Andrew V. Kossenkov

Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A -mutated cancers

ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers.The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K–AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

Gene Expression Profiles in Peripheral Blood Mononuclear Cells Can Distinguish Patients with Non–Small Cell Lung Cancer from Patients with Nonmalignant Lung Disease

Early diagnosis of lung cancer followed by surgery presently is the most effective treatment for non-small cell lung cancer (NSCLC). An accurate, minimally invasive test that could detect early disease would permit timely intervention and potentially reduce mortality. Recent studies have shown that the peripheral blood can carry information related to the presence of disease, including prognostic information and information on therapeutic response. We have analyzed gene expression in peripheral blood mononuclear cell samples including 137 patients with NSCLC tumors and 91 patient controls with nonmalignant lung conditions, including histologically diagnosed benign nodules. Subjects were primarily smokers and former smokers. We have identified a 29-gene signature that separates these two patient classes with 86% accuracy (91% sensitivity, 80% specificity). Accuracy in an independent validation set, including samples from a new location, was 78% (sensitivity of 76% and specificity of 82%). An analysis of this NSCLC gene signature in 18 NSCLCs taken presurgery, with matched samples from 2 to 5 months postsurgery, showed that in 78% of cases, the signature was reduced postsurgery and disappeared entirely in 33%. Our results show the feasibility of using peripheral blood gene expression signatures to identify early-stage NSCLC in at-risk populations.

Lineage and species-specific long noncoding RNAs during erythro-megakaryocytic development

Mammals express thousands of long noncoding (lnc) RNAs, a few of which are known to function in tissue development. However, the entire repertoire of lncRNAs in most tissues and species is not defined. Indeed, most lncRNAs are not conserved, raising questions about function. We used RNA sequencing to identify 1109 polyadenylated lncRNAs expressed in erythroblasts, megakaryocytes, and megakaryocyte-erythroid precursors of mice, and 594 in erythroblasts of humans. More than half of these lncRNAs were unannotated, emphasizing the opportunity for new discovery through studies of specialized cell types. Analysis of the mouse erythro-megakaryocytic polyadenylated lncRNA transcriptome indicates that ~75% arise from promoters and 25% from enhancers, many of which are regulated by key transcription factors including GATA1 and TAL1. Erythroid lncRNA expression is largely conserved among 8 different mouse strains, yet only 15% of mouse lncRNAs are expressed in humans and vice versa, reflecting dramatic species-specificity. RNA interference assays of 21 abundant erythroid-specific murine lncRNAs in primary mouse erythroid precursors identified 7 whose knockdown inhibited terminal erythroid maturation. At least 6 of these 7 functional lncRNAs have no detectable expression in human erythroblasts, suggesting that lack of conservation between mammalian species does not predict lack of function.

Stem Cells with Neural Crest Characteristics Derived from the Bulge Region of Cultured Human Hair Follicles

In this study, we demonstrate that we can isolate stem cells (SCs) with neural crest characteristics from the bulge area of cultured human hair follicles (HFs). These SCs can proliferate in situ and form spheroid structures attached to the bulge area of HFs, and they express immature neural crest cell markers but not differentiation markers. An expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. These human SCs are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. In addition, these SCs show differentiation potential toward mesenchymal lineages, and they can be differentiated into adipocyte, chondrocyte, and osteocyte lineages. Neuronal differentiation of these cells induces global gene expression changes with a significantly increased expression of neuron-associated genes. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. The presence of SCs with neural crest characteristics in HFs may offer new opportunities for the use of these cells in regenerative medicine.

sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

LS-NMF: A modified non-negative matrix factorization algorithm utilizing uncertainty estimates

BackgroundNon-negative matrix factorisation (NMF), a machine learning algorithm, has been applied to the analysis of microarray data. A key feature of NMF is the ability to identify patterns that together explain the data as a linear combination of expression signatures. Microarray data generally includes individual estimates of uncertainty for each gene in each condition, however NMF does not exploit this information. Previous work has shown that such uncertainties can be extremely valuable for pattern recognition.ResultsWe have created a new algorithm, least squares non-negative matrix factorization, LS-NMF, which integrates uncertainty measurements of gene expression data into NMF updating rules. While the LS-NMF algorithm maintains the advantages of original NMF algorithm, such as easy implementation and a guaranteed locally optimal solution, the performance in terms of linking functionally related genes has been improved. LS-NMF exceeds NMF significantly in terms of identifying functionally related genes as determined from annotations in the MIPS database.ConclusionUncertainty measurements on gene expression data provide valuable information for data analysis, and use of this information in the LS-NMF algorithm significantly improves the power of the NMF technique.

Landscape of the mitochondrial Hsp90 metabolome in tumours

Reprogramming of tumor cell metabolism contributes to disease progression and resistance to therapy, but how this process is regulated on the molecular level is unclear. Here we report that Heat Shock Protein 90 (Hsp90)-directed protein folding in mitochondria controls central metabolic networks in tumor cells, including the electron transport chain, citric acid cycle, fatty acid oxidation, amino acid synthesis, and cellular redox status. Specifically, mitochondrial Hsp90, but not cytosolic Hsp90, binds and stabilizes the electron transport chain Complex II subunit succinate dehydrogenase-B, maintaining cellular respiration under low-nutrient conditions, and contributing to hypoxia-inducible factor-1α-mediated tumorigenesis in patients carrying succinate dehydrogenase-B mutations. Thus, Hsp90-directed proteostasis in mitochondria regulates tumor cell metabolism, and may provide a tractable target for cancer therapy.

The Rac1 splice form Rac1b promotes K-ras-induced lung tumorigenesis

Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. Although expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis.

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients

PMN-MDSC in cancer patients can be distinguished from neutrophils by a genomic signature and by expression of the LOX-1 receptor. Stressing myeloid-derived suppressor cells in cancer Immunotherapies for cancer have shown promising results in part because they overcome the suppressive effects of the tumor microenvironment on immune cells. Condamine et al. now report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) can be distinguished from neutrophils in the same cancer patient by the expression of the lipid metabolism–related molecule lectin-type oxidized LDL receptor-1 (LOX-1). LOX-1–expressing neutrophils were nearly undetectable in healthy individuals but were found prominently in tumor tissues. Moreover, exposing neutrophils from healthy individuals to endoplasmic reticulum stress resulted in up-regulation of LOX-1 and increased suppressive function. These data support the specific targeting of LOX-1–expressing PMN-MDSC for cancer immunotherapy. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are important regulators of immune responses in cancer and have been directly implicated in the promotion of tumor progression. However, the heterogeneity of these cells and the lack of distinct markers hamper the progress in understanding the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation, we determined that low-density PMN-MDSC and high-density neutrophils from the same cancer patients had a distinct gene profile. The most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Unexpectedly, low-density lipoprotein (LDL) was one of the most increased regulators, and its receptor oxidized LDL receptor 1 (OLR1) was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor-1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5 to 15% of total neutrophils in cancer patients and 15 to 50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1− counterparts, LOX-1+ neutrophils had gene signature, potent immunosuppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSCs. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSCs. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insights into the biology and potential therapeutic targeting of these cells.

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion

Significance Despite the promise of personalized cancer medicine, most molecular therapies produce only modest and short-lived patient gains. In addition to drug resistance, it is also possible that tumors adaptively reprogram their signaling pathways to evade therapy-induced “stress” and, in the process, acquire more aggressive disease traits. We show here that small-molecule inhibitors of PI3K, a cancer node and important therapeutic target, induce transcriptional and signaling reprogramming in tumors. This involves the trafficking of energetically active mitochondria to subcellular sites of cell motility, where they provide a potent, “regional” energy source to support tumor cell invasion. Although this response may paradoxically increase the risk of metastasis during PI3K therapy, targeting mitochondrial reprogramming is feasible, and could provide a novel therapeutic strategy. Molecular therapies are hallmarks of “personalized” medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, “spatiotemporal” mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.