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

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.

Recent studies have demonstrated that epigenetic modifications are deeply involved in neurogenesis; however, the precise mechanisms remain largely unknown. To determine the role of UTX (also known as KDM6A), a demethylase of histone H3K27, in neural development, we generated Utx-deficient mice in neural stem/progenitor cells (NSPCs). Since Utx is an X chromosome-specific gene, the genotypes are sex-dependent; female mice lose both Utx alleles (Utx

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.

Nagase and Inoue et al. generated a novel Asxl1 mutant mouse model to mimic clonal hematopoiesis and myelodysplastic syndromes caused by ASXL1 mutations and elucidated the effects of mutant versus wild-type ASXL1 on hematopoiesis, gene expression, and chromatin state., Additional sex combs like 1 (ASXL1) is frequently mutated in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). Although loss of ASXL1 promotes hematopoietic transformation, there is growing evidence that ASXL1 mutations might confer an alteration of function. In this study, we identify that physiological expression of a C-terminal truncated Asxl1 mutant in vivo using conditional knock-in (KI) results in myeloid skewing, age-dependent anemia, thrombocytosis, and morphological dysplasia. Although expression of mutant Asxl1 altered the functions of hematopoietic stem cells (HSCs), it maintained their survival in competitive transplantation assays and increased susceptibility to leukemic transformation by co-occurring RUNX1 mutation or viral insertional mutagenesis. KI mice displayed substantial reductions in H3K4me3 and H2AK119Ub without significant reductions in H3K27me3, distinct from the effects of Asxl1 loss. Chromatin immunoprecipitation followed by next-generation sequencing analysis demonstrated opposing effects of wild-type and mutant Asxl1 on H3K4me3. These findings reveal that ASXL1 mutations confer HSCs with an altered epigenome and increase susceptibility for leukemic transformation, presenting a novel model for CHIP.

Chronic myelomonocytic leukemia (CMML) is a hematological malignancy characterized by uncontrolled proliferation of dysplastic myelomonocytes and frequent progression to acute myeloid leukemia (AML). We identified mutations in the Cbl gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant Cbl in CMML pathogenesis, we generated conditional knockin mice for Cbl that express wild-type Cbl in a steady state and inducibly express CblQ367P, a CMML-associated Cbl mutant. CblQ367P mice exhibited sustained proliferation of myelomonocytes, multilineage dysplasia, and splenomegaly, which are the hallmarks of CMML. The phosphatidylinositol 3-kinase (PI3K)-AKT and JAK-STAT pathways were constitutively activated in CblQ367P hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. Gem, a gene encoding a GTPase that is upregulated by CblQ367P, enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, Evi1, a gene encoding a transcription factor, was found to cooperate with CblQ367P and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of CblQ367P-bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant Cbl-induced CMML and propose a possible molecular targeting therapy for mutant Cbl-carrying CMML patients.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played 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 hematopoietic stem cells declined 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 analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

E2A-PBX1 is a chimeric gene product detected in t(1;19)-bearing acute lymphoblastic leukemia (ALL) with B-cell lineage. To investigate the leukemogenic process, we generated conditional knock-in (cKI) mice for E2A-PBX1, in which E2A-PBX1 is inducibly expressed under the control of the endogenous E2A promoter. Despite the induced expression of E2A-PBX1, no hematopoietic disease was observed, strongly suggesting that additional genetic alterations are required to develop leukemia. To address this possibility, retroviral insertional mutagenesis was used. Virus infection efficiently induced T-cell, B-cell, and biphenotypic ALL in E2A-PBX1 cKI mice. Inverse PCR identified eight retroviral common integration sites, in which enhanced expression was observed in the Gfi1, Mycn, and Pim1 genes. In addition, it is of note that viral integration and overexpression of the Zfp521 gene was detected in one tumor with B-cell lineage; we previously identified Zfp521 as a cooperative gene with E2A-HLF, another E2A-involving fusion gene with B-lineage ALL. The cooperative oncogenicity of E2A-PBX1 with overexpressed Zfp521 in B-cell tumorigenesis was indicated by the finding that E2A-PBX1 cKI, Zfp521 transgenic compound mice developed B-lineage ALL. Moreover, upregulation of ZNF521, the human counterpart of Zfp521, was found in several human leukemic cell lines bearing t(1;19). These results indicate that E2A-PBX1 cooperates with additional gene alterations to develop ALL. Among them, enhanced expression of ZNF521 may play a clinically relevant role in E2A fusion genes to develop B-lineage ALL.

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (EedΔ/Δ) mice. EedΔ/Δ mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in EedΔ/Δ HSPCs and consistently, EedΔ/Δ HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other and these conflicting events would lead to abnormal differentiation and functional defect of EedΔ/Δ HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.