Purpose: The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. Experimental Design: We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. Results: We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. Conclusions: Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

Chronic inflammation accompanies obesity and limits subcutaneous white adipose tissue (WAT) expandability, accelerating the development of insulin resistance and type 2 diabetes mellitus. MicroRNAs (miRNAs) influence expression of many metabolic genes in fat cells, but physiological roles in WAT remain poorly characterized. Here, we report that expression of the miRNA in subcutaneous WAT corresponds with insulin sensitivity in obese mice and humans. To examine the hypothesis that restoration of expression in WAT improves insulin sensitivity, we injected adenovirus (Adv) expressing into the subcutaneous fat pad of diabetic mice. Exogenous expression in the subcutaneous WAT depot of obese mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. High-throughput proteomic profiling and RNA-Seq suggested that targets the transcription factor STAT1 to limit the actions of the proinflammatory cytokine interferon-γ (IFN-γ) that would otherwise restrict WAT expansion and decrease insulin sensitivity. We further demonstrated that opposes the actions of IFN-γ, suggesting an important role for in defending adipocytes against proinflammatory cytokines that reduce peripheral insulin sensitivity. Together, our data identify a critical molecular signaling axis, elements of which are involved in uncoupling obesity from metabolic dysfunction.

RUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET–proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network–based Cellular Signatures 1000–connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1.

First-generation bromodomain extra-terminal protein (BETP) inhibitors (BETi) (e.g., OTX015) that disrupt binding of BETP BRD4 to chromatin transcriptionally attenuate AML-relevant progrowth and prosurvival oncoproteins. BETi treatment induces apoptosis of AML BPCs, reduces in vivo AML burden and induces clinical remissions in a minority of AML patients. Clinical efficacy of more potent BETis, e.g., ABBV-075 (AbbVie, Inc.), is being evaluated. Venetoclax and A-1210477 bind and inhibit the antiapoptotic activity of BCL2 and MCL1, respectively, lowering the threshold for apoptosis. BETi treatment is shown here to perturb accessible chromatin and activity of enhancers/promoters, attenuating MYC, CDK6, MCL1 and BCL2, while inducing BIM, HEXIM1, CDKN1A expressions and apoptosis of AML cells. Treatment with venetoclax increased MCL1 protein levels, but cotreatment with ABBV-075 reduced MCL1 and Bcl-xL levels. ABBV-075 cotreatment synergistically induced apoptosis with venetoclax or A-1210477 in patient-derived, CD34+ AML cells. Compared to treatment with either agent alone, cotreatment with ABBV-075 and venetoclax was significantly more effective in reducing AML cell-burden and improving survival, without inducing toxicity, in AML-engrafted immune-depleted mice. These findings highlight the basis of superior activity and support interrogation of clinical efficacy and safety of cotreatment with BETi and BCL2 or MCL1 inhibitor in AML.

Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolar secondary septation and vascular growth. Exposure to high concentrations of oxygen (hyperoxia) contributes to the development of BPD. The male sex is considered an independent risk factor for the development of BPD. The reasons underlying sexually dimorphic outcomes in premature neonates are not known. We hypothesized that sex-specific modulation of biological processes in the lung under hyperoxic conditions contributes to sex-based differences. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia [95% [Formula: see text], postnatal day (PND) 1-5: saccular stage of lung development] and euthanized on PND 7 or 21. Pulmonary gene expression was studied using RNA-Seq on the Illumina HiSeq 2500 platform. Analysis of the pulmonary transcriptome revealed differential sex-specific modulation of crucial pathways such as angiogenesis, response to hypoxia, inflammatory response, and p53 pathway. Candidate genes from these pathways were validated at the mRNA level by qPCR. Analysis also revealed sex-specific differences in the modulation of crucial transcription factors. Focusing on the differential modulation of the angiogenesis pathway, we also showed sex-specific differential activation of α-regulated genes using ChIP-qPCR and differences in expression of crucial genes () modulating angiogenesis. We demonstrate the translational relevance of our findings by showing that our murine sex-specific differences in gene expression correlate with those from a preexisting human BPD data set. In conclusion, we provide novel molecular insights into differential sex-specific modulation of the pulmonary transcriptome in neonatal hyperoxic lung injury and highlight angiogenesis as one of the crucial differentially modulated pathways.

The promising activity of BET protein inhibitors (BETi’s) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

There is an unmet need to overcome nongenetic therapy-resistance to improve outcomes in AML, especially post-myeloproliferative neoplasm (MPN) secondary (s) AML. Studies presented describe effects of genetic knockout, degradation or small molecule targeted-inhibition of GFI1/LSD1 on active enhancers, altering gene-expressions and inducing differentiation and lethality in AML and (MPN) sAML cells. A protein domain-focused CRISPR screen in LSD1 (KDM1A) inhibitor (i) treated AML cells, identified BRD4, MOZ, HDAC3 and DOT1L among the codependencies. Our findings demonstrate that co-targeting LSD1 and one of these co-dependencies exerted synergistic in vitro lethality in AML and post-MPN sAML cells. Co-treatment with LSD1i and the JAKi ruxolitinib was also synergistically lethal against post-MPN sAML cells. LSD1i pre-treatment induced GFI1, PU.1 and CEBPα but depleted c-Myc, overcoming nongenetic resistance to ruxolitinib, or to BETi in post-MPN sAML cells. Co-treatment with LSD1i and BETi or ruxolitinib exerted superior in vivo efficacy against post-MPN sAML cells. These findings highlight LSD1i-based combinations that merit testing for clinical efficacy, especially to overcome nongenetic therapy-resistance in AML and post-MPN sAML.

Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ;240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.