Renee Read

Drosophila Brat and Human Ortholog TRIM3 Maintain Stem Cell Equilibrium and Suppress Brain Tumorigenesis by Attenuating Notch Nuclear Transport

Cancer stem cells exert enormous influence on neoplastic behavior, in part by governing asymmetric cell division and the balance between self-renewal and multipotent differentiation. Growth is favored by deregulated stem cell division, which enhances the self-renewing population and diminishes the differentiation program. Mutation of a single gene in Drosophila, Brain Tumor (Brat), leads to disrupted asymmetric cell division resulting in dramatic neoplastic proliferation of neuroblasts and massive larval brain overgrowth. To uncover the mechanisms relevant to deregulated cell division in human glioma stem cells, we first developed a novel adult Drosophila brain tumor model using brat-RNAi driven by the neuroblast-specific promoter inscuteable Suppressing Brat in this population led to the accumulation of actively proliferating neuroblasts and a lethal brain tumor phenotype. brat-RNAi caused upregulation of Notch signaling, a node critical for self-renewal, by increasing protein expression and enhancing nuclear transport of Notch intracellular domain (NICD). In human glioblastoma, we demonstrated that the human ortholog of Drosophila Brat, tripartite motif-containing protein 3 (TRIM3), similarly suppressed NOTCH1 signaling and markedly attenuated the stem cell component. We also found that TRIM3 suppressed nuclear transport of active NOTCH1 (NICD) in glioblastoma and demonstrated that these effects are mediated by direct binding of TRIM3 to the Importin complex. Together, our results support a novel role for Brat/TRIM3 in maintaining stem cell equilibrium and suppressing tumor growth by regulating NICD nuclear transport. Cancer Res; 76(8); 2443-52. ©2016 AACR.

CDK5 Inhibition Resolves PKA/cAMP-Independent Activation of CREB1 Signaling in Glioma Stem Cells

SUMMARY Cancer stem cells promote neoplastic growth, in part by deregulating asymmetric cell division and enhancing self-renewal. To uncover mechanisms and potential therapeutic targets in glioma stem cell (GSC) self-renewal, we performed a genetic suppressor screen for kinases to reverse the tumor phenotype of our Drosophila brain tumor model and identified dCdk5 as a critical regulator. CDK5, the human ortholog of dCdk5 (79% identity), is aberrantly activated in GBMs and tightly aligned with both chromosome 7 gains and stem cell markers affecting tumor-propagation. Our investigation revealed that pharmaceutical inhibition of CDK5 prevents GSC self-renewal in vitro and in xenografted tumors, at least partially by suppressing CREB1 activation independently of PKA/cAMP. Finally, our TCGA GBM data analysis revealed that CDK5, stem cell, and asymmetric cell division markers segregate within non-mesenchymal patient clusters, which may indicate preferential dependence on CDK5 signaling and sensitivity to its inhibition in this group.

Abstract A02: A kinome-wide RNAi screen in Drosophila glia and human GBM models reveals Stk17A drives neoplastic glial proliferation and migration

Glioblastoma multiforme (GBM), the most common primary malignant brain tumor, are highly proliferative, diffusely invasive, and incurable by current therapies. Genetic and molecular analyses reveal that GBMs frequently harbor activating mutations in the EGFR receptor tyrosine kinase (EGFR) and Pi-3 kinase (PI3K) signaling pathways. While the ability of these mutations to drive gliomagenesis has been verified in mouse models, current data also indicate that EGFR and PI3K signaling cooperates with as yet unknown factors to drive its invasive pathology and therapeutic resistance in GBM. To identify such factors, we performed a cross-species, multidisciplinary genetic screen in both Drosophila melanogaster and mammalian GBM model systems designed to identify novel genes that augment EGFR- and PI3K- dependent neoplasia. Our results in both Drosophila and GBM cell culture and animal model systems indicate that Stk17A, a cytoplasmic serine-threonine kinase subject to copy-gain and overexpression in GBM, is necessary for GBM cell proliferation and invasion. Our data also illustrate that overexpression of Stk17A in combination with oncogenic EGFR mutations is sufficient to promote increased glial cell proliferation and invasion. Furthermore, in our GBM model systems, we find that Stk17A overexpression stimulates increased phosphorylation and activity of non-muscle myosin II regulatory light chain (MRLC), a known Stk17A substrate and key regulator of the cell cycle and cellular motility, which itself is required for neoplastic glial transformation. Finally, oncogenomic analysis of GBMs and other high-grade gliomas reveal that Stk17A becomes overexpressed in association with EGFR-PI3K mutations, and Stk17A copy-gain and overexpression are significantly associated with poor prognosis in patients. Together, these mechanistic insights indicate that Stk17A potentiates the oncogenenic effects of EGFR- and PI3K-driven tumors through the upregulation of MLRC activity, and suggest that Stk17A may serve as a novel therapeutic target for EGFR-PI3K dependent GBMs and high-grade gliomas. Current work is continuing to examine the molecular function of Stk17A in the context of GBM, normal glia, and neural stem cells in order to further our understanding of how Stk17A contributes to tumor pathology. Citation Format: Joanna Wardwell-Ozgo, Colleen Mosley, Harley Kornblum, Renee Read. A kinome-wide RNAi screen in Drosophila glia and human GBM models reveals Stk17A drives neoplastic glial proliferation and migration. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A02.