Abstract A017: IGF2BP1 is essential for ETO2/GLIS2 leukemogenesis via stabilization of primitive myeloid transcriptional programs

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Abstract Certain subtypes of pediatric leukemia continue to have dismal cure rates despite advances in targeted therapy. One such disease is acute megakaryoblastic leukemia (AMKL) with a rearrangement of ETO2/GLIS2 (also known as CBFA2T3/GLIS2.) Based on our preliminary data utilizing CRISPR/Cas9 screens in vivo in patient-derived xenografts, we have nominated IGF2BP1 as a potential vulnerability in ETO2/GLIS2-rearranged AMKL. For our discovery phase, we utilized a patient-derived E2G2r AMKL which successfully engrafts in NSGS mice. We created a stable Cas9-expressing subclone using lentiviral transduction followed by FACS enrichment and limiting dilution transplants. We then generated a custom library targeting the top 100 overexpressed genes between E2G2r leukemias versus other leukemia PDXs which we previously characterized, including eight transcription factors with motifs enriched in differentially expressed genes, as well as eight pan-essential positive controls. Mice were sacrificed at humane endpoints, and spleens and bone marrow underwent PCR amplification for gRNA sequences followed by sequencing and analysis with MAGeCK-VISPR (Wei L, et al. Genome Biol 2015). We observed the dropout of 38 genes with a false discovery rate < 0.25, including all positive control gRNAs. Among the most significantly depleted genes was the RNA binding protein IGF2BP1. We subsequently characterized phenotypic changes using shRNAs in the E2G2r cell line M07e and ETO2/GLIS2-transformed CD34+ umbilical cord blood cells. Notably, we observed impaired cell growth with IGF2BP1 knockdown in both cell lines as well as the parent xenograft. We also generated M07e lines expressing a degron (FKBPF36V, aka “dTAG”, Nabet et al. Nat Chem Biol 2018) knocked in at the endogenous IGF2BP1 locus and observed similar phenomena. Through flow cytometry of M07e following IGF2BP1 depletion, we observed changes in the leukemic immunophenotype as well, suggesting perturbation of megakaryocyte differentiation potential. To determine the mechanism underlying our observations, we performed enhanced UV crosslinking with IP and sequencing (Blue SM et al. Nat Protoc 2022) against both IGF2BP1 and m6A residues based on the putative role of IGF2BP1 as an m6A reader. We observed binding of IGF2BP1 predominantly in 3’UTRs, as well as the presence of m6A-modified bases at IGF2BP1 binding sites. Gene ontology (GO) analysis of IGF2BP1-bound transcripts demonstrated enrichment for GO terms pertaining to embryonic hematopoiesis and megakaryocyte maturation/development. We further evaluated global changes in gene expression following IGF2BP1 loss, and performed RNAseq 72 hours following shRNA-mediated knockdown. GO analysis showed that downregulated genes were highly enriched for E2F-mediated transcriptional programs. We hypothesize that IGF2BP1 regulates myeloid developmental transcripts essential for E2G2r leukemogenesis, likely via stabilization in an m6A-dependent fashion, and thus may represent a selective therapeutic target. Work is ongoing to further elucidate this proposed mechanism. Citation Format: Jason Clark, Mark Wunderlich, Jing Chen, Daniel Starczynowski, Lynn Lee. IGF2BP1 is essential for ETO2/GLIS2 leukemogenesis via stabilization of primitive myeloid transcriptional programs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A017.