Purpose: Epigenetic deregulation is deeply implicated in the pathogenesis of bladder cancer. KDM6A (Lysine (K)-specific demethylase 6A) is a histone modifier frequently mutated in bladder cancer. However, the molecular mechanisms of how KDM6A deficiency contributes to bladder cancer development remains largely unknown. We hypothesized that clarification of the pathogenic mechanisms underlying KDM6A-mutated bladder cancer can help in designing new anticancer therapies. Experimental Design: We generated mice lacking Kdm6a in the urothelium and crossed them with mice heterozygous for p53, whose mutation/deletion significantly overlaps with the KDM6A mutation in muscle-invasive bladder cancer (MIBC). In addition, BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine), a cigarette smoke-like mutagen, was used as a tumor-promoting agent. Isolated urothelia were subjected to phenotypic, pathologic, molecular, and cellular analyses. The clinical relevance of our findings was further analyzed using genomic and clinical data of patients with MIBC. Results: We found that Kdm6a deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused bladder cancer in cooperation with p53 haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human bladder cancer samples with decreased KDM6A expression also showed activated proinflammatory pathways. Notably, dual inhibition of IL6 and chemokine (C-C motif) ligand 2, upregulated in response to Kdm6a deficiency, efficiently suppressed Kdm6a-deficient bladder cancer cell growth. Conclusions: Our findings provide insights into multistep carcinogenic processes of bladder cancer and suggest molecular targeted therapeutic approaches for patients with bladder cancer with KDM6A dysfunction.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. Here, we show that UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of Compass-like and SWI/SNF complexes, plays an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in HSCs declines with age and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation (ChIP) analyses coupled with RNA-seq data indicated that UTX contributes to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging, via both demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged iPS-induced neuronal cells showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drive inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer which upregulates transcription of EVI1. Here we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 which is frequently present in inv(3)/t(3;3) AML and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly co-occurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of six amino acids at the 3' end of the second Zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent co-occurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

<p>Supplementary Information: Tables S1, S2, S3, S4 (gene set enrichment analyses); Figures S1, S2, S3, S4</p>

<p>Supplementary Information: Tables S1, S2, S3, S4 (gene set enrichment analyses); Figures S1, S2, S3, S4</p>