KDM6A Loss Research Articles

Kdm6a deficiency in microglia/macrophages epigenetically silences Lcn2 expression and reduces photoreceptor dysfunction in diabetic retinopathy

Diabetic retinopathy (DR) is one of the leading causes of severe visual impairment worldwide. However, the role of adaptive immune inflammation driven by microglia/macrophages in DR is not yet well elucidated. Kdm6a is a histone demethylase that removes the trimethyl groups of histones H3K27 and plays important biological roles in activating target genes. To elucidate the role of Kdm6a in microglia/macrophages in diabetic retinas, we established diabetic animal models with conditional knockout mice to investigate the impacts of Kdm6a deficiency. The RNA-seq analysis, mass spectrum examination, immunohistochemistry and detection of enzyme activities were used to elucidate the effect of Kdm6a deletion on gene transcription in microglia/macrophages. The expression of Kdm6a was increased in the retinas of diabetic mice compared to the control group. Loss of Kdm6a in microglia/macrophages ameliorated the diabetes-induced retinal thickness decrease, inflammation, and visual impairment. Kdm6a in microglia/macrophages regulated Lcn2 expression in a demethylase activity-dependent manner and inhibited glycolysis progression in photoreceptor cells through Lcn2. These results suggest that Kdm6a in microglia/macrophages aggravated diabetic retinopathy by promoting the expression of Lcn2 and impairing glycolysis progression in photoreceptor cells.

104 Loss of Kdm6a and Trp53 drives the development of squamous cell skin cancer in mice

Cutaneous squamous cell carcinoma (cSCC) has among the highest mutation burdens of all cancers, reflecting its association with the mutagenic effects of ultraviolet light. Though mutations in established, cancer-relevant genes like TP53 and NOTCH1 are prevalent in cSCC, they are tolerated in clinically normal skin and are insufficient to cause skin cancer. Whether epigenetic regulators cooperate in the development of squamous cell carcinoma is just starting to be defined. KDM6A encodes a histone H3K27me2/me3 demethylase that is frequently mutated in cSCC and other cancers.

MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER

You have accessJournal of UrologyCME1 May 2022MP06-10 KDM6A DEFICIENCY IN UROTHELIUM PRODUCES A DISTINCT IMMUNE PHENOTYPE IN A MOUSE MODEL OF BLADDER CANCER Ao Zhang, Hong Qiu, Timothy Chan, and Byron Lee Ao ZhangAo Zhang More articles by this author , Hong QiuHong Qiu More articles by this author , Timothy ChanTimothy Chan More articles by this author , and Byron LeeByron Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 6A (KDM6A) is a chromatin modifier that is frequently mutated in bladder cancer and causes epigenetic dysregulation. Recently immune check point inhibition has shown great clinical promise in bladder cancer. Because epigenetic signature has been shown to predict response to immunotherapy, here we assessed if loss of KDM6A leads to alterations of tumor immune microenvironment in a mouse bladder cancer model. METHODS: Kdm6a urothelium conditional knockout (cKO) mice were generated by crossing Kdm6aflox/flox mice with Upk3aCre mice that express Cre recombinase in the urothelium. Littermate control mice were of the same genetic background but intact Kdm6a gene. Mice at 8 weeks of age (three in each group) were fed with drinking water containing 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Bladders were harvested after 4 weeks and analyzed with H&E staining. Single cell RNA sequencing was performed on urothelium from control and Kdm6A cKO mice without BBN treatment. RESULTS: After treatment with BBN for 4 weeks, there was marked edema and inflammatory changes with increased infiltration of immune cells in H&E stained bladder sections in Kdm6a cKO mice compared to Kdm6a intact controls (Figure A). While urothelium from control mice maintained grossly unchanged tissue architecture, urothelium from cKO mice exhibited cytologic atypia with loss of organized cell layer. Comparison of single cell RNA sequencing profile revealed enrichment of immune cell population in cKO mice (Figure B, cluster 12), which was consistent with histological findings. CONCLUSIONS: Loss of KDM6A in the urothelium creates a distinct immune microenvironment in the BBN mouse bladder cancer model. Whether this immune cell-rich microenvironment affects response to immune check point inhibition warrants further investigation. Source of Funding: This work was supported in part by the 2021 Urology Care Foundation Residency Research Award Program and the Kahlert Foundation © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e80 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ao Zhang More articles by this author Hong Qiu More articles by this author Timothy Chan More articles by this author Byron Lee More articles by this author Expand All Advertisement PDF DownloadLoading ...

Kdm6a deficiency restricted to mouse hematopoietic cells causes an age- and sex-dependent myelodysplastic syndrome-like phenotype.

Kdm6a/Utx, a gene on the X chromosome, encodes a histone H3K27me3 demethylase that has an orthologue on the Y chromosome (Uty) (Zheng et al. 2018). We previously identified inactivating mutations of Kdm6a in approximately 50% of mouse acute promyelocytic leukemia samples; however, somatic mutations of KDM6A are more rare in human AML samples, ranging in frequency from 2-15% in different series of patients, where their role in pathogenesis is not yet clear. In this study, we show that female Kdm6aflox/flox mice (with allele inactivation initiated by Vav1-Cre in hematopoietic stem and progenitor cells (HSPCs) have a sex-specific phenotype that emerges with aging, with features resembling a myelodysplastic syndrome (MDS). Female Kdm6a-knockout (KO) mice have an age-dependent expansion of their HSPCs with aberrant self-renewal, but they did not differentiate normally into downstream progeny. These mice became mildly anemic and thrombocytopenic, but did not develop overt leukemia, or die from these cytopenias. ChIP-seq and ATAC-seq studies showed only minor changes in H3K27me3, H3K27ac, H3K4me, H3K4me3 and chromatin accessibility between Kdm6a-WT and Kdm6a-KO mice. Utilizing scRNA-seq, Kdm6a loss was linked to the transcriptional repression of genes that mediate hematopoietic cell fate determination. These data demonstrate that Kdm6a plays an important role in normal hematopoiesis, and that its inactivation may contribute to AML pathogenesis.

Abstract PR-008: Kdm6 demethylases are critical regulators of pancreatic cancer initiation, progression and subtype specification

Abstract Aberrant epigenetic regulation is a hallmark of pancreatic cancer. Since changes in histone methylation are potentially reversible, they present an attractive target for therapeutic intervention. Kdm6a and Kdm6b belong to a family of histone demethylases specific for lysine 27 of histone 3 and have been implicated in multiple types of cancer, including pancreatic ductal adenocarcinoma (PDA). We have gathered data from the extensive investigation of a mouse model of PDA in which either Kdm6b or both Kdm6a and Kdm6b have been deleted with concomitant activation of oncogenic KrasG12D restricted to adult acinar cells. While prior work had demonstrated an important role for Kdm6a in PDA formation, our data now show that the closely related Kdm6b is implicated in PDA tumorigenesis as well. Partial or complete loss of Kdm6b results in accelerated acinar-to-ductal metaplasia (ADM) and formation of pancreatic intraepithelial neoplasia (PanIN) with increased inflammation and fibrosis. Accelerated ADM formation was also observed ex vivo upon culturing acinar cells in the absence of oncogenic Kras, indicating that Kdm6b activity is critical for the maintenance of acinar identity and suppresses the early stages of pancreatic neoplasia development. However, only mice heterozygous for Kdm6b continue to exhibit a reduced overall median survival compared to KrasG12D alone, while complete loss of Kdm6b results in a similar overall median survival compared to KrasG12D. This suggests a requirement for oncogenic Kdm6b function during later progression of PanIN to invasive cancer. Conversely, concomitant loss of Kdm6a and Kdm6b protects against ADM and PanIN formation in vivo and ex vivo and double mutants display a trend towards an increased overall median survival compared to mice carrying only the KrasG12D mutation. Strikingly, the resulting tumors of Kdm6a/Kdm6b deficient mice exhibit features of an extremely rare subtype of sarcomatoid pancreatic cancer found in human patients. Taken together, this suggests that Kdm6 demethylase function is required during the initiation and progression of PDA. Furthermore, targeting Kdm6a/Kdm6b with specific inhibitors at distinct stages might provide opportunities for novel treatment avenues for pancreatic cancer. Citation Format: Laura Leonhardt, Lucia Y. Li, David I. Berrios, Sudipta Ashe, Audrey M. Hendley, Grace E. Kim, Matthias Hebrok. Kdm6 demethylases are critical regulators of pancreatic cancer initiation, progression and subtype specification [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-008.

KDM6 Demethylases Integrate DNA Repair Gene Regulation: Loss of KDM6A Sensitizes AML to PARP Inhibition and Potentiates with BCL2 Blockade

Acute myeloid leukemia (AML) is a heterogeneous, aggressive hematological malignancy with dismal prognosis where limited targeted therapies are currently available. Poly-(ADP-ribose)-polymerase (PARP) inhibition has emerged as an important therapeutic arsenal to target homologous recombination-deficient tumors. However, molecular understanding of PARP blockade in the context of epigenetic derangements and transcriptional plasticity in human elderly AML pathogenesis remains unexplored. KDM6 proteins are H3K27 demethylases that critically regulate chromatin architecture in multi-cellularity and tumorigenesis (Tran, Mol Cell Biol 2020). KDM6A escapes X-chr inactivation, and Utx-/- female mice spontaneously develop aging associated myeloid leukemia (Gozdecka, Nat Genet 2018; Sera, Blood 2021). In addition, KDM6A loss of function mutation is implicated in AML relapse (Stief, Leukemia 2020). In contrast, KDM6B primarily exerts an oncogenic function in heme-malignancies. Together, KDM6A and KDM6B play cell type-specific function in leukemia, and KDM6 proteins and associated signaling emerge as important focal point for developing molecular targeted therapy.We identify that KDM6 demethylase activity critically regulates DNA damage repair (DDR) gene expression program in AML. Transcriptome analysis indicated a significant downregulation of expression of DDR genesets in both KDM6A deficient human AML and Utx -/- pre-leukemic cells. Lentiviral shRNA screening performed in response to low-dose γ-irradiation in AML stem cells, revealed a radioprotective function of KDM6A. Expression of KDM6s is regulated by genotoxic stress in a time-dependent manner, and deficiency of JmjC catalytic function impaired DDR transcriptional activation and compromised repair potential. Mechanistically, quantitative ChIP experiments also revealed co-operation between KDM6A and SWI/SNF facilitating dynamic chromatin remodeling at TSS/promoter to induce DDR gene expression. To interrogate changes in chromatin accessibility we performed ATAC-seq analysis in KDM6 deficient AML. Motif enrichment highlighted that while KDM6A depletion led to reduced chromatin access to 140 transcription factors (TFs), only 56 TF binding sites showed increased accessibility. Overall, changes in chromatin accessibility, associated with a reduced binding of DDR regulatory TFs in KDM6 deficient AML, account for a compromised DDR function.In agreement with these findings an array of KDM6 deficient AML cells were more sensitive to PARP inhibition, and pre-clinical mice models xenotransplanted with KDM6A loss of function AML line showed an increased susceptibility to PARP blockade in vivo. FLT3-ITD positive AML with a lower KDM6A expression was more sensitive to olaparib. In addition, olaparib administration significantly reduced bone marrow engraftment of patient-derived xenografts of KDM6A-mutant primary AML. Interestingly, KDM6A expression is upregulated in venetoclax-resistant monocytic-AML compared to venet-sensitive primitive-AML. Using venet responsive isogenic lines we demonstrated that attenuation of KDM6 function increased mitochondrial activity, intracellular ROS levels, de-repressed BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6 loss resulted in transcriptional repression of BCL2A1, commonly associated with venet resistance (Zhang, Nat Cancer 2020). Corroborating these results, dual targeting of PARP with BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing primary AML apoptosis, and KDM6A loss further enhanced this synergism.In sum, our study illustrates a molecular mechanistic rationale in support for a novel combination targeted therapy for AML, and posit KDM6A as a molecular determinant for therapeutic efficacy. Intriguingly, KDM6A functions as a gatekeeper of BCL2 and BCL2A1 expression. Similar to TET2 although bi-allelic Utx loss causes evolution to myeloid neoplasms, minimal KDM6 activity is important for survival of human AML cells. KDM6s have been implicated in solid tumors, and both PARP and BCL2 inhibitors are being tested in cancer patients, underscoring a wider scope of application. To conclude, KDM6A unfolds to be a central regulator for susceptibility of AML to both PARP and BCL2 inhibition, expanding the possibility to characterize effective combination targeted therapy for AML in clinical settings. DisclosuresMinden: Astellas: Consultancy. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

Background & AimsInactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development.MethodsWe performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis.ResultsLoss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial–mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata.ConclusionsLoss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

The Role of Kdm6a in Malignant Hematopoiesis

Our preliminary studies identified recurrent, somatic mutations in Kdm6a in a mouse model of acute promyelocytic leukemia (APL). Putative inactivating mutations of Kdm6a (primarily small deletions) occurred in approximately 50% of mouse APL samples, making these the most common acquired progression events in this model. We found somatic mutations of KDM6A in 4 of 200 acute myeloid leukemia (AML) samples. However, in a cohort of 42 patients with APL, we identified no somatic alterations in KDM6A . To better understand the role of Kdm6a in malignant hematopoiesis, we intercrossed our Kdm6a conditional knockout (KO) x Vav1 - Cre mice with a “knock-in” mouse model of APL (Ctsg-PML-RARA mice). To determine whether Kdm6a inactivation cooperates with PML - RARA, we performed a tumor watch that included all genotypes of Kdm6a conditional KO female and male triple cross mice. After 18 months of observation, we found that the latency of APL was significantly decreased in all three cohorts of Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (Figure 1). This finding was most pronounced in female homozygous Kdm6a KO triple cross mice, which had a median survival of 161 days versus 365 days in the control cohort (P < 0.01 by log-rank test). The Kdm6a -null mice developed the typical phenotype of mouse APL, including splenomegaly, myeloproliferation and leukemic cells that coexpressed Gr-1 and Kit. The triple cross APL cells remained sensitive to all- trans retinoic acid (ATRA) and arsenic trioxide ex vivo . Compared with 56% penetrance in Ctsg-PML-RARA mice, female homozygous and male hemizygous Kdm6a KO x Vav1 - Cre x Ctsg-PML-RARA mice developed APL with 100% penetrance. We fully characterized pre-leukemic hematopoiesis in young (6 to 8-weeks-old) Kdm6a conditional KO x Vav1 - Cre x Ctsg-PML-RARA mice (compared to appropriate littermate controls) to define the underlying mechanism(s) by which Kdm6a inactivation contributes to leukemogenesis. Like our double cross Kdm6a conditional KO x Vav1 - Cre mice, the loss of Kdm6a expression in young female homozygous triple cross mice caused mild thrombocytopenia and splenomegaly. These young mice had neutrophilia and lymphopenia, which the female Kdm6a -null double cross mice only acquired with age. We observed a trend for increased numbers of colonies in a myeloid serial replating assay that was also specific to this cohort. Next, we demonstrated that this cohort had significant decreases in the Kit+Lin-Sca-1+ (KLS) cell, short-term hematopoietic stem cell (ST-HSC), and multipotent progenitor (MPP) hematopoietic stem and precursor compartments. This recapitulated the findings from the young, double cross Kdm6a -null female mice but was more pronounced. We found a concomitant increase in granulocyte-macrophage progenitors (GMPs) and decrease in megakaryocyte-erythroid progenitors (MEPs) that was also specific for the female Kdm6a -null triple cross cohort. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged (1-year-old) female Kdm6a -null double cross mice and aged controls. Unsupervised hierarchical clustering revealed a clear distinction between cohorts. We identified 1520 differentially expressed genes by an ANOVA analysis (fold change > 2, P < 0.05) between the Kdm6a -null versus WT aged SLAM cells. The list of differentially expressed genes included Pml (downregulated in Kdm6a -null SLAM cells with fold change of 2.35) and Flt3 (upregulated in Kdm6a -null SLAM cells with fold change of 2.32). Expression changes of these two genes may be associated with the mechanism of how inactivation of Kdm6a cooperates with PML-RARA in our mouse model. Pml haploinsuffiency has been show to modestly cooperate with PML-RARA in mouse models of APL, and FLT3 mutations are the most common secondary mutations in APL. In an effort to further elucidate the mechanism by which loss of Kdm6a contributes to malignant hematopoiesis, we have completed RNA-Seq, ATAC-Seq, anti-histone H3 (monomethyl K4) ChIP-Seq, and anti-histone H3 (trimethyl K27) ChIP-Seq studies on mouse APL samples derived from Ctsg-PML-RARA mice that developed spontaneous leukemias (n = 3 Kdm6a -WT samples versus n = 3 Kdm6a -mutant samples). Analysis of this data set is now underway. In sum, our data have demonstrated a functional role of Kdm6a in malignant hematopoiesis and have improved our understanding of its subsequent connection to leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

KDM6A, a histone demethylase, regulates stress hematopoiesis and early B-cell differentiation

Histone methylases and demethylases regulate gene expression programs in hematopoiesis. The molecular function ofthe demethylase KDM6Ain normal hematopoiesis and, in particular, for the hematopoietic stem and progenitor cell (HSPC) compartment remains only partially understood. Female but not male Kdm6a-/- HSPCs were functionally impaired in adoptive transfer experiments as well as upon proliferative stress induced by 5-fluorouracil. Loss of Kdm6a affected primarily early B cells and erythroid and myeloid progenitor cells with respect to both number and function. Global gene expression analyses revealed a shared altered gene signature in Kdm6a-/- pro-B and pre-B cells that is also present in HSPCs, supporting that altered B-cell differentiation in Kdm6a-/- animals is already initiated in HSPCs. Interestingly, loss of KDM6A did not affect the global level of methylation of H3K27, its presumed target, in hematopoietic cells. Our data indicate a critical role for KDM6A in the regulation of hematopoietic differentiation and differentiation-specific gene expression programs, with a prominent role in early B-cell differentiation that is likely independent of H3K27 methylation status.

KDM6A Controls Genes Modulating Immune Surveillance in Multiple Myeloma

Background: In multiple myeloma (MM), inactivating mutations and loss of the histone demethylase KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) locus are found in up to 50% of patients and are associated with poorer prognosis. KDM6A belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex activates enhancers by adding activation marks on histones and removing methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. The H3K27me3 methyltransferase EZH2 that catalyzes the reverse reaction of KDM6A, is often overexpressed in MM and this also correlates with poor prognosis. As well, KMT2C, KMT2D and SWI/SNF components of its complex are all found significantly mutated in MM which emphasize the importance of enhancer deregulation in myeloma. Aim: Our goal is to understand how loss of KDM6A affects gene transcription networks and enhancer function to affect MM pathogenesis or progression. Method: We explored the transcriptional consequences of KDM6A deficiency in patient tumors using the gene expression dataset included within the Multiple Myeloma Research Foundation (MMRF) Researcher Gateway and in MM cell lines using data from the Cancer Cell Line Encyclopedia (CCLE). We further modeled the loss of KDM6A in female MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology and by re-expressing KDM6A in cells null for KDM6A. Using ChIP-sequencing analysis in isogenic KDM6A knockout or replete cell lines, we identified KDM6A binding site in MM and explored how KDM6A deficiency affects chromatin structure to regulate a transcriptional network. Results: Epithelial-to-mesenchymal-transition (EMT)-related pathways were downregulated in low KDM6A expressing tumor from the MMRF patient cohort. In both male and female patients, low expression of KDM6A was associated with decreased expression of genes involved in immune recognition. In MM cell lines, we observed a significant positive correlation between KDM6A expression level and expression of the transcriptional regulators of Major Histocompatibility complex I and II (MHCII and MCHII), NLRC5 and CIITA, respectively. With ChIP-sequencing analysis we find that KDM6A directly binds NLRC5 and CIITA genes, among many other genes involved in immune surveillance. KDM6A null MM cells displayed upregulation of pathways promoting EMT and downregulation of genes regulating immune function, such as HLA-A/B/C, CCL3/5, HHLA2 and ITGAL. Moreover, re-expression of KDM6A in KDM6A null cells upregulated the expression of these gene along with surface expression of MHCI proteins. Conclusion: Together, our data demonstrates that KDM6A modulates EMT and directly regulates expression of major regulators of immune surveillance. KDM6A deficiency may drive MM towards an EMT program to promote systemic spread and facilitate escape from the immune surveillance. Disclosures No relevant conflicts of interest to declare.

The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

Abstract P055: Smooth Muscle Cell-specific Conditional Knockout Of Lysine (K)-specific Demethylase 6a Causes Endothelial Dysfunction, Increased Blood Pressure And Arterial Stiffness

Arterial stiffening is the progressive loss of elasticity of the large vessels resulting from age. Increased arterial stiffening has been correlated with hypertension and other cardiovascular co-morbidities. KDM6A is a histone 3 lysine demethylase whose activity increases expression of target genes via removal of silencing methylation marks on lysine 27 of histone 3 (H3K27). The precise role of H3K27me3, or its absence in aortic vascular smooth muscle cells (VSMCs), remains to be investigated. The objectives of this study were to determine the effects of loss of KDM6A on arterial stiffness, blood pressure, and vascular cell function. We generated VSMC-specific Kdm6a knockout mice of C57BL/6 background and confirmed knockout by western blot. Knockout of Kdm6a resulted in increased resting systolic blood pressure and pulse wave velocity in male mice. Pulse wave velocity (PWV) is the gold-standard method for determination of arterial stiffness. PWVs of the knockout mice were significantly increased compared to controls four weeks after Kdm6a knockout (Controls 2.62 + 0.69 mm/ms; KO 3.13 + 0.30 mm/ms; P=0.0228). Systolic blood pressure was directly obtained for each mouse via carotid cannulation. The systolic blood pressures of knockout mice were significantly increased. Mesenteric arteries were isolated from three control and three Kdm6a knockout mice and mounted onto a wire myograph. Vessels were pre-constricted with norepinephrine and vasodilation was measured by treatment with increasing doses of acetylcholine and sodium nitroprusside to assess endothelial and VSMC function. Interestingly, despite Kdm6a knockout in VSMCs, vasodilation of VSMCs was not different between control and knockout arteries, but relaxation in response to acetylcholine, which stimulates the release of nitric oxide from endothelial cells, was decreased. This suggests endothelial dysfunction. These results indicate that loss of KDM6A in VSMCs leads to elevated systolic blood pressure and arterial stiffness.

Comparative meta-analysis of Kabuki syndrome with and without hyperinsulinaemic hypoglycaemia.

Kabuki syndrome (KS), caused by pathogenic variants in KMT2D or KDM6A, is associated with hyperinsulinaemic hypoglycaemia (HH) in 0.3%-4% of patients. We characterized the clinical, biochemical and molecular data of children with KS and HH compared to children with KS without HH in a multicentre meta-analysis. Data of seven new and 17 already published children with KS and HH were compared to 373 recently published KS patients without HH regarding molecular and clinical characteristics. Seven new patients were identified with seven different pathogenic variants in KDM6A (n=4) or KMT2D (n=3). All presented with HH on the first day of life and were responsive to diazoxide. KS was diagnosed between 9months and 14years of age. In the meta-analysis, 24 KS patients with HH had a significantly higher frequency of variants in KDM6A compared to 373 KS patients without HH (50% vs 11.5%, P<.001), and KDM6A-KS was more likely to be associated with HH than KMT2D-KS (21.8% vs. 3.5%, P<.001). Sex distribution and other phenotypic features did not differ between KS with and without HH. The higher incidence of HH in KDM6A-KS compared to KMT2D-KS indicates that KDM6A loss of function variants predispose more specifically to beta cell dysfunction compared to KMT2D variants. As difficulties to assign syndromic characteristics to KS in early infancy often lead to delayed diagnosis, genetic testing for KS should be considered in children with HH, especially in the presence of other extrapancreatic/syndromic features.

Abstract C22: Loss of KDM6A promotes pancreatic cancer progression by upregulating epithelial-mesenchymal transition pathway

Abstract Metastasis is one of the main reasons for poor survival in patients with pancreatic ductal adenocarcinoma (PDA). However, the molecular mechanism of PDA metastasis remains elusive. Recent studies failed to find metastasis-specific genetic mutations, and thus suggested that aberrant epigenetic regulation may be one of the crucial mechanisms. Many epigenetic regulator genes are altered in PDA, including KDM6A, a histone H3K27 demethylase, which has been identified by whole-genomic mutational analyses as a driver of PDA. Recent studies have shown that loss of KDM6A induces aggressive squamous-like PDA by activing superenhancers and promotes metastasis. However, the underlying mechanism by which KDM6A promotes metastasis remains elusive. Using stable KDM6A knockout PDA cell lines established by CRISPR/Cas9 system and siRNA, we knocked out or knocked down the expression of KDM6A in PDA cells. We also established an inducible KDM6A re-expressed PDA cell line that has homozygous KDM6A deletion. A novel Bru-seq technology was used to determine global changes of nascent RNA synthesis in KDM6A-null cells. Cell migration and invasion was assessed in vitro. To study the impact of KDM6A loss in vivo, we crossed KDM6Afl/fl mice with the well-established Ptf1aCre; Kras G12D; p53 R172H (KPC) PDA mouse model. Bru-seq data have shown that epithelial-mesenchymal transition (EMT) pathway is highly upregulated in KDM6A knockout cells and downregulated in KDM6A re-expressed PDA cells. EMT is a well-known process that is involved in tumor metastasis. KDM6A knockout cells demonstrated decreased cell proliferation but increased cell migration and invasion with mesenchymal cell morphology changes in vitro. Hallmark epithelial marker E-cadherin was significantly downregulated in KDM6A knockout cells, supporting the EMT process. Furthermore, genetic mouse model showed that mice that lose KDM6A expression developed PDA and metastasis much earlier than KPC mice. The morphology of the PDA developed in KDM6A-null KPC mice is more sarcomatoid or mesenchymal compared to PDA in KPC mice. Immunohistochemistry analysis also demonstrated loss of epithelial marker E-cadherin and gain of mesenchymal marker vimentin in tumor cells. In summary, these results suggest that loss of KDM6A promotes PDA development and metastasis by upregulating EMT signaling. These data linked epigenetic regulation to EMT signaling and provided insights into the role of KDM6A in PDA development. These novel findings also provided foundation for developing new therapeutic strategies. Citation Format: Sivakumar Jeyarajan, Zhujun Yi, Shanqiao Wei, Shuang Lu, Hong Kim, Michelle Paulsen, Karan Bedi, Mats Ljungman, Jiaqi Shi. Loss of KDM6A promotes pancreatic cancer progression by upregulating epithelial-mesenchymal transition pathway [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C22.

Abstract 2883: Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor

Abstract BACKGROUND: Rhabdoid tumor (RT) is an aggressive pediatric malignancy defined by the genetic deletion of SMARCB1 (BAF47/SNF5/INI1), a subunit of the SWI/SNF chromatin remodeling complex. SMARCB1 loss redistributes the SWI/SNF complex to favor a proliferative non-differentiated cellular state. Nevertheless, while EZH2, the catalytic subunit of PRC2, is an established, synthetically lethal therapeutic target, other vulnerabilities that result from this imbalance between the SWI/SNF and PRC2 complexes are relatively unexplored. METHODS: In a screen of multiple pediatric cancer cell lines, we identified a heightened sensitivity of RT cells to mithramycin (MMA), a small molecule transcription inhibitor. We hypothesized the hypersensitivity of rhabdoid tumor to mithramycin was due to the disruption of SWI/SNF and PRC2 dynamics. Here, we describe an epigenetic mechanism of action for mithramycin in rhabdoid tumor. We characterize KDM6A, a H3K27me3 histone demethylase, as a novel therapeutic vulnerability in rhabdoid tumor with live cell imaging, western blot, small molecule inhibition, and chromatin immunoprecipitation (ChIP) sequencing. We identify SWI/SNF eviction from chromatin as a novel mechanism of action for mithramycin with biochemical fractionation, ChIP-seq, and ATAC-seq. Finally, we demonstrate these findings in vivo using RT xenograft models. RESULTS: Mithramycin induced apoptosis and a striking increase in H3K27me3, the catalytic mark of PRC2, in rhabdoid tumor cell lines in a dose and time-dependent manner. This amplification of H3K27me3 preceded suppression of cellular proliferation and induction of apoptosis suggesting H3K27me3 amplification may drive the apoptotic phenotype. In addition, knockdown of KDM6A, a H3K27me3 histone demethylase, potentiates the effects of MMA providing further evidence that KDM6A loss and H3K27me3 amplification lead to apoptosis. We have identified SP1 as the transcription factor driving KDM6A and mithramycin interferes with SP1-dependent activation of KDM6A by evicting SWI/SNF from chromatin. We are currently working on the mechanism of action for mithramycin in rhabdoid tumor cells, including H3K27me3 ChIP-seq and ATAC-seq to elucidate global transcription and chromatin changes. We have recapitulated these results in vivo with xenograft mouse models. CONCLUSIONS: Mithramycin inhibits KDM6A, a histone demethylase, and SWI/SNF leading to apoptosis through the amplification of H3K27me3. Eviction of SWI/SNF from chromatin is a novel mechanism of action for mithramycin. Overall, this study indicates KDM6A and SWI/SNF loss are novel therapeutic vulnerabilities in rhabdoid tumor and mithramycin may be a promising clinical candidate for the treatment of rhabdoid tumor. Citation Format: Maggie H. Chasse, Elissa Boguslawski, Katie Sorensen, Ian Beddows, Kristin Rybski, Zachary Madaj, Susan M. Kitchen-Goosen, Patrick J. Grohar. Mithramycin inhibits KDM6A (UTX) and SWI/SNF to induce apoptosis in malignant rhabdoid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2883.

Loss of KDM6A confers drug resistance in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

Loss of KDM6A characterizes a poor prognostic subtype of human pancreatic cancer and potentiates HDAC inhibitor lethality.

Although genomic analysis have recently discovered the malignant subtype of human pancreatic ductal adenocarcinoma (PDAC) characterized by frequent mutations of histone demethylase KDM6A, the biological and molecular roles still remain obscure. We herein elucidated the clinical and biological impacts of KDM6A deficiency on human PDAC and identified the therapeutic potential by pathological and molecular evaluation. Immunohistochemical analysis suggested that loss of KDM6A in cancerous tissues was an independent prognostic factor for both recurrence-free and overall survival in the 103 tumor specimens surgically resected from patients with PDAC. We established KDM6A knocked out cells by using the CRISPR/Cas9 system and KDM6A-expressed cells by doxycycline-inducible system from each two human PDAC cell lines, respectively. KDM6A knockout enhanced aggressive traits of human PDAC cell lines, whereas KDM6A overexpression suppressed them. Microarray analysis revealed reduced expression of 22 genes including five well-known tumor suppressors, such as CDKN1A, and ChIP-PCR analysis displayed depleted enrichment of histone H3 lysine 27 acetylation (H3K27ac) at the promoter regions of the five candidates. The epigenetic alterations were induced by the impaired recruitment of histone acetyltransferase p300, which cooperatively interacted with KDM6A. Consistent with these results, the KDM6A knockout cells demonstrated higher vulnerability to histone deacetylase (HDAC) inhibitors through the reactivation of CDKN1A in vitro and in vivo than the KDM6A wild-type. In conclusion, KDM6A exhibited essential roles in human PDAC as a tumor suppressor and KDM6A deficiency could be a promising biomarker for unfavorable outcome in PDAC patients and a potential surrogate marker for response to HDAC inhibitors.

The Complex Role of KDM6A in B-Cell Development and Function

Loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell lymphoid malignancies, including B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma. Germline inactivating mutations in KDM6A cause a neurodevelopmental disorder called Kabuki syndrome that is associated with recurrent infections and hypogammaglobulinemia.1 The role of KDM6A in normal B-cell development and function, as well as how the somatic loss of KDM6A contributes to B-cell malignancies, has not been completely defined.To address this issue, we generated a conditional knockout mouse of the KDM6A gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate KDM6A in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) male and female KDM6A conditional KO mice. We found a significant shift from lymphoid to myeloid differentiation in the bone marrow and peripheral blood of these mice. Young, female KDM6A-null mice had mild splenomegaly. Their spleens had an increased number of neutrophils (Gr-1+CD11b+ cells) and erythrocyte progenitors (CD71+Ter119+ cells) and a decreased number of B-cells (B220+ cells). These changes became more pronounced with age and were specific to the female, homozygous KDM6A knockout mice. Furthermore, analysis of B-cell maturation showed that the loss of KDM6A was associated with decreased immature (B220+IgM+ cells) and mature, resting B-cells (B220+IgD+ cells) in the spleen. Similar changes were present in the bone marrow (decreased B220+IgM+ cells and B220+CD19+ cells) and peripheral blood (decreased B220+IgM+, B220+IgD+ and B220+CD19+ cells).Early B-cell development is also altered in KDM6A-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female KDM6A-null mice bone marrow. Next, we performed flow cytometry to catergorize the Hardy fractions of early B-cell development on bone marrow isolated from young, female KDM6A-null mice. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D, which was likely indicative of an incomplete block in B-cell differentiation after the Fraction A stage. When bulk bone marrow cells isolated from young, female KDM6A-null mice were plated in methylcellulose supplemented with interleukin-7, we observed a significantly decreased colony formation compared with bone marrow cells isolated from wildtype littermates. This pre-B lymphoid progenitor cell plating phenotype was expected given the flow cytometry results of decreased B-cell progenitors outlined above.We examined the effect of the loss of KDM6A expression on germinal center (GC) formation in the spleen following immunization with NP-CGG (4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Ratio 16). Two weeks after NP-CGG immunization, we observed a significant decrease in follicular B-cells (FO) and a significant increase in GC B-cells as compared to wildtype littermates (Figure 1). The result is significant as GC B-cells are thought to be the cell-of-origin of follicular and DLBCL.To determine if inactivation of KDM6A affected antibody production, we measured IgM, IgG, IgE and IgA levels by ELISA from serum isolated from young, female KDM6A-null mice. Results revealed higher levels of IgM and lower levels of IgG in serum from KDM6A-null mice, which is suggestive of a class switch recombination (CSR) defect. Concordant with this result, we observed that the loss of KDM6A impaired CSR to IgG1 in splenic B cells after in vitro stimulation for three days with lipopolysaccharide (LPS), an anti-CD180 antibody and interleukin-4. Moreover, we observed a striking defect in the production of plasma cells from KDM6A-null B-cells after LPS stimulation. Taken together, our data shows that KDM6A plays an important, but complex, role in B-cell development and that loss of KDM6A impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies.Stagi S, et al. Epigenetic control of the immune system: a lesson from Kabuki syndrome. Immunol Res. 2016; 64(2):345-359. DisclosuresNo relevant conflicts of interest to declare.

Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM.We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM.Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. DisclosuresLicht:Celgene: Research Funding.

Loss of KDM6A Confers Drug Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML.In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells.To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1.To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity.In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML. DisclosuresHiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.