Abstract 5136: Development of a highly specific NAPRT monoclonal antibody for the detection of tumors sensitive to pharmacological inhibition of NAMPT

Abstract Introduction: A key component of targeting synthetic lethality in cancer is identifying specific biomarkers, such as sources of NAD+ synthesis, that differentiate between healthy and diseased tissue. In mammals, NAD+ can be synthesized from nicotinamide via nicotinamide phosphoribosyltransferase (NAMPT) or from nicotinic acid (niacin, NA) via nicotinic acid phosphoribosyltransferase (NAPRT). NAPRT loss has been identified across tumor indications as a synthetic lethal sensitizer to small molecule inhibitors of NAMPT. Given the reliance of cancer cells on NAD+ synthesis, blocking NAD+ production is a compelling target for anti-cancer therapeutics. However, identifying tumors with NAPRT loss has been a challenge as NAPRT can be expressed by as many as 14 transcripts, only some of which code for the active enzyme. This complicates transcript-based analyses and necessitates proteomic assessment. Here, we have developed a mouse monoclonal antibody (4A5D7) to specifically detect active NAPRT and to identify indications enriched for NAPRT loss in patients through an IHC-based assay. We further confirm that loss of NAPRT makes cells susceptible to NAMPT inhibition, and ultimately show that our antibody can identify patient-derived xenograft (PDX) models that respond to NAMPT inhibition. Methods We utilized a hybridoma system to produce antibodies against epitopes in the NAPRT active site; clones were subsequently screened for their ability to selectively recognize active NAPRT. After identifying clone 4A5D7, we validated our proprietary antibody against FFPE specimens across multiple cancer indications (TNBC, NSCLC, SCLC, CRC, gastric, ovarian, and prostate). Stained samples were scored by a pathologist and assessed for percent of positive tumor cells, nuclear and/or cytoplasmic staining, and staining intensity. CCLE and TCGA datasets were mined for indications enriched for suspected NAPRT-loss and sensitivity to NAMPT inhibitors. We then selected cell lines and pan-tumor PDX tissue microarrays (TMAs) from indications of interest and characterized samples by western blot and IHC to determine NAPRT status. Cell lines and PDX tumor tissue were exposed to increasing ranges of NAMPTi in the presence or absence of NAPRT-substrate NA to validate our approach. Results: We validated that our proprietary NAPRT monoclonal antibody clone 4A5D7 only detects functional NAPRT and demonstrated improved performance over previous antibodies. We used our antibody to characterize cell lines and PDX tumor samples, and subsequently show that NAPRT-null cell lines and tumor samples are susceptible to NAMPT inhibition, while samples with functional NAPRT can be rescued by NA. Conclusion: Our proprietary antibody can identify NAPRT null tumors and identify samples that will respond to NAMPT inhibition, a critical advancement for patient selection in clinical trials. Citation Format: Josh Spurrier, Emmanuel S. Burgos, Mark R. Lundquist, Keiichiro Tanaka, Samuel Brook, Matt Gozzi, Jae-Sung Yi, Joseph Edmonds, Mitch Raponi. Development of a highly specific NAPRT monoclonal antibody for the detection of tumors sensitive to pharmacological inhibition of NAMPT abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5136.