Glioma Stem Cells Are Sensitized to BCL-2 Family Inhibition by Compromising Histone Deacetylases.

Glioblastoma (GBM) remains an incurable disease with an extremely high five-year recurrence rate. We studied apoptosis in glioma stem cells (GSCs) in response to HDAC inhibition (HDACi) combined with MEK1/2 inhibition (MEKi) or BCL-2 family inhibitors. MEKi effectively combined with HDACi to suppress growth, induce cell cycle defects, and apoptosis, as well as to rescue the expression of the pro-apoptotic BH3-only proteins BIM and BMF. A RNAseq analysis of GSCs revealed that HDACi repressed the pro-survival BCL-2 family genes MCL1 and BCL-XL. We therefore replaced MEKi with BCL-2 family inhibitors and observed enhanced apoptosis. Conversely, a ligand for the cancer stem cell receptor CD44 led to reductions in BMF, BIM, and apoptosis. Our data strongly support further testing of HDACi in combination with MEKi or BCL-2 family inhibitors in glioma.

Relationships between gene expression and behavior in mice in response to systemic modulation of the O-GlcNAcylation pathway.

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA), which removes the O-GlcNAc modification from proteins, has been explored in mouse models of amyloid-beta and tau pathology. However, the O-GlcNAcylation-dependent link between gene expression and neurological behavior remains to be explored. Using chronic administration of Thiamet G (TG, an OGA inhibitor) in vivo, we used a protocol designed to relate behavior with the transcriptome and selected biochemical parameters from the cortex of individual animals. TG-treated mice showed improved working memory as measured using a Y-maze test. RNA sequencing analysis revealed 151 top differentially expressed genes with a Log2fold change >0.33 and adjusted p-value <0.05. Top TG-dependent upregulated genes were related to learning, cognition and behavior, while top downregulated genes were related to IL-17 signaling, inflammatory response and chemotaxis. Additional pathway analysis uncovered 3 pathways, involving gene expression including 14 cytochrome c oxidase subunits/regulatory components, chaperones or assembly factors, and 5 mTOR (mechanistic target of rapamycin) signaling factors. Multivariate Kendall correlation analyses of behavioral tests and the top TG-dependent differentially expressed genes revealed 91 statistically significant correlations in saline-treated mice and 70 statistically significant correlations in TG-treated mice. These analyses provide a network regulation landscape that is important in relating the transcriptome to behavior and the potential impact of the O-GlcNAC pathway.

ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors.

Metastatic urothelial carcinoma is generally incurable with current systemic therapies. Chromatin modifiers are frequently mutated in bladder cancer, with ARID1A-inactivating mutations present in about 20% of tumors. EZH2, a histone methyltransferase, acts as an oncogene that functionally opposes ARID1A. In addition, PI3K signaling is activated in more than 20% of bladder cancers. Using a combination of in vitro and in vivo data, including patient-derived xenografts, we show that ARID1A-mutant tumors were more sensitive to EZH2 inhibition than ARID1A WT tumors. Mechanistic studies revealed that (a) ARID1A deficiency results in a dependency on PI3K/AKT/mTOR signaling via upregulation of a noncanonical PI3K regulatory subunit, PIK3R3, and downregulation of MAPK signaling and (b) EZH2 inhibitor sensitivity is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. We show that PIK3IP1 inhibited PI3K signaling by inducing proteasomal degradation of PIK3R3. Furthermore, ARID1A-deficient bladder cancer was sensitive to combination therapies with EZH2 and PI3K inhibitors in a synergistic manner. Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K and revealed mechanistic insights into the role of noncanonical PI3K constituents in bladder cancer biology.

PVT1 is a stress-responsive lncRNA that drives ovarian cancer metastasis and chemoresistance.

Metastatic growth of ovarian cancer cells into the peritoneal cavity requires adaptation to various cellular stress factors to facilitate cell survival and growth. Here, we demonstrate the role of PVT1, one such stress induced long non-coding RNA, in ovarian cancer growth and metastasis. PVT1 is an amplified and overexpressed lncRNA in ovarian cancer with strong predictive value for survival and response to targeted therapeutics. We find that expression of PVT1 is regulated by tumor cells in response to cellular stress, particularly loss of cell-cell contacts and changes in matrix rigidity occurring in a YAP1-dependent manner. Induction of PVT1 promotes tumor cell survival, growth, and migration. Conversely, reducing PVT1 levels robustly abrogates metastatic behavior and tumor cell dissemination in cell lines and syngeneic transplantation models in vivo. We find that reducing PVT1 causes widespread changes in the transcriptome leading to alterations in cellular stress response and metabolic pathways including doxorubicin metabolism, which impacts chemosensitivity. Together, these findings implicate PVT1 as a promising therapeutic target to suppress metastasis and chemoresistance in ovarian cancer.

PIK3CA missense mutations promote glioblastoma pathogenesis, but do not enhance targeted PI3K inhibition.

BACKGROUND: Glioblastoma (GBM) is the most common adult primary brain tumor. Multimodal treatment is empiric and prognosis remains poor. Recurrent PIK3CA missense mutations (PIK3CAmut) in GBM are restricted to three functional domains: adaptor binding (ABD), helical, and kinase. Defining how these mutations influence gliomagenesis and response to kinase inhibitors may aid in the clinical development of novel targeted therapies in biomarker-stratified patients. METHODS: We used normal human astrocytes immortalized via expression of hTERT, E6, and E7 (NHA). We selected two PIK3CAmut from each of 3 mutated domains and induced their expression in NHA with (NHARAS) and without mutant RAS using lentiviral vectors. We then examined the role of PIK3CAmut in gliomagenesis in vitro and in mice, as well as response to targeted PI3K (PI3Ki) and MEK (MEKi) inhibitors in vitro. RESULTS: PIK3CAmut, particularly helical and kinase domain mutations, potentiated proximal PI3K signaling and migration of NHA and NHARAS in vitro. Only kinase domain mutations promoted NHA colony formation, but both helical and kinase domain mutations promoted NHARAS tumorigenesis in vivo. PIK3CAmut status had minimal effects on PI3Ki and MEKi efficacy. However, PI3Ki/MEKi synergism was pronounced in NHA and NHARAS harboring ABD or helical mutations. CONCLUSION: PIK3CAmut promoted differential gliomagenesis based on the mutated domain. While PIK3CAmut did not influence sensitivity to single agent PI3Ki, they did alter PI3Ki/MEKi synergism. Taken together, our results demonstrate that a subset of PIK3CAmut promote tumorigenesis and suggest that patients with helical domain mutations may be most sensitive to dual PI3Ki/MEKi treatment.