Project 3: Genomics of secondary AML progression. The long-term goal of this project is to define the mechanisms underlying subclone expansion during progression from myelodysplastic syndromes (MDS) to secondary AML (sAML). Approximately 30% of patients with MDS, the most common adult myeloid malignancy in the US, progress to a rapidly fatal secondary AML. We have shown that progression from MDS to sAML is characterized by persistence of founding clone mutations, and expansion of one or more subclones. Preliminary data from our group and others suggest that an acquired mutation in a signaling gene (e.g., NRAS) or a myeloid transcription factor (e.g., RUNX1) may contribute to subclone and blast expansion in up to 50% of MDS patients. What drives subclone expansion in the remaining 50% of patients is not yet clear. We hypothesize that alternative genetic (i.e., non-coding or structural variants) or epigenetic alterations may also contribute to progression. We will identify acquired alterations that drive subclone expansion in this project. In Specific Aim 1, we will define the genomes and transcriptomes of rising subclones during progression from MDS to sAML. We will perform enhanced whole genome sequencing on sample trios (skin, MDS, and sAML bone marrow) to comprehensively define the clonal architecture of samples, including rising subclones during progression. In parallel, we will perform single-cell RNA-sequencing (scRNA-seq) on MDS/sAML paired samples to identify the expression signatures of subclones that evolve to cause sAML. By sequencing samples with and without known subclonal driver gene mutations, we will test whether all rising subclones have dysregulated expression of genes or pathways that control normal myeloid maturation. These studies will be further informed by the data collected on de novo and TP53 mutated AML samples in projects 1, 2, and 4. Collectively, these studies should define drivers of subclonal expansion that could potentially be targeted to prevent sAML progression. In Specific Aim 2, we will functionally validate mutations that contribute to subclone expansion during progression from MDS to sAML. The genes that cooperate with founding clone mutations to drive subclone expansion are not always known. We hypothesize that common MDS-initiating mutations in epigenetic modifier and spliceosome genes may ?prime? a hematopoietic stem/progenitor cell (HSPC), and make it more susceptible to progression by cooperating mutations; this in turn suggests that the order of mutation acquisition may be important for sAML pathogenesis. We will test the importance of subclone mutations by introducing them into primary mouse HSPCs that harbor a founding clone mutation using viral over-expression, or CRISPR/Cas9 technology for loss-of-function and monitor self-renewal, proliferation, and clonal expansion in recipient mice. We predict that only specific combinations of mutations--acquired in the correct order--will cooperate to induce clonal expansion. We will test the efficacy of drugs targeting the founding clone and/or the subclone using preclinical mouse and patient-derived xenograft models.