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Previously, we identified SETD2 loss-of-function mutations in 22% of MLL-rearranged (MLLr) acute leukemia patients, implicating a mechanism for cooperativity between SETD2 mutations and MLL fusions. However, the detailed mechanism of how SETD2-H3K36me3 downregulation accelerates MLLr leukemia remains unclear. Here, we show that in MLLr leukemia, both H3K79me2 and H3K36me3 are aberrantly elevated and co-enriched in a group of genes. SETD2 inactivation leads to a global reduction of H3K36me3 and a further elevation of H3K79me2, but does not change the expression of known MLL fusion target genes. Instead, this pattern of histone changes is associated with transcriptional deregulation of a novel set of genes; downregulating tumor suppressors (for example, ASXL1) and upregulating oncogenes (for example, ERG). Taken together, our findings reveal a global crosstalk between the oncogenic DOT1L-H3K79me2 axis and the tumor suppressive SETD2-H3K36me3 axis in gene regulation, provide molecular insights into how SETD2 mutations accelerate MLLr leukemogenesis through differential regulation of additional tumor suppressors and oncogenes.

Both membrane-proximal and truncation mutations in CSF3R have recently been reported to drive the onset of chronic neutrophilic leukemia (CNL). Here we show that although truncation mutation alone can not induce leukemia, both proximal and compound mutations (proximal and truncation mutations on same allele) are leukemogenic with a disease latency of 90 and 23 days, respectively. Comparative whole-genome expression profiling and biochemical experiments revealed that induced expression of Mapk adaptor protein Ksr1 and enhanced Mapk signaling are crucial to leukemogenesis by CSF3R proximal and compound mutants. Moreover, inhibition of Mek1/2 by trametinib alone is sufficient to suppress leukemia induced by both CSF3R proximal and ruxolitinib-resistant compound mutations. Together, these findings elucidate a Mapk-dependent mechanism of CSF3R-induced pathogenesis, and they establish the rationale for clinical evaluation of MEK1/2 inhibition in CNL.

PURPOSE: To evaluate cellular protein changes in response to treatment with an approved drug, ibrutinib, in cells expressing normal or mutated Granulocyte-Colony Stimulating Factor Receptor (G-CSFR). G-CSFR mutations are associated with some hematological malignancies. Previous studies showed the efficacy of ibrutinib (a Bruton’s tyrosine kinase inhibitor) in mutated G-CSFR leukemia models but did not addressed broader signaling mechanisms. EXPERIMENTAL DESIGN: A label-free quantitative proteomics workflow to evaluate the cellular effects of ibrutinib treatment was established. This included 3 biological replicates of normal and mutated G-CSFR expressed in a mouse progenitor cell (32D cell line) with and without ibrutinib treatment. RESULTS: The proteomics dataset showed about 1,000 unique proteins quantified with nearly 400 significant changes (p value < 0.05), suggesting a highly dynamic network of cellular signaling in response to ibrutinib. Importantly, the dataset was very robust with coefficients of variation (CVs) for quantitation at 13.0-20.4% resulting in dramatic patterns of protein differences among the groups. CONCLUSIONS AND CLINICAL RELEVANCE: This robust data set is available for further mining, hypothesis generation, and testing. A detailed understanding of the restructuring of the proteomics signaling cascades by ibrutinib in leukemia biology will provide new avenues to explore its use for other related malignancies.

MLL partial tandem duplication (MLL-PTD) is found in 5-8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. Mll-PTD knock-in mouse model shows that Mll-PTD HSPCs exhibited a proliferative advantage over wild-type HSPCs, and The ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, Mll-PTD HSPCs never develop MDS or s-AML in primary or transplanted recipients, suggesting that additional genetic and/or epigenetic defects are necessary for transformation. Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in MDS/AML patients, which suggest a potential cooperation for transformation between these two mutations. Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/- background and assessed HSPCs of Mll-PTD/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and Mll-PTD/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LK and LSK cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT controls. FLCs of DH also showed dominant expansion in both serial competitive and non-competitive BMT assays compared to WT controls. The DH derived ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from Mll-PTD mice. However, DH HSPCs do not develop MDS or s-AML in primary or in BMT recipient mice. We further generated Mll-PTD/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia one month after pI-pC injection, and developed pancytopenia 2-4 months later. The complete blood count exhibited increased MCV, RDW and severe anemia. All these Mll-PTD/Runx1Δ/Δ mice died of MDS induced complications within 7-8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in Mll-PTD/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Citation Format: Yue Zhang, Xiao-mei Yan, Goro Sashida, Ai-Li Chen, Xing-Hui Zhao, Susan P. Whitman, Michael A. Caligiuri, Zhi-Jian Xiao, Harry Leighton Grimes, Gang Huang. Modeling MLL-PTD related Myelodysplastic Syndromes (MDS) in mouse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3855. doi:10.1158/1538-7445.AM2013-3855