Supported by R01CA254354: Targeting the Metabolic Regulator SIRT5 in Acute Myeloid Leukemia (AML) we have reported that the lysine deacylase SIRT5 is a druggable metabolic target in AML (Yan et al. Blood Cancer Discov. 2021;2(3):266-287). Our new preliminary data suggest that the role of SIRT5 may extend to acute lymphoblastic leukemia (ALL), including relapsed/refractory (R/R) ALL. ALL is the most common cancer in children. After a peak at age 9, incidence initially declines, but then rises steadily after the age of 30. Most ALL cases originate from B-lymphoid precursor cells (B-ALL), but 15% are of T cell origin (T-ALL). Aggressive chemotherapy has greatly improved outcome in children, but often at the expense of long-term functional impairment. In contrast, long-term survival in adults is <50%, reflecting high-risk genotypes and higher treatment- related mortality. Chimeric antigen receptor (CAR) T cells targeting B cell antigens are effective in a subset of R/R B-ALL, but resistance often develops due to loss of the target antigen. Salvage therapies for RR T-ALL are ALL survival and reduce functional impairment in survivors. While ALL is genetically heterogeneous, most cases exhibit a differentiation block at the level of a B or T cell precursor. There is evidence that energy restriction creates a barrier to malignant transformation as B cell precursors undergo somatic hypermutation during antibody diversification. Mutational inactivation of B lineage transcription factors disables this metabolic brake and allows malignant transformation by oncogenes such as BCR-ABL1. Similar mechanisms may operate in T cell precursors during T cell receptor diversification. Our discovery that SIRT5 is a key regulator of AML metabolism and the reported interplay between metabolic reprogramming and malignant transformation in ALL, we asked whether SIRT5 dependence extends to ALL. We find that many ALL cell lines, including drug-resistant lines, are sensitive to SIRT5 knockdown (KD), prompting us to hypothesize that SIRT5 is required for maintaining ALL metabolism. Our Aims are: (1) Determine the effects of SIRT5 disruption on ALL cell lines and primary cells and correlate with global changes in energy metabolism. We will use shRNA, CRISPR/Cas9 and small molecule inhibitors to disrupt SIRT5 in ALL cells, correlate effects on growth and viability with changes in energy metabolism, and test whether disrupting SIRT5 is synergistic with other ALL therapeutics. (2) Identify SIRT5- regulated pathways and biomarkers of SIRT5 dependency in ALL. We will subject ALL cells to RNA and DNA sequencing and metabolomic profiling and identity features associated with SIRT5 dependency. (3) Determine whether SIRT5 disruption reduces leukemia burden and improves survival in mouse models of ALL. We will test the effect of SIRT5 disruption in genetic ALL models and patient-derived xenografts. Metabolic plasticity limits the efficacy of inhibiting individual metabolic pathways. To ci...