Cooperative Effect of Haploinsufficient ASXL1 and NF1 in Acute Myeloid Leukemia Transformation

Objective: The advent of high-throughput whole genome or exome sequencing has led to the identification of almost all genetic aberrations in acute myeloid leukemia (AML). These genetic aberrations have been classified into at least three classes (class I: activate signal transduction pathways and confer a proliferation advantage; class II: affect transcription factors and impair hematopoietic differentiation; class III: concern with epigenetic regulation. AML development is considered to be a multistep process that requires the collaboration of at least two classes of mutations, the so-called "two-hit model". Additional Sex Combs Like 1 (ASXL1) gene, encoding polycomb protein ASXL1, is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. Hyperactive RAS signaling is also common in myeloid malignancies, which is caused by mutations in the RAS genes or an inactivation of the neurofibromatosis (NF1) gene. The objective of this project is to determine the cooperative effects of NF1 loss (class I) and ASXL1 loss (class III) on the AML development using a novel mouse model with combined haploinsufficiency of Asxl1 and Nf1.

Methods: In the current study, we intercrossed Asxl1^+/- mice with Nf1^+/- mice and characterized the hematopoietic features of Asxl1^+/-;Nf1^+/- mice in vivo. A serial analyses were used for hematopoietic analysis, including peripheral blood counts, blood smear, morphology, flow cytometry and histology. In vivo tumor transfer was performed to evaluate the malignant nature of the infiltrated cells in moribund/diseased compound Asxl1^+/-;Nf1^+/- mice. To define the molecular mechanisms by which haploinsufficient Asxl1 and Nf1 cooperate in promoting AML transformation, western blotting was performed to examine the dysregulated histone modifications and RNA-seq was performed to survey the differentially expressed genes in each genotype of mice.

Results: A worse survival rate was seen in Asxl1^+/-;Nf1^+/- mice as compared to all other three groups of control mice, including WT, Nf1^+/-, and Asxl1^+/- mice. WT and Nf1^+/- mice remain disease free till the end of the observed period of time (17 months), while a proportion of aged Asxl1^+/- mice developed MDS-like diseases as characterized by multilineage cytopenias and dysplasia, which is consistent with our previous report (Wang et al. Blood 2014). Interestingly, haploinsufficiency of both Asxl1 and Nf1 results in progressive and lethal AML, as reflected by >20% of blasts in the bone marrow. Asxl1^+/-;Nf1^+/- mice also exhibited an elevated white blood cell counts with anemia and thrombocytopenia, as well as spleno-hepatomegaly. Infiltration of immatured myeloid cells was promident in the spleens and livers of Asxl1^+/-;Nf1^+/- mice. Tumor transfer assay revealed that the leukemia was transferable to the sub-lethal irradiated recipients, and the secondary transplanted mice displaying a similar phenotype to the primary AML mouse as early as 7 weeks after the tumor transfer. Furthermore, biochemistry study revealed a reduction of the global levels of H3K27me3 in Asxl1^+/- c-Kit⁺ cells. Heterozygous of Nf1 in Asxl1^+/- c-Kit⁺ cells further diminished globle level of H3K27me3. Interestingly, the mRNA expression of Jarid2, an accessory component of polycomb repressive complex-2 (PRC2), was significantly lower in c-Kit⁺ cells of Asxl1^+/-;Nf1^+/- mice than that in c-Kit⁺cells of other genotypes of mice. The reduced level of Jarid2 may result in further reduction in recruitment of the PRC2 component to the chromatin, dminishing H3K27me3 level.

Conclusion: Mutations of NF1, encoding neurofibromin that functions as a negative regulator of RasGTP, are found co-exist with ASXL1 mutations in patients with myeloid malignancies. Our data indicate that combined haploinsufficiency of ASXL1 and NF1 trigger AML transformation through an additive effects to reduce H3K27me3 levels. Together, our data reinforce the impact of combined genetic abnormalities in class I and class III genes in the progression of myeloid malignancies into AML.