Identification of Nuclear NAD+ Salvage As a Therapeutic Vulnerability in B-Lymphoid Malignancies

Background and Significance: Reflecting collateral damage from immunoglobulin V(D)J recombination, hypermutation and class switch recombination, B-cells are ~500-times more prone to oncogenic transformation than other cell types. Approaches for targeted elimination of B-cells as the root-cause of disease have been highly effective in the treatment of B-ALL (e.g. CD19 CAR-T cells) and mature B-cell lymphoma (rituximab, ibrutinib), yet therapy resistance necessitates the identification of additional B-cell selective therapeutics. For this reason, we developed an interactive computational tool (lymphoblasts.org) to identify B-cell selective pathway vulnerabilities. Given that B-cell depletion is generally well tolerated, we expect that new approaches targeting B-cell-exclusive vulnerabilities will help to reduce overall toxicity of current chemotherapy-based regimens. Developing a tool to identify cell-type specific pathway dependencies: Compound screening data from CTD and GDSC databases, together with gene-dependency scores from CRISPR and RNAi screens, were re-analyzed and differential sensitivity between B-ALL and solid tumor cell lines calculated. Integration of differential compound sensitivity, gene dependency, and pathway annotations identified B-ALL specific perturbation vulnerabilities and provides insight into their potential mechanisms. Most notably, B-ALL cell lines were found to be dramatically more sensitive to perturbation of the NAD+ salvage pathway than solid tumors. B-ALL cell lines were particularly sensitive to loss or inhibition of NAMPT - the rate limiting enzyme in NAD+ salvage. Three NAMPT inhibitors demonstrate favorable PK/PD and safety profiles in clinical trials (NCT04281420, NCT04914845, NCT03921879), making them strong candidates for drug repurposing. NAD+ salvage deficiency in B-ALL causes metabolic dysfunction: To model NAD+ salvage deficiency in B-ALL, we transformed pre-B cells from Namptfl/fl mice with BCR-ABL1 and NRASG12D oncogenes. Nampt deletion impaired competitive fitness, colony-forming ability, and cell cycle progression. B-cell-specific Nampt-KO mice (Namptfl/fl x Mb1-Cre) showed near-complete ablation of B-lymphopoiesis. Nampt-KO significantly reducing FCCP-stimulated oxygen consumption rate, indicative of mitochondrial dysfunction. Metabolomic studies revealed dysregulation of metabolites involved in amino acid, pyrimidine metabolism, and the TCA cycle. NAD+ salvage dependency is compartmentalized to the nucleus: While NAMPT is universally required for NAD salvage, three isoforms of NMNAT1-3 are specific to the nucleus, cytosol, mitochondria respectively. Only loss of NMNAT1 was found to be B-cell specific, and genetic models of Nmnat1 deletion in B-ALL phenocopied Nampt-KO. Conversely, Nmnat2 deletion or Cebpa-mediated myeloid-reprogramming showed reduced toxicity, thus NAD+ salvage dependency is specific to the nuclear compartment of B-cells. Shut-down of de novo NAD+ synthesis and nuclear NAD+ utilization sensitizes B-ALL: Isotope tracing of NAD biosynthetic pathways demonstrated that the de novo pathway of NAD+ synthesis is shut down in B-ALL but not myeloid cell lines. Chemogenomic CRISPR screening in FK866-treated NALM6 cells revealed that loss of nuclear NAD+ and ATP utilizing enzymes involved in epigenetic regulation, protein deacetylation and DNA damage response, rescues the effect of NAMPT inhibition. In keeping, the NAD-utilizing NNMT pathway was repressed in B-ALL and forced re-activation deepened toxicity of Nampt loss. These results suggest that B-ALL cells are sensitive to NAMPT inhibition due to nuclear NAD+ deficiency coupled with repression of de novo NAD synthesis. Conclusions: Our computational drug repurposing platform prioritizes B-cell specific vulnerabilities for exploitation. We validate the NMNAT1-NAMPT nuclear NAD-salvage axis as a therapeutic vulnerability in B-ALL and identify the likely underlying mechanism. Future research will extend pre-clinical testing of the three FDA-approved NAMPT inhibitors for repurposing to target therapy resistant B-ALL.