Building a translational cancer dependency map for The Cancer Genome Atlas.

Cancer dependency maps have accelerated the discovery of tumor vulnerabilities that can be exploited as drug targets when translatable to patients. The Cancer Genome Atlas (TCGA) is a compendium of 'maps' detailing the genetic, epigenetic and molecular changes that occur during the pathogenesis of cancer, yet it lacks a dependency map to translate gene essentiality in patient tumors. Here, we used machine learning to build translational dependency maps for patient tumors, which identified tumor vulnerabilities that predict drug responses and disease outcomes. A similar approach was used to map gene tolerability in healthy tissues to prioritize tumor vulnerabilities with the best therapeutic windows. A subset of patient-translatable synthetic lethalities were experimentally tested, including PAPSS1/PAPSS12 and CNOT7/CNOT78, which were validated in vitro and in vivo. Notably, PAPSS1 synthetic lethality was driven by collateral deletion of PAPSS2 with PTEN and was correlated with patient survival. Finally, the translational dependency map is provided as a web-based application for exploring tumor vulnerabilities.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation.

Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Drug targeting of NR4A nuclear receptors for treatment of acute myeloid leukemia.

NR4As are AML tumor suppressors that are frequently silenced in human acute myeloid leukemia (AML). Despite their potential as novel targets for therapeutic intervention, mechanisms of NR4A silencing and strategies for their reactivation remain poorly defined. Here we show that NR4A silencing in AML occurs through blockade of transcriptional elongation rather than epigenetic promoter silencing. By intersection of NR4A-regulated gene signatures captured upon acute, exogenous expression of NR4As in human AML cells with in silico chemical genomics screening, we identify several FDA-approved drugs including dihydroergotamine (DHE) that reactivate NR4A expression and regulate NR4A-dependent gene signatures. We show that DHE induces NR4A expression via recruitment of the super elongation complex to enable elongation of NR4A promoter paused RNA polymerase II. Finally, DHE exhibits AML selective NR4A-dependent anti-leukemic activity in cytogenetically distinct human AML cells in vitro and delays AML progression in mice revealing its potential as a novel therapeutic agent in AML.

Glucocorticoids Inhibit Oncogenic RUNX1-ETO in Acute Myeloid Leukemia with Chromosome Translocation t(8;21).

Acute myeloid leukemia (AML) is a major blood cancer with poor prognosis. New therapies are needed to target oncogene-driven leukemia stem cells, which account for relapse and resistance. Chromosome translocation t(8;21), which produces RUNX1-ETO (R-E) fusion oncoprotein, is found in ~13% AML. R-E dominance negatively inhibits global gene expression regulated by RUNX1, a master transcription factor for hematopoiesis, causing increased self-renewal and blocked cell differentiation of hematopoietic progenitor cells, and eventually leukemia initiation. Methods: Connectivity-Map followed by biological activity testing were used to identify candidate compounds that can inhibit R-E-mediated gene transcription. Molecular mechanistic studies were also performed. Results: Glucocorticoid drugs, such as betamethasone and dexamethasone, were found to exhibit potent and selective in vitro and in vivo activities against R-E leukemia, as well as strong synergy when combined with chemotherapeutics. Microarray analysis showed that treatment with glucocorticoids significantly inhibited R-E's activity and reactivated that of RUNX1. Such gene expression changes caused differentiation and apoptosis of R-E leukemia cells. Our studies also show a possible molecular mechanism for the targeted therapy. Upon treatment with a glucocorticoid drug, more glucocorticoid receptor (GR) was translocated into the nucleus and bound to DNA, including promoters of RUNX1 target genes. GR was found to associate with RUNX1, but not R-E. This interaction increased binding of RUNX1 to DNA and reduced that of R-E, shifting to a RUNX1 dominance. Conclusion: Glucocorticoid drugs represent a targeted therapy for AML with chromosome translocation t(8:21). Given their high activity, favorable human pharmacokinetics as well as synergy with chemotherapeutics, glucocorticoids could be clinically useful to treat R-E AML.

Lipopolysaccharide stimulates platelets through an IL-1ß autocrine loop.

LPS activates platelets through TLR4, aiding productive sepsis, with stimulated splicing and translation of stored heteronuclear pro-IL-1ß RNA. Although the IL-1R type 1 (IL-1R1) receptor for IL-1 shares downstream components with the TLR4 receptor, platelets are not known to express IL-1R1, nor are they known to respond to this cytokine. We show by flow cytometry and Western blotting that platelets express IL-1R1, and that IL-1ß and IL-1α stimulate heteronuclear I-1ß splicing and translation of the newly made mRNA in platelets. Platelets also respond to the IL-1ß they make, which is exclusively associated with shed microparticles. Specific blockade of IL-1R1 with IL-1R antagonist suppressed platelet stimulation by IL-1, so IL-1ß stimulates its own synthesis in an autocrine signaling loop. Strikingly, IL-1R antagonist inhibition, pharmacologic or genetic suppression of pro-IL-1ß processing to active cytokine by caspase-1, or blockade of de novo protein synthesis also blocked LPS-induced IL-1ß mRNA production. Robust stimulation of platelets by LPS therefore also required IL-1ß amplification. Activated platelets made IL-1ß in vivo as IL-1ß rapidly accumulated in occluded murine carotid arteries by posttranscriptional RNA splicing unique to platelets. We conclude that IL-1ß is a platelet agonist, that IL-1ß acts through an autocrine stimulatory loop, that an IL-1ß autocrine loop is required to amplify platelet activation by LPS, and that platelets immobilized in occlusive thrombi are activated over time to produce IL-1ß. IL-1 is a new platelet agonist that promotes its own synthesis, connecting thrombosis with immunity.