Novel Small Molecule Inhibitor Against IGF2BP3 with Potent Anti-Leukemic Activity

Background: RNA binding proteins have emerged as promising therapeutic targets in cancer because of their role as important mediators of oncogenesis. The RNA binding protein IGF2BP3 is an oncofetal protein expressed during fetal development, absent or lowly expressed in adult tissues, and strongly re-expressed in cancer cells. IGF2BP3 has been found to be overexpressed in approximately 15-20 % of all cancer types, including glioblastoma, pancreatic cancer, soft tissue sarcoma, and colon cancer. Our group previously showed that IGF2BP3 was specifically overexpressed in MLL-AF4 translocated B-cell acute lymphoblastic leukemia (B-ALL) and is critical to the development of MLL-AF4 leukemia. We previously showed using a Igf2bp3 KO mouse that while IGF2BP3 is not required for normal hematopoiesis, depletion of IGF2BP3 significantly increased the survival of mice with MLL-AF4 leukemia and significantly decreased their leukemic burden. Therefore, these observations made by our group and others have established that IGF2BP3 is a potential therapeutic target for MLL-rearranged B-ALL and has the potential to be a target in multiple other cancer types, and therefore, is a promising target for the development of next generation therapeutics.

Methods and Results: Development of small molecule inhibitors against RNA binding proteins have been challenging due to limited structural information and highly disordered structures. In our present study, we developed an assay system leveraging the RNA binding function of IGF2BP3 using time-resolved Förster resonance energy transfer (TR-FRET) technology for high-throughput screening of compounds. Briefly, we created an IGF2BP3-GFP fusion construct, overexpressed it in 293T cells and purified the fusion protein using M2-FLAG resin. The purified protein was further incubated with biotin-labeled M6A-modified RNA oligonucleotides saturated with terbium-labeled streptavidin. The protein-RNA complex was excited at 350 nM, resulting in GFP fluorescence due to TR-FRET. Using the TR-FRET assay, we screened >190,000 compounds and identified 417 compounds as hits, with a hit percentage of 0.23%. We further confirmed the hits on cell-based counter-screen on WT and IGF2BP3 KO SEM cells and found IGF2BP3-specific activity in 11 of the compounds. We identified the compound I3IN-002 as the most promising hit and further characterized it in the following downstream studies.

We performed IC50 experiments on multiple cell lines including B-ALL cell lines with MLL-AF4 translocation and found an IC50 of ~2.0 µM in SEM, RS4;11 cells, 5.0 µM in murine bone marrow derived Lin^- MLL- Af4 and OCI-AML 8227 cells. Further, I3IN-002 treatment induced apoptosis and cell death in SEM and RS4;11 cells. Treatment of murine Lin^- MLL-Af4 cells led to the reduced colony forming units and decrease in CD34+ cKit+ population, suggesting reduction in stem cell population due to compound treatment. In vivo study performed on C57Bl6/J mice transplanted with Lin^- MLL-Af4 cells and treated with I3IN-002/ vehicle showed reduced leukemia expansion and decreased leukemic burden in the treatment group compared to vehicle control. On-target activity of the I3IN-002 was assessed by performing RNA immunoprecipitation assay and found reduced enrichment of IGF2BP3 targets such as CDK6, HOXA9 and BCL2. Next, we studied change in the gene expression by RNA-seq experiment, by treating WT cells with I3IN-002 and DMSO carrier control and IGF2BP3 KO cells treated with DMSO. Using hypergeometric testing, we found extremely significant common pathway enrichment between I3IN-002 treatment and IGF2BP3 KO. On-target activity was further assessed by performing cellular thermal shift assay (CETSA). We found that binding of the compound to the IGF2BP3 protein stabilizes the protein in thermal denaturation step. Lastly, we tested the activity of I3IN-002 on MLL-AF4 PDX cells and found a significant decrease in the proliferation of cells with treatment.

Conclusion: We report the successful identification and development of a novel small molecule inhibitor that targets IGF2BP3 protein and inhibits the proliferation of leukemic cells resulting in reduced leukemic burden in the leukemia mice model.