The deep biological characterization of Acute Myeloid Leukemia harboring recurrent genetic alterations to tailor novel target therapies

Despite recent improvements in Acute Myeloid Leukemia (AML) survival rates, the percentage of relapsed/refractory AML remains too high. Improved clinical outcomes will arise with a better characterization of the molecular consequences of the genetic lesions driving each AML subtype: this project will focus on mutations affecting the FLT3 and ASXL1 genes. FLT3 mutations (FLT3-mut) are present in about 1/3 of de novo AML and their prognostic role have been described several years ago, thus several FLT3 inhibitors (FLT3i) are currently part of routine clinical practice. By analyzing legacy RNA-seq data from the BEAT-AML Master Trial we documented an association between FLT3-mut and AML differentiation state and then between FLT3-mut and a cold/less-infiltrated AML immune microenvironment but with an intact Effector T-cell state. Secondly, by enrolling two cohorts of FLT3-mut AML patients treated real-life with FLT3-i based therapies, we showed how the achievement of response relies on immune transcriptomic pathways activation, related to T-/NK-cell activity in regard to Gilteritinib salvage and to innate immunity in patients treated upfront with intensive chemotherapy plus Midostaurin. ASXL1 mutations are frequently detected in elderly people without any evident hematological disease, representing a common subtype of Clonal Hematopoiesis (CH), associated with an increased risk of myeloid malignancies (MM). Developing a target drug to halt the progression of ASXL1-mut CH toward MM/AML would be a major breakthrough in hematology. In this project we first built an in-vitro model of ASXL1-mut CH by using CRISPR Cas9 genome editing technology on 32D murine myeloblast-like cell line. Then we evaluated phenotypic differences induced by ASXL1-mut, without documenting evident changes in cell proliferation, morphology and differentiation state. We then performed a genome-wide CRISPR Cas9 drop-out screen on isogenic 32D cell line clones and identified genetic vulnerabilities associated with ASXL1-mut, which may represent candidate for target drug development, deserving further investigation.