Functional analysis of gene interactions in ASXL1 mutated Acute Myeloid Leukemia using CRISPRa/i screenings

Acute Myeloid Leukemia (AML) presents a complex therapeutic challenge largely driven by its genetic heterogeneity. Among the genes recurrently mutated in AML, Additional Sex Combs-Like 1 (ASXL1) is notable for its association with adverse clinical outcomes. To better understand the contributions of ASXL1 mutations to leukemogenesis, I established a complementary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation/interference (CRISPRa/i) screening platform in murine interleukin-3 (IL-3) dependent 32Dcl3 myeloid progenitor cells stably expressing wildtype, mutant (Asxl1Y588X), or control Asxl1 constructs. By withdrawing IL-3, I aimed to identify the ASXL1-linked genetic networks that enable growth factor-independent proliferation, thereby shedding light on the mechanisms by which ASXL1 mutations confer a proliferative advantage in AML. To validate the CRISPRa/i technology, I first characterized the 32Dcl3 cells using pooled sgRNA libraries targeting apoptosis- and cancer-related genes, successfully identifying Colony-Stimulating Factor 2 (Csf2) as a key dependency marker. Building on this validation, I then screened Asxl1 wildtype, mutant, and control cells and identified multiple proliferation-associated genes. Among these, Acidic leucine-rich nuclear phosphoprotein 32 family member B (Anp32b) stood out for exhibiting distinct, growth-promoting effects in both wildtype and mutant Asxl1-expressing 32Dcl3 cells. These findings confirmed the functionality of the CRISPRa/i platform and provided physiologically relevant expression insights not captured by conventional CRISPR knockout methods. Despite methodological constraints, including a limited number of replicates and the strong selection pressure imposed by IL-3 withdrawal, these results offer a robust foundation for identifying genetic interactions and dependencies in ASXL1-mutated AML. Moving forward, candidate genes and pathways highlighted in these screens can be further validated in vitro and in vivo, ultimately advancing our understanding of AML pathogenesis and guiding the development of targeted therapies.