Abstract Myeloproliferative neoplasms (MPNs) are clonal myeloid malignancies which are initiated by somatic mutations in hematopoietic stem and progenitor cells. Of the classic MPNs, the disease myelofibrosis (MF) is characterized by the shortest median survival as well as progressive anemia, splenomegaly, and constitutional symptoms. Somatic JAK2V617F mutations are observed in approximately 60% of MF patients; however, murine studies have shown that JAK2V617F mutations are not sufficient to induce MF in the absence of additional mutations. Notably, the most common mutations that co-occur with JAK2V617F in MF are in the polycomb gene Additional Sex Combs Like-1 (ASXL1). Importantly, ASXL1 mutations are the strongest predictor of poor overall survival in MF. Given the role of ASXL1 mutations in MF pathogenesis and outcome, we hypothesized that loss of ASXL1 and JAK2V617F would collaborate to modulate epigenetic regulation and facilitate hematopoietic disease transformation. Previous studies have shown that JAK2V617F can translocate to the nucleus and phosphorylate histone H3 on tyrosine 41 (H3Y41), allowing for regulation of specific target genes including JAK2 itself. We used RNA interference to knock down ASXL1 in two JAK2V617F-mutant leukemic cell lines, SET2 and UKE1, which revealed that global and locus-specific H3Y41 phosphorylation levels increased after ASXL1 silencing leading to increased expression of JAK2/H3Y41 target genes. To evaluate whether H3Y41 levels were changed in vivo as well, we crossed a Jak2V617F knock-in mouse to our Asxl1 hematopoietic conditional knockout allele. Heterozygous expression of Jak2V617F and heterozygous deletion of Asxl1 driven by Vav-Cre hastened myeloid expansion and decreased overall survival in primary mice consistent with in vivo cooperativity between Jak2V617F and Asxl1. Multiparameter flow cytometric staining revealed that Vav+ Jak2V617F/+ Asxl1f/+ mice have an expansion of immature erythrocytes in their bone marrow as compared to Vav+ Jak2V617F/+ mice, suggestive of impaired hematopoietic differentiation, which was more severe in Vav+ Jak2V617 VF/+ Asxl1 f/f mice. We then used the Mx1-Cre transgene to induce Jak2V617F and delete Asxl1 in the adult hematopoietic compartment. Mx1-Cre+ Jak2V617F/+ Asxl1f/f mice developed severe, progressive anemia, erythroid precursor expansion in the peripheral blood, bone marrow and spleen, and increased disease burden in primary mice and serially transplanted mice. Importantly, mice expressing Jak2V617F/+ and concomitant Asxl1 loss/haploinsufficency have increased Jak2 protein expression, and increased elevated expression of H3Y41 target genes consistent with coordinate dysregulation of chromatin state by JAK2V617F and ASXL1 loss. These findings suggest ASXL1 loss contributes to myeloid transformation in part through modulation of H3Y41 phosphorylation levels, leading to amplified JAK2 expression, increased JAK-STAT signaling, and disease progression. Citation Format: Lindsay M. LaFave, Young Rock Chung, Matt Keller, Maria Kleppe, Ann Mullally, Omar Abdel-Wahab, Ross L. Levine. Coordinate regulation of chromatin state by JAK2 and ASXL1 mutations in myeloid malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A09.