Abstract A021: Evolutionary trajectories and single cell methylation analysis of DNMT3A and TET2 mutant acute myeloid leukemia

Abstract Rationale: Leukemic evolution occurs through gradual clonal selection at early and late stages of disease development. The early stage of this evolutionary process is called clonal hematopoiesis (CH) and is typified by expansion, and increased self-renewal capacity, of non-malignant, somatically mutated hematopoietic stem and progenitor cells (HSPCs). CH-mutant genes result in either genomic hypomethylation through loss of DNMT3A, or hypermethylation either by genetic loss of TET2 or its’ inhibition by the oncometabolite 2-HG generated by mutant IDH1/IDH2. Leukemic transformation is often associated with subsequent mutagenesis in a broader range of genes including mitogenic signaling genes that confer increased proliferative capacity (FLT3, NRAS). The stepwise acquisition of mutations generates a polyclonal “ecosystem” of clones with increasing clonal diversity indicative of disease progression and poor prognosis. The goal of this study is to 1) identify genetic evolutionary trends associated with distinct CH-related mutations in DNMT3A and TET2 and 2) implement innovative single cell DNA sequencing protocols to identify genotype-selective DNA methylation alterations at single cell resolution. Approach: We compiled patients diagnosed with de novo AML at the University of Pennsylvania that had multiple genomic profiling studies, a cohort comprising 182 patients. All patients were profiled at diagnosis, with n=119 at first remission and n=76 at first relapse. 84.6% of patients received induction chemotherapy with combination anthracycline and nucleoside analog therapy. To investigate the genomic changes in AML disease course, we calculated the change in variant allele frequencies (VAFs) for all identified variants, evaluating matched samples from individual patients across time. We infer phylogenetic relationships from matched genomic profiles across timepoints in individual patients using CALDER. Finally, we performed single cell DNA methylation sequencing on 3 patients (n=4000 cells) using the Single Cell Methylation Kit from Scale Biosciences. This is a combinatorial indexing-based whole genome bisulfite sequencing based approach. Results: Comparing VAFs between diagnosis and relapse for DNMT3Amut vs TET2mut AML, we observed that FLT3 and NPM1 variants were similarly split between increased and decreased VAFs at relapse vs diagnosis. NRAS mutations were both gained and lost in DNMT3Amut samples between diagnosis and relapse, but no NRAS mutations were identified at all in TET2mut samples. When inferring phylogeny associations, we found DNMT3Amut samples were more likely to undergo subclonal swapping from diagnosis to relapse, whereas TET2mut samples relapsed with a more stable mutation profile. Analyses are currently underway for single cell DNA methylation data. Conclusions: Our analyses suggest that DNMT3A and TET2 have distinct sets of co-occurring genetic alterations at diagnosis and relapse. Early CH-associated mutations preceding transformation can shape the evolutionary trajectories of AML throughout disease development. Citation Format: Nisargbhai Shah, Ryan Cho, Priya Velu, Selina Luger, Martin Carroll, Jennifer Morrissette, Michael Bowman, Robert Bowman. Evolutionary trajectories and single cell methylation analysis of DNMT3A and TET2 mutant acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A021.