Deciphering inv3/t(3;3) Myeloid Leukemia Vulnerabilities via Proteogenomics and N-terminal Proteomics

PROJECT SUMMARY The overall objective of this proposal is to develop and apply novel proteogenomic approaches to identify therapeutic vulnerabilities of chromosome 3 rearranged myeloid leukemias. Chromosome 3 inversions or transversions (inv3/t(3;3)) leukemias result in aberrant overexpression of oncogenic transcription factor MECOM (MDS1 and EVI1 complex locus, specifically a truncated isoform EVI1) and decreased expression of tumor suppressor GATA2. Inv3/t(3;3) abnormal myeloid leukemia patients have no targeted precision therapies as they are resistant to standard of care regimens like Tyrosine Kinase Inhibitors (TKIs), and so experience dismal 5- year survival rates of <5%. Consequently, there is an unmet need for new approaches to therapeutically target inv3/t(3;3)-driven leukemias. Canonically, MECOM is critical in regulating hematopoiesis, however, alternative transcripts of MECOM drive oncogenesis when overexpressed as a result of chromosomal rearrangements. In addition to oncogenic MECOM, I hypothesize that the inv3/t(3;3) abnormality leads to the production of additional leukemia specific proteoforms that contribute to the aggressive nature of this disease. Here, I will test this hypothesis by comparing inv3/t(3;3) positive and negative myeloid leukemia cell lines with healthy cord-blood derived hematopoietic progenitor cells using novel proteogenomic approaches. To test this hypothesis I will: identify inv3/t(3;3) specific genes by combining RNA-Seq and bulk proteomics (Aim 1), utilize PacBio long read sequencing with proteomics to identify novel protein coding alternative transcripts (Aim 2) and capture inv3/t(3;3) unique post-translational processes through utilization of my N-terminal proteomics methodology termed N- SPyCE (neo-N-terminal SP3 Pyridine Carboxaldehyde Enrichment) (Aim 3). In exciting preliminary data, I have already identified several key candidates for MECOM-affected genes, including HLA-DPB1, IL16, SPP1, CD74 and ALDH1A1. Pathway analysis of my bulk proteomic data revealed a striking correlation between inv3/t(3;3) harboring cells and increased cathepsin-mediated protease activity. I hypothesize that there are specific cathepsin cleaved substrates that are specific effectors of inv3/t(3;3) biology. Overall, this project proposes to utilize proteogenomics to detect disease specific transcript isoforms, to validate their expression at the protein level and to investigate leukemia specific proteoforms as therapeutically relevant.