Cooperative Epigenetic Regulation By ASXL1 and NF1 Loss on Leukemogenesis

Introduction:Additional sex combs-like 1 (ASXL1) is frequently mutated in a wide range of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasm, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). Notably, ASXL1 mutations are generally associated with the poor clinical outcomes. We and others have established Asxl1 mouse models and demonstrated that loss of Asxl1 leads to MDS-like disease, which can transform to myeloid leukemia in aged mice. These studies suggest that additional mutations may cooperate with Asxl1 loss to induce the leukemia transformation. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Methods: To identify cooperating events with ASXL1 mutations in myeloid malignancies, we recruited a cohort of 138 ASXL1 mutated patients and performed targeted exome sequencing. We then characterized the hematopoietic features using mouse models. A serial hematopoietic phenotypic analyses were used, including peripheral blood counts, flow cytometry, colony assay, morphology and transplantation assays. To decipher the molecular mechanisms by which loss of Asxl1 and Nf1 cooperate in promoting myeloid leukemia transformation, we performed RNA-seq and ChIP-seq to identify the differentially expressed genes and their associated histone modifications in four genotypes of mice, WT, Asxl1+/-, Nf1+/-, and Asxl1+/-;Nf1+/- mice. Finally, the leukemic mice were treated with pharmacologic inhibitors targeting both MAPK pathway and BET bromodomain in vivo as a proof-of-concept approach. Results: We analyzed the gene mutation profiles of 138 ASXL1 mutated patients based on targeted sequencing and found that 35 of these patients have gene mutations involving in RAS/MAPK signaling pathway, including NF1, NRAS, KRAS, PTPN11 or CBL. The incidence of AML was significantly higher in patients with RAS pathway mutations (48.6%) than in the cases without RAS pathway mutations (29.1%, p = 0.036, chi-square test). These data suggest that concomitant mutations of ASXL1 and RAS pathway genes associate with poor prognosis in myeloid malignancies. To validate the functional significance of cooperative mutations of ASXL1 and NF1, a negative regulator of the RAS signaling pathway, in the disease progression of myeloid leukemia, we generated Asxl1+/-;Nf1+/- and Mx1Cre;Asxl1fl/fl;Nf1fl/fl (Asxl1Δ/Δ;Nf1Δ/Δ) mice and performed hematopoietic phenotypic