Infant KMT2A::AFF1 acute lymphoblastic leukemia stem cell model reveals early impact on the proteome

Infants with acute lymphoblastic leukemia (iALL) are at high risk of early relapse, rapid progression, and poor survival despite intensive therapies. Rearrangement of KMT2A ( KMT2A -r) is an established feature of these cases, creating a fusion oncogene that leads to epigenomic dysregulation. Genomic studies have failed to find collaborating oncogenic drivers that would explain why infants have such a high risk of relapse compared to older patients with the same rearrangement. To expand our understanding of KMT2A - driven leukemogenesis, we applied global proteomic profiling to identify potential nongenomic drivers that could provide novel therapeutic targets.For this study, we performed proteomics with our human inducible pluripotent stem cell model (iPSC) of KMT2A- r iALL. We have engineered this model with a human KMT2A- r transgene construct (rearranged with the most common partner AFF1 ) incorporated with an established doxycycline (dox) regulated platform. These cells tightly express the KMT2A::AFF1 transgene only in response to dox treatment. We have developed functional hematopoietic stem cells (HSPC) that we have induced with dox to express KMT2A- r and represent the preleukemic state. With replicates of dox-induced KMT2A::AFF1 HSPC and of non-dox control, we generated proteomic profiles via data independent acquisition (DIA) using the timsTOF HT (Bruker). Data were searched in DIA-NN using the Bruker spectral library and human protein database downloaded from Uniprot on 05-05-2024. Data analysis was performed in R 4.3.3 including differential expression analysis using limma 3.58.1, pathway enrichment using gProfiler 0.2.3, and network analysis using STRING 12.0.Differential expression analysis identified 149 proteins with higher and 381 proteins with lower expression in KMT2A- r HSPC as compared to the control (p <0.05, |log2FC| >1). Histone binding is a top upregulated pathway of proteins with the most significance (adjusted p <0.05), which is what we would expect with induction of a epigenomic regulator. Pathways downregulated in KMT2A- r HSPC largely involve the extracellular environment (for example, cell adhesion mediator activity, collagen binding, extracellular matrix organization). STRING analysis of the downregulated proteins also demonstrates a prominence of extracellular factors (i.e. MFAP4, L1CAM, COL5A1). Comparison to bulk RNA sequencing on the model at the same timepoint reveals protein expression correlates with gene expression about half of the time. Phosphoproteomics has been performed and is currently being analyzed for further comparison. Preleukemic KMT2A- r HSPC demonstrate early changes in the proteome that suggest the extracellular environment may play a key role in leukemogenesis. With further validation and testing of these factors, we will aim to identify essential drivers that may provide new therapeutic targets for this devastating disease.