Aurora kinase A inhibition reverses the Warburg effect and elicits unique metabolic vulnerabilities in glioblastoma

Aurora kinase A (AURKA) has emerged as a drug target for glioblastoma (GBM). However, resistance to therapy remains a critical issue. By integration of transcriptome, chromatin immunoprecipitation sequencing (CHIP-seq), Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), proteomic and metabolite screening followed by carbon tracing and extracellular flux analyses we show that genetic and pharmacological AURKA inhibition elicits metabolic reprogramming mediated by inhibition of MYC targets and concomitant activation of Peroxisome Proliferator Activated Receptor Alpha (PPARA) signaling. While glycolysis is suppressed by AURKA inhibition, we note an increase in the oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO), which was accompanied by an increase of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). Combining AURKA inhibitors with inhibitors of FAO extends overall survival in orthotopic GBM PDX models. Taken together, these data suggest that simultaneous targeting of oxidative metabolism and AURKAi might be a potential novel therapy against recalcitrant malignancies

CLONING AND CHARACTERIZATION OF EXON EDITED HOTSPOT 1 ROD REGION DYSTROPHIN PROTEINS

Duchenne Muscular Dystrophy, DMD, one of the most common fatal genetic diseases, is caused by the absence of dystrophin protein. This protein is coded by the largest gene in the human genome, which has 79 exons and spans 2.4 Mbp. DMD affects 1 in 3600 boys and the average life expectancy of patients is 25 years. The most common type of defects leading to DMD are large exonic deletions that juxtapose remaining exons of incompatible phases, causing a frameshift. Inevitably this introduces a nonsense mutation (i.e. a stop codon) in the frame shifted exons and thus protein truncation. A related but milder condition called Becker’s Muscular Dystrophy, BMD, results when deletions are in-frame and so allow for the production of some, albeit modified, dystrophin protein. While there is no treatment available for DMD the recent clinical trials involving exon skipping suggest this will be highly promising treatment option. Exon skipping therapy is a strategy that uses anti-sense oligonucleotide analogs, AONs, to induce skipping of additional exons during mRNA maturation and thus restore the reading frame. This allows dystrophin translation to continue to its normal C-terminus, albeit with an internal deletion edit corresponding to both the original deletion, and newly skipped exon, which results in expression of protein thus converting the severe form of DMD to a milder form BMD. Frameshift causing deletions require exons that begin and end in different phases, and DMD deletions thus are clustered where such exons are located in, two so called ‘hotspots’ in the gene. The first of these occur in the region from exon 11-22 and is known as Hotspot 1. In many cases, DMD defects that are amenable to exon skip repair can be repaired in two or more alternative fashions by skipping of alternative exons. It is thought that the differences in severity of BMD, can range from being nearly as debilitating as DMD, to xi nearly benign, are at least in part related to the nature of the defect and its impact on the protein’s structure. Thus we are producing 5 different exon edited proteins from hotspot 1 rod region and assessing them for structure and stability as compared to unskipped fully functional dystrophin, to determine which edits hold the most promise of producing a well formed and stable edit.M.S. in Biology, December 201

EZH2 Inhibition Sensitizes IDH1R132H-Mutant Gliomas to Histone Deacetylase Inhibitor

Isocitrate Dehydrogenase-1 (IDH1) is commonly mutated in lower-grade diffuse gliomas. The IDH1R132H mutation is an important diagnostic tool for tumor diagnosis and prognosis; however, its role in glioma development, and its impact on response to therapy, is not fully understood. We developed a murine model of proneural IDH1R132H-mutated glioma that shows elevated production of 2-hydroxyglutarate (2-HG) and increased trimethylation of lysine residue K27 on histone H3 (H3K27me3) compared to IDH1 wild-type tumors. We found that using Tazemetostat to inhibit the methyltransferase for H3K27, Enhancer of Zeste 2 (EZH2), reduced H3K27me3 levels and increased acetylation on H3K27. We also found that, although the histone deacetylase inhibitor (HDACi) Panobinostat was less cytotoxic in IDH1R132H-mutated cells (either isolated from murine glioma or oligodendrocyte progenitor cells infected in vitro with a retrovirus expressing IDH1R132H) compared to IDH1-wild-type cells, combination treatment with Tazemetostat is synergistic in both mutant and wild-type models. These findings indicate a novel therapeutic strategy for IDH1-mutated gliomas that targets the specific epigenetic alteration in these tumors

Ribosomal protein S11 influences glioma response to TOP2 poisons.

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas, the most malignant of all primary brain tumors in adults, is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, and 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of proapoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers

Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic and targetable tissue states

Abstract Glioblastoma (GBM) diffusely infiltrates the brain and intermingles with non-neoplastic brain cells, including astrocytes, neurons and microglia/myeloid cells. This complex mixture of cell types forms the biological context for therapeutic response and tumor recurrence. We used single-nucleus RNA sequencing and spatial transcriptomics to determine the cellular composition and transcriptional states in primary and recurrent glioma and identified three compositional ‘tissue-states’ defined by cohabitation patterns between specific subpopulations of neoplastic and non-neoplastic brain cells. These tissue-states correlated with radiographic, histopathologic, and prognostic features and were enriched in distinct metabolic pathways. Fatty acid biosynthesis was enriched in the tissue-state defined by the cohabitation of astrocyte-like/mesenchymal glioma cells, reactive astrocytes, and macrophages, and was associated with recurrent GBM and shorter survival. Treating acute slices of GBM with a fatty acid synthesis inhibitor depleted the transcriptional signature of this pernicious tissue-state. These findings point to therapies that target interdependencies in the GBM microenvironment

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration