Modulating Androgen Receptor-Driven Transcription in Prostate Cancer with Selective CDK9 Inhibitors.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

AP-1 activity induced by co-stimulation is required for chromatin opening during T cell activation.

Activation of T cells is dependent on the organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naive T cells within 5 h of activation. These newly opened regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at >70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced the expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1-dependent elements with risk loci for multiple immune diseases, including multiple sclerosis, inflammatory bowel disease, and allergic disease. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.

Abnormal Peyer patch development and B-cell gut homing drive IgA deficiency in Kabuki syndrome.

BACKGROUND: Kabuki syndrome (KS) is commonly caused by mutations in the histone-modifying enzyme lysine methyltransferase 2D (KMT2D). Immune dysfunction is frequently observed in individuals with KS, but the role of KMT2D in immune system function has not been identified. OBJECTIVE: We sought to understand the mechanisms driving KS-associated immune deficiency (hypogammaglobulinemia [low IgA], splenomegaly, and diminished immunization responses). METHODS: We performed a comprehensive evaluation of humoral immunity and secondary lymphoid tissues in an established KS (Kmt2d+/ßGeo) mouse model and validated select findings in a patient with KS. RESULTS: Compared with wild-type littermates, Kmt2d+/ßGeo mice demonstrated deficiencies in multiple B-cell lineages and reduced serum IgA and elevated IgM levels across multiple ages. The bone marrow, spleen, and intestine of Kmt2d+/ßGeo mice contained diminished numbers of IgA-secreting cells, while elevated germinal center B cells were found in the mesenteric lymph node and Peyer patches. Kmt2d+/ßGeo mice have decreased size and numbers of Peyer patches, a finding confirmed in human samples. We identified deficiency of Itgb7 RNA and protein expression, a gene encoding an adhesion protein that mediates intestinal homing, and we demonstrated KMT2D-dependent control of ITGB7 expression in a human cell line. CONCLUSIONS: Kmt2d haploinsufficiency has broad deleterious effects on B-cell differentiation, specifically hampering gut lymphocyte homing and IgA+ plasma cell differentiation. Intestinal lymphoid defects caused by ITGB7 deficiency have not previously been recognized in KS, and these results provide new mechanistic insights into the pathogenesis of KS-associated immune deficiency.

IRF2 is a master regulator of human keratinocyte stem cell fate.

Resident adult epithelial stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. The stem cell potential of human epidermal keratinocytes is retained in vitro but lost over time suggesting extrinsic and intrinsic regulation. Transcription factor-controlled regulatory circuitries govern cell identity, are sufficient to induce pluripotency and transdifferentiate cells. We investigate whether transcriptional circuitry also governs phenotypic changes within a given cell type by comparing human primary keratinocytes with intrinsically high versus low stem cell potential. Using integrated chromatin and transcriptional profiling, we implicate IRF2 as antagonistic to stemness and show that it binds and regulates active cis-regulatory elements at interferon response and antigen presentation genes. CRISPR-KD of IRF2 in keratinocytes with low stem cell potential increases self-renewal, migration and epidermis formation. These data demonstrate that transcription factor regulatory circuitries, in addition to maintaining cell identity, control plasticity within cell types and offer potential for therapeutic modulation of cell function.

Microbiota-sensitive epigenetic signature predicts inflammation in Crohn's disease.

Altered response to the intestinal microbiota strongly associates with inflammatory bowel disease (IBD); however, how commensal microbial cues are integrated by the host during the pathogenesis of IBD is not understood. Epigenetics represents a potential mechanism that could enable intestinal microbes to modulate transcriptional output during the development of IBD. Here, we reveal a histone methylation signature of intestinal epithelial cells isolated from the terminal ilea of newly diagnosed pediatric IBD patients. Genes characterized by significant alterations in histone H3-lysine 4 trimethylation (H3K4me3) showed differential enrichment in pathways involving immunoregulation, cell survival and signaling, and metabolism. Interestingly, a large subset of these genes was epigenetically regulated by microbiota in mice and several microbiota-sensitive epigenetic targets demonstrated altered expression in IBD patients. Remarkably though, a substantial proportion of these genes exhibited H3K4me3 levels that correlated with the severity of intestinal inflammation in IBD, despite lacking significant differential expression. Collectively, these data uncover a previously unrecognized epigenetic profile of IBD that can be primed by commensal microbes and indicate sensitive targets in the epithelium that may underlie how microbiota predispose to subsequent intestinal inflammation and disease.

FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy.

FOXF1, a member of the forkhead box family of transcription factors, has been previously shown to be critical for lung development, homeostasis, and injury responses. However, the role of FOXF1 in lung regeneration is unknown. Herein, we performed partial pneumonectomy, a model of lung regeneration, in mice lacking one Foxf1 allele in endothelial cells (PDGFb-iCre/Foxf1 fl/+ mice). Endothelial cell proliferation was significantly reduced in regenerating lungs from mice deficient for endothelial Foxf1. Decreased endothelial proliferation was associated with delayed lung regeneration as shown by reduced respiratory volume in Foxf1-deficient lungs. FACS-sorted endothelial cells isolated from regenerating PDGFb-iCre/Foxf1 fl/+ and control lungs were used for RNAseq analysis to identify FOXF1 target genes. Foxf1 deficiency altered expression of numerous genes including those regulating extracellular matrix remodeling (Timp3, Adamts9) and cell cycle progression (Cdkn1a, Cdkn2b, Cenpj, Tubb4a), which are critical for lung regeneration. Deletion of Foxf1 increased Timp3 mRNA and protein, decreasing MMP14 activity in regenerating lungs. ChIPseq analysis for FOXF1 and histone methylation marks identified DNA regulatory regions within the Cd44, Cdkn1a, and Cdkn2b genes, indicating they are direct FOXF1 targets. Thus FOXF1 stimulates lung regeneration following partial pneumonectomy via direct transcriptional regulation of genes critical for extracellular matrix remodeling and cell cycle progression.

Genomic integration of Wnt/ß-catenin and BMP/Smad1 signaling coordinates foregut and hindgut transcriptional programs.

Digestive system development is orchestrated by combinatorial signaling interactions between endoderm and mesoderm, but how these signals are interpreted in the genome is poorly understood. Here we identified the transcriptomes of Xenopus foregut and hindgut progenitors, which are conserved with mammals. Using RNA-seq and ChIP-seq we show that BMP/Smad1 regulates dorsal-ventral gene expression in both the endoderm and mesoderm, whereas Wnt/ß-catenin acts as a genome-wide toggle between foregut and hindgut programs. Unexpectedly, ß-catenin and Smad1 binding were associated with both transcriptional activation and repression, with Wnt-repressed genes often lacking canonical Tcf DNA binding motifs, suggesting a novel mode of direct repression. Combinatorial Wnt and BMP signaling was mediated by Smad1 and ß-catenin co-occupying hundreds of cis-regulatory DNA elements, and by a crosstalk whereby Wnt negatively regulates BMP ligand expression in the foregut. These results extend our understanding of gastrointestinal organogenesis and of how Wnt and BMP might coordinate genomic responses in other contexts.

Molecular chaperone GRP78 enhances aggresome delivery to autophagosomes to promote drug resistance in multiple myeloma.

Despite the clinical benefit of the proteasome inhibitor bortezomib, multiple myeloma (MM) patients invariably relapse through poorly defined mechanisms. Myeloma cells inevitably develop chemoresistance that leads to disease relapse and patient-related deaths. Studies in tumor cell lines and biopsies obtained from patients refractory to therapy have revealed that myeloma cells adapt to stress by inducing expression of glucose-regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone with anti-apoptotic properties. Treatment of myeloma cells with bortezomib increased GRP78 levels and activated GRP78-dependent autophagy. Expression profiling indicated that GRP78-encoding HSPA5 was significantly upregulated in bortezomib-resistant cells. Co-treatment with the anti-diabetic agent metformin suppressed GRP78 and enhanced the anti-proliferative effect of bortezomib. Bortezomib treatment led to GRP78 co-localization with proteotoxic protein aggregates, known as aggresomes. Pharmacologic suppression, genetic ablation or mutational inactivation of GRP78 followed by bortezomib treatment led to the accumulation of aggresomes but impaired autophagy and enhanced anti-myeloma effect of bortezomib. GRP78 was co-immunoprecipitated with the KDEL receptor, an ER quality control regulator that binds proteins bearing the KDEL motif to mediate their retrieval from the Golgi complex back to the ER. Taken together, we demonstrate that inhibition of GRP78 functional activity disrupts autophagy and enhances the anti-myeloma effect of bortezomib.

Correlation of long non-coding RNA expression with metastasis, drug resistance and clinical outcome in cancer.

The therapeutic response and clinical outcome of patients diagnosed with the same cancer type and that receive identical treatment is highly variable to reflect the genetic heterogeneity within tumor cells. Non-coding RNAs (ncRNAs) are recently discovered molecules that regulate eukaryotic gene expression and represent a significant advance towards a better understanding of the mechanisms that govern cellular growth. NcRNAs are essential for the proper regulation of cell proliferation and survival under physiologic conditions and are deregulated in many pathologies, e.g., human cancers. NcRNAs have been associated with cancer diagnosis, staging, treatment response, metastasis and survival and include distinct subtypes, e.g., long ncRNAs (lncRNAs) and microRNAs (miRNAs). LncRNAs have been linked to essential growth-promoting activities and their deregulation contributes to tumor cell survival. A prominent example is the Hox transcript antisense intergenic lncRNA, HOTAIR, that cooperates with the polycomb repressive complex to reprogram chromatin organization. HOTAIR expression is deregulated in a spectrum of cancers and HOTAIR expression correlates with patient survival. Here, we highlight emerging evidence that supports a role for lncRNAs in cancer with implications for the development of novel diagnostics and therapeutics.

The brain microenvironment negatively regulates miRNA-768-3p to promote K-ras expression and lung cancer metastasis.

The brain microenvironment promotes metastasis through mechanisms that remain elusive. Co-culture of lung cancer cells with astrocytes - the most abundant cell type within the metastatic brain niche - lead to downregulation of miRNA-768-3p which drives K-ras expression and key signaling pathways, enhances cell viability and promotes chemotherapeutic resistance. Vector-based forced expression of miRNA-768-3p complementary sequence or a chemically-engineered miRNA-768-3p inhibitor recapitulated the astrocyte effect to increase tumor cell viability. The miRNA-768-3p inhibitor targeted the K-ras 3'-UTR as demonstrated by increased luminescence from a luciferase reporter and strikingly increased the K-ras protein and the downstream effectors ERK1/2 and B-Raf. miRNA-768-3p was reduced in patient brain metastases compared to normal brain tissue and was lower in patient tissue from brain metastases compared to same-patient primary tumour tissue. The brain microenvironment negatively regulates miRNA-768-3p to enhance K-ras and promote metastasis. We propose that therapeutic replacement of the metastasis suppressor miRNA-768-3p holds clinical promise.