the absence of a detectable DNA damage response. Here we demonstrate that senescent glioblastoma cells exhibit an aberrant centrosome morphology. This was observed in basal levels of senescence, in p21-induced senescence, and in PKCι depletion-induced senescence. In addition, senescent glioblastoma cells are polyploid, Ki-67 negative and arrest at the G1/S checkpoint, as determined by expression of cell cycle regulatory proteins. These markers are all consistent with cells that have undergone mitotic slippage. Failure of the spindle assembly checkpoint to function properly can lead to mitotic slippage, resulting in the premature exit of mitotic cells into the G1 phase of the cell cycle. Although in G1, these cells have the replicated DNA and centrosomal phenotype of a cell that has entered mitosis and failed to divide. Overall, we demonstrate that PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma cells. To our knowledge, this is the first evidence of markers of mitotic slippage directly in senescent cells by co-staining for senescence-associated ß-galactosidase and immunofluorescence markers in the same cell population. We suggest that markers of mitotic slippage be assessed in future studies of senescence to determine the extent of mitotic slippage in the induction of cellular senescence.

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1.

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Induction of premature senescence by hsp90 inhibition in small cell lung cancer.

BACKGROUND: The molecular chaperone Hsp90 is a promising new target in cancer therapy and selective Hsp90 inhibitors are currently in clinical trials. Previously these inhibitors have been reported to induce either cell cycle arrest or cell death in cancer cells. Whether the cell cycle arrest is reversible or irreversible has not generally been assessed. Here we have examined in detail the cell cycle arrest and cell death responses of human small cell lung cancer cell lines to Hsp90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: In MTT assays, small cell lung cancer cells showed a biphasic response to the Hsp90 inhibitors geldanamycin and radicicol, with low concentrations causing proliferation arrest and high concentrations causing cell death. Assessment of Hsp90 intracellular activity using loss of client protein expression showed that geldanamycin concentrations that inhibited Hsp90 correlated closely with those causing proliferation arrest but not cell death. The proliferation arrest induced by low concentrations of geldanamycin was not reversed for a period of over thirty days following drug removal and showed features of senescence. Rare populations of variant small cell lung cancer cells could be isolated that had additional genetic alterations and no longer underwent irreversible proliferation arrest in response to Hsp90 inhibitors. CONCLUSIONS/SIGNIFICANCE: We conclude that: (1) Hsp90 inhibition primarily induces premature senescence, rather than cell death, in small cell lung cancer cells; (2) small cell lung cancer cells can bypass this senescence through further genetic alterations; (3) Hsp90 inhibitor-induced cell death in small cell lung cancer cells is due to inhibition of a target other than cytosolic Hsp90. These results have implications with regard to how these inhibitors will behave in clinical trials and for the design of future inhibitors in this class.