Expression of Major Enzymes in NAD+ Synthesis in Patients with Multiple Myeloma
Authors: Crone Lara , Cutler Samuel , Boudreau Jeanette , Trottier Amy , Rahmani Mahboubeh 
Published: December 2024

Introduction
While advancements in treatments have improved patient outcomes, multiple myeloma (MM) remains incurable, highlighting a need for novel therapeutic targets. Nicotinamide Dinucleotide (NAD+) is a key metabolite which is upregulated in cancers to meet the increased energy demands of the malignant cells. NAD+ is synthesized through one of three pathways: the salvage pathway, the Preiss-Handler pathway, and the de novo synthesis pathway. The key rate-limiting enzymes in these pathways are NAMPT, NAPRT, and IDO respectively. In vitro and in vivo studies have demonstrated that NAD+ is upregulated in MM via the salvage pathway and that inhibition of that pathway decreases cancer cell viability. Less is known about the expression of these specific pathways in MM patients and how they might inform a patient's therapeutic response. With this study, we aimed to determine whether markers of NAD+ synthesis could serve as a prognostic biomarker in patients with MM.
Methods
Archived bone marrow biopsy samples from MM patients (n = 64) in the Nova Scotia Health/Dalhousie University Myeloma Tumour Bank were stained using Opal multiplex immunohistochemistry (IHC; Akoya Biosciences). Opal IHC enables the staining of multiple markers within a single tissue sample. The panel of markers included NAMPT, NAPRT, IDO, and plasma cell marker CD138. Biopsy images were analyzed and scored based on expression intensity at a single-cell resolution using InForm, an automated image analysis software package. Each NAD+ synthesis enzyme per sample was assigned an H-score, a semi-quantitative score based on proportions of relative expression intensity. The processing of IHC expression data and statistical analyses were conducted using R-Studio. The correlation between CD138+ plasma cell proportion by Opal IHC and plasma cell burden assessed at time of diagnosis was conducted using Spearman's rank correlation coefficient. The impact of enzyme expression in CD138+ cells on MM patient survival was assessed using Cox proportional hazards ratio models and Kaplan-Meier survival analyses.
Results
The detection of CD138+ cells in MM samples by Opal IHC was consistent with plasma cell burden estimates done by traditional IHC at time of diagnosis (ρ = 0.789, p &lt; 2.2e-16). Across all MM samples, there was little IDO colocalized with CD138, with no significant impact on patient survival outcomes. NAMPT was expressed in the majority of CD138+ cells in MM patients (n = 55 [86%] vs. n = 9 [14%]). However, expression did not significantly associate with overall survival (OS; p = 0.14). Positive expression of NAPRT in CD138+ cells (n = 37) was associated with worse OS, with a median OS of 30 months compared to a median OS of 60 months in samples with negative NAPRT expression (n = 27; p = 0.013). Higher intensity of NAPRT expression correlated with worse OS independent of NAMPT expression within the sample (HR: 6.72, p = 0.00015). The median OS for intermediate NAPRT expression was 8 months (n = 8) compared to 33 months for low NAPRT expression (n = 29) and 60 months for negative NAPRT expression (n = 27; p = 0.00038).
Conclusion
NAPRT expression in CD138+ cells defines a subset of MM patients that have a poor prognosis, with increasing intensity portending shorter overall survival. Targeting these pathways could offer novel therapeutic opportunities for improving survival in MM patients. Future research should focus on validating these biomarkers and exploring targeted inhibitors of NAPRT as potential treatments.