DDRE-21. THE FUNCTION OF MITOCHONDRIAL OSMR IN GLIOMA STEM CELL RESPIRATION AND GLIOBLASTOMA PATHOGENESIS

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. We have discovered that OSMR translocates to the mitochondria and regulates oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.

EGFR blockade in GBM brain tumor stem cells synergizes with JAK2/STAT3 pathway inhibition to abrogate compensatory mechanisms in vitro and in vivo

Background The EGFR pathway is frequently mutated in glioblastoma (GBM). However, to date, EGFR therapies have not demonstrated efficacy in clinical trials. Poor brain penetration of conventional inhibitors, lack of patient stratification for EGFR status, and mechanisms of resistance are likely responsible for the failure of EGFR-targeted therapy. We aimed to address these elements in a large panel of molecularly diverse patient-derived GBM brain tumor stem cells (BTSCs). Methods In vitro growth inhibition and on-target efficacy of afatinib, pacritinib, or a combination were assessed by cell viability, neurosphere formation, cytotoxicity, limiting dilution assays, and western blotting. In vivo efficacy was assessed with mass spectrometry, immunohistochemistry, magnetic resonance imaging, and intracranial xenograft models. Results We show that afatinib and pacritinib decreased BTSC growth and sphere-forming capacity in vitro. Combinations of the 2 drugs were synergistic and abrogated the activation of STAT3 signaling observed upon EGFR inhibition in vitro and in vivo. We further demonstrate that the brain-penetrant EGFR inhibitor, afatinib, improved survival in EGFRvIII mt orthotopic xenograft models. However, upregulation of the oncogenic STAT3 signaling pathway was observed following afatinib treatment. Combined inhibition with 2 clinically relevant drugs, afatinib and pacritinib, synergistically decreased BTSC viability and abrogated this compensatory mechanism of resistance to EGFR inhibition. A significant decrease in tumor burden in vivo was observed with the combinatorial treatment. Conclusions These data demonstrate that brain-penetrant combinatorial therapies targeting the EGFR and STAT3 signaling pathways hold therapeutic promise for GBM.

TMIC-56. ROLE OF HUMAN BRAIN TUMOR STEM CELLS-DERIVED EXTRACELLULAR VESICLES ON THE PHENOTYPIC TRANSDIFFERENTIATION OF HUMAN NEURAL PROGENITOR CELLS

Glioblastoma (GBM) is the most aggressive of all the brain tumors with a median patient survival less than 15 months. Despite of surgical resection, radiotherapy, and chemotherapy, recurrence rate is almost 100%. A great percentage of GBM tumors (~60%) infiltrate and contact the ventricular-subventricular zone (V-SVZ). Interestingly, these tumors are the most aggressive, and invariably lead to higher distal recurrence rates, shorter time to tumor progression, and lower overall survival of the patient. The reason for this role of V-SVZ-proximity on the outcome of GBM patients is unknown. We suggest that a potential explanation is the interaction of GBM with the V-SVZ. This region is the largest neurogenic niche in the adult brain where neural stem cells (NSCs) give rise to newborn neuroblasts that migrate toward the olfactory bulb. In GBM there is a cell subpopulation called brain tumor stem cells (BTSCs) with NSCs-like characteristics, but with added potential for tumor initiation, recurrence and invasiveness. Tumor microenvironment plays an important role in migration and invasion process. In the present work, we used the total exosome isolation kit to purify Extracellular Vesicles (EVs) from human primary cultures of BTSCs. We determined that BTSCs-derived EVs contain specific information that is transfer to primary cultures of human Neural Progenitors Cells (NPCs) modulating their proliferation rate, cell viability, and migration. In addition, we identify that NPCs taken up BTSCs-derived EVs and significantly increase the expression levels of stemness-related genes such as Nestin, Nanog, and Sox2, suggesting that a phenotypic transdifferentiation is being carry out. These results support our hypothesis that GBM modulate the tumor microenvironment close to the V-SVZ by releasing EVs that target cellular components in this region and promote their phenotypic transformation, highlighting that NPCs biology changes in the context of tumor environment.

STEM-21. INVESTIGATING DOT1L AS AN EPIGENETIC VULNERABILITY IN BRAIN TUMOR STEM CELLS

Glioblastoma (GBM), the most common and aggressive primary adult brain cancer, is thought to be driven by a small subpopulation of brain tumor stem cells (BTSCs). BTSCs exhibit shared properties with normal stem cells such as self-renewal and multilineage differentiation. These stem cell properties have been proposed to underlie GBM tumorigenicity, treatment evasion and contribute to tumor heterogeneity. To investigate the biology underlying the stem cell properties of GBM, we compared gene essentiality profiles for a panel of BTSCs, fetal neural stem cells and non-GBM cell lines using a CRISPR Cas9 knockout library. Interestingly, from these screens, we identified the histone methyltransferase disrupter of telomeric silencing-1-like (DOT1L) as an essential gene for the growth of BTSCs and fetal neural stem cells but not for non-GBM cell lines. DOT1L is the only known histone methyltransferase responsible for histone 3 lysine 79 methylation, an epigenetic mark associated with active gene transcription. The role of this epigenetic regulator in BTSCs was investigated in depth using EPZ-5676, a clinically relevant small molecule inhibitor. Short-term treatment with EPZ-5676 in BTSCs showed minimal effects on cell viability but led to striking morphological changes, increased neuronal and astrocytic differentiation and a reduction in self-renewal. Longer treatment periods with EPZ-5676 led to a decrease in BTSC proliferation and an increase in apoptosis. Furthermore, BTSCs pretreated with EPZ-5676 led to slowed orthotopic tumor growth and improved overall survival in a SCID mouse model. Overall, these findings suggest DOT1L epigenetically regulates GBM stem cell properties and tumor growth. We are further investigating the mechanisms underlying DOT1L regulation of gene expression in BTSCs with the goal of improving the field’s understanding of epigenetics and the therapeutic implications of targeting epigenetic processes in GBM.

SLUG Directs the Precursor State of Human Brain Tumor Stem Cells

In glioblastoma (GBM), brain tumor stem cells (BTSCs) encompass heterogenous populations of multipotent, self-renewing, and tumorigenic cells, which have been proposed to be at the root of therapeutic resistance and recurrence. While the functional significance of BTSC heterogeneity remains to be fully determined, we previously distinguished relatively quiescent stem-like precursor state from the more aggressive progenitor-like precursor state. In the present study, we hypothesized that progenitor-like BTSCs arise from stem-like precursors through a mesenchymal transition and drive post-treatment recurrence. We first demonstrate that progenitor-like BTSCs display a more mesenchymal transcriptomic profile. Moreover, we show that both mesenchymal GBMs and progenitor-like BTSCs are characterized by over-activated STAT3/EMT pathways and that SLUG is the primary epithelial to mesenchymal transition (EMT) transcription factor directly regulated by STAT3 in BTSCs. SLUG overexpression in BTSCs enhances invasiveness, promotes inflammation, and shortens survival. Importantly, SLUG overexpression in a quiescent stem-like BTSC line enhances tumorigenesis. Finally, we report that recurrence is associated with SLUG-induced transcriptional changes in both BTSCs and GBM patient samples. Collectively, our findings show that a STAT3-driven precursor state transition, mediated by SLUG, may prime BTSCs to initiate more aggressive mesenchymal recurrence. Targeting the STAT3/SLUG pathway may maintain BTSCs in a quiescent stem-like precursor state, delaying recurrence and improving survival in GBM.