Metabolic Drivers of VDJ Diversity in B-ALL: Implications for MRD Detection and Relapse - PROJECT SUMMARY Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and a leading cause of pediatric cancer mortality. Despite modern multi-agent chemotherapy achieving ~85% long-term survival in children, outcomes for relapsed ALL remain poor, and survival for adults is substantially worse. Minimal residual disease (MRD), measured by high-throughput sequencing (HTS) of immunoglobulin heavy chain (IgH) rearrangements, is the most powerful prognostic factor used in clinical risk stratification. Yet two major gaps remain: first, the biology of the residual cells themselves and their leukemia-initiating capacity are poorly understood; and second, some patients relapse despite undetectable MRD, underscoring the limitations of current methods. Preliminary work from our group demonstrates that subclonal IgH rearrangements with V-DJ diversity are common in ALL, limit MRD detectability, and correlate with poor outcomes. These VDJ-diverse subclones are enriched for activated mTOR signaling and mitochondrial/pyrimidine metabolism, suggesting that relapse biology is tied to both immunogenomic and metabolic states. We hypothesize that VDJ diversity reflects a biologically distinct state that supports persistence and relapse, and that targeting its metabolic underpinnings may eliminate these resistant populations. The proposed studies will test this hypothesis across three integrated aims. Aim 1 will define prognostic parameters of VDJ diversity by analyzing Ig HTS data from more than 1,700 patients treated on Phase 3 clinical trials and with standard of care immunotherapies. These analyses will establish the prevalence of VDJ diversity, its relationship to clinical and molecular features, and its prognostic impact on outcomes. Aim 2 will identify relapse-permissive cellular states based on VDJ rearrangements by performing multimodal single-cell profiling (Tapestri DNA+ADT, CITE-seq, and CyTOF) in 25 paired diagnosis-relapse samples. These studies will determine whether VDJ-diverse subclones are enriched at relapse and whether they exhibit metabolic programs marked by mTOR activation, glycolysis, oxidative phosphorylation, and pyrimidine synthesis. Aim 3 will determine the leukemia-initiating potential of VDJ subclones under metabolic stress. Using patient-derived xenografts, limiting dilution assays, and targeting of mitochondrial metabolism, we will test how perturbing mitochondrial metabolism remodels VDJ clonal composition and impacts leukemia-initiating subclones. Performing these studies in immunodeficient mice is essential for assessing human leukemiainitiating capacity, as the functional definition of leukemic stem cells requires demonstration of disease initiation and propagation in vivo, which cannot be replicated with in vitro culture models alone. By integrating large clinical cohorts, high-resolution single-cell analysis, and functional in vivo assays, this project will establish the clinical and biologic importance of VDJ diversity, define its mechanistic link to metabolism, and evaluate therapeut ic strategies to target relapse-permissive states. The anticipated outcome is a transformative framework that refines risk stratification, improves MRD interpretation, and nominates metabolism-directed therapies that may prevent relapse.
