UCLA

MLL-rearranged (MLL-r) leukemias are a clinically challenging and biologically unique subtype of leukemias associated with a poor prognosis and are characterized by their MLL-fusion proteins (MLL-FPs) that drive leukemia through epigenetic dysregulation and cooperation with downstream regulatory mechanisms. While novel therapeutic strategies for MLL-rearranged leukemias have been primarily directed at epigenetic dysregulation and concurrent downstream activating mutations or kinases, post-transcriptional gene regulation mechanisms have recently emerged as important mediators in MLL-FP leukemogenesis and have the potential to be potent combinatorial therapeutic targets. Our group has previously identified and studied the RNA binding protein, insulin-like growth factor 2 binding protein 3 (IGF2BP3) as a critical regulator of MLL-AF4 leukemogenesis. The goal of this dissertation is to investigate the mechanisms by which IGF2BP3 supports MLL-AF4 leukemogenesis and to explore its therapeutic potential by investigating combined inhibition of menin-MLL and IGF2BP3 in models of MLL-AF4 driven leukemia, in a novel combinatorial therapeutic strategy of targeting leukemia at the transcriptional and post-transcriptional level.We studied the combined effects of targeting menin-MLL and IGF2BP3 in MLL-AF4 leukemia using human B-acute lymphoblastic leukemia (B-ALL) cell lines and murine hematopoietic stem and progenitor (HSPCs) immortalized by MLL-Af4 expression in vitro and in vivo. In our studies, we harnessed the versatility of these immortalized HSPCs, derived from bone marrow of Cas9-EGFP mice, using their abilities to be readily modified by single-guide RNA introduction and to provide functional readouts on leukemic initiating cell function and number in vitro and in vivo using endpoint colony formation assays and transplantation. We found that IGF2BP3 knockdown via CRISPR/Cas9-mediated deletion sensitized human B-ALL cells with MLL-AF4 fusion and murine MLL-Af4 HSPCs to treatment with multiple commercially available menin-MLL inhibitors, showing anti-leukemic effects of decreased cell growth and increased apoptosis in vitro and negative effects on leukemic initiating cells by decreased colony formation in endpoint colony formation assays in vitro and by decreased leukemic engraftment in transplantation experiments in vivo. With regards to possible underlying mechanism, detailed evaluation of colony morphologies, histopathology, and RNA sequencing data all showed a consistent shift towards increased differentiation with IGF2BP3 knockdown and menin-MLL inhibition. While additional gene expression analyses and molecular studies are ongoing, we have noted significant overlap in the differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown, in biologically relevant pathways, confirming that IGF2BP3 and MLL-Af4 closely interact and functionally cooperate to regulate gene expression in MLL-AF4 driven leukemia. Lastly, in our in vivo MLL-Af4 leukemia model, IGF2BP3 depletion demonstrated a greater effect on increasing survival and delaying leukemia progression than ex vivo MI-503 treatment at the chosen micromolar dose, highlighting IGF2BP3 as a potent and promising therapeutic target. In summary, our studies confirm a role for IGF2BP3 as an oncogenic amplifier of MLL- AF4 driven leukemia, with open questions regarding molecular mechanisms and role in leukemic stem cell function, and highlight its therapeutic potential, while suggest a promising and novel combinatorial approach to targeting leukemia at the transcriptional and post-transcriptional level.