CD38-Induced Metabolic Dysfunction Primes Multiple Myeloma Cells for NAD+-Lowering Agents

Abstract

Cancer cells fuel growth and energy demands by increasing their NAD⁺ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD⁺-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD⁺ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD⁺-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD⁺-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

Keywords: NAD+ biosynthetic pathway; NAD+-lowering agents; cancer metabolism; mitochondrial disfunction; multiple myeloma; oxidative stress.

Figure 1

CD38 enzymatic activity affects NAD⁺ intracellular level and influences anti-MM activity of NAD⁺-depleting agents. (A) A panel of MM cell lines (HMCLs) were analyzed for CD38 and NAMPT protein levels by WB (top panel) or quantitative flow cytometry (bottom panel). One representative experiment is shown. (B) NAD⁺ content and CD38 enzymatic activity (GDP-ribosyl cyclase) were measured in indicated HMCLs. (C) NAD⁺ content and CD38 surface level (based on arbitrary units, as detailed in the Supplementary Materials) were measured in CD138+ cells derived from MM patients. (D) Relative expression of CD38 surface protein plotted versus FK866 cytotoxicity IC₅₀ values: box plot showing cumulative results of MTS assays. (E) H929 cell line was lentivirally transduced with empty pLV and CD38-overexpressing pLV (CD38 OE) and then treated with increasing doses of FK866 (0–10 nM) for 96 h. Cell viability was measured with an MTS assay and presented as a percentage of control. Data are presented as mean ± S.D (n = 3) (* p ≤ 0.05, **** p ≤ 0.0001; unpaired t test).

Figure 2

Anti-MM synergistic effects of the NAMPT inhibitor FK866 combined with CD38 inducers. (A) Indicated HMCLs were incubated with 10 nM ATRA for 24, 48, or 72 h and then analyzed by flow cytometry. The panel shows the fold increase in CD38 median fluorescence intensity (MFI) compared with control. (B) Apoptotic cell death assessed with flow cytometry analysis after Annexin V/PI staining in a panel of HMCLs treated with FK866 (3 nM), ATRA (3 nM) or their combination for 96 h. (C) CD138⁺ cells collected from three MM patients or PBMCs derived from one MM patient and three healthy donors (HDs) were treated with indicated doses of FK866 (3 nM), ATRA (1 nM) and their combination for 72 h. Cell viability was measured by MTS assay. Data in B and C are presented as mean ± S.D (n = 3). (ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; unpaired t test).

Figure 3

FK866 transcriptomic changes among CD38-overexpressing MM patients confers survival advantage. (A) Heatmap showing FK866 activity signature expression in NDMM patients derived from the CoMMpass dataset: a group of patients with gene expression in accordance with FK866 treatment is highlighted in the green rectangle (FK866 treated-like). (B) Kaplan–Meyer curves of the overall survival probability for FK866 treated-like patients were divided in quartiles for their expression of CD38. (FK866dn, signature created by FK866 down-regulated genes; FK866up, signature created by FK866 upregulated genes; CD38low, CD38 expression bottom quartile; CD38high, CD38 expression top quartile).

Figure 4

NAD⁺ depletion accounts for the enhanced sensibility of CD38-upregulated MM cells to NAMPT inhibitor FK866. (A,B) LP1 cells infected with lentiviruses overexpressing wild-type CD38 (CD38OE) or empty vector were assayed for their NAD⁺ content (A) and CD38 enzymatic activity (GDP-ribosyl cyclase) (B). In the same cells, NAD⁺ content (C) without FK866 was also measured. Data are presented as mean ± S.D. (n = 3). (** p ≤ 0.01, *** p ≤ 0.001; unpaired t-test). (D,E) Intracellular NAD⁺ level and CD38 enzymatic activity (GDP-ribosyl cyclase) were measured in the H929 cell line after 48 h of treatment with ATRA (1 nM) (D), LEN (5 µM), or POM (2.5 µM) (E) alone or with FK866 (1 nM).

Figure 5

NAD⁺ and Nicotinic Acid supplementation abolishes the activity of co-treatment in MM cells. Viability of LP1 (A) and H929 (B) cells treated as indicated with ATRA (1 or 3 nM), FK866 (2 or 3 nM), and their combination for 96 h in the presence or absence of NAD⁺ (1 mM) or NA (10 µM). Cell viability was measured with MTS assay and presented as a percentage of control. The results are a mean ± SD of triplicate samples (**** p ≤ 0.0001; unpaired t-test).

Figure 6

Metabolic reprogramming elicited by CD38-overexpression identifies a novel druggable vulnerability in MM cells. (A) Cellular and mitochondrial NAD⁺ contents were measured in H929 CD38 OE or control cells and normalized to the protein content of each fraction. (B) Oxygen consumption, (C) activity of Fo-F1 ATP synthase, (D) energy status expressed as ATP/AMP ratio, and (E) oxidative phosphorylation efficiency as P/O ratio were measured in H929 control and CD38 OE cells. Data are presented as the mean ± SD of three different experiments. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; unpaired t-test).

Figure 7

Mitochondrial dynamic shift underlies an organelle-specific dysfunction triggered by CD38 upregulation. (A) Mitochondrial complexes (I, II, III, IV) activities were measured in H929 control and CD38 OE cells at baseline and following treatment with FK866 (2 nM). (B) TEM image of H929 control and CD38 OE cells displaying elongated mitochondria. Scale bars: 200 nm. Average mitochondrial length quantified in μm. Data are presented as mean ± S.D (n = 3). (*** p ≤ 0.001, **** p ≤ 0.0001; unpaired t-test).

Figure 8

The oxidative stress triggered by energetic depletion is crucial for tested drugs combination. Mitochondrial superoxide anions were detected by immunofluorescence (A) and flow cytometry (B) using MitoSOX, in H929 cells lentivirally transduced with pLVempty vector or pLV CD38 OE. (C) Oxidative stress marker (MDA) and activities of antioxidant enzymes (Catalase, Glutathione reductase-GR) were measured in control and CD38 OE cells. Data are presented as mean ± S.D (n = 3). (* p = 0.05, *** p = 0.001, **** p ≤ 0.0001; unpaired t-test).

Figure 9

NAD⁺-depleting agents’ treatment could be beneficial for oxidative stress-prone MM patients. (A) Heatmap displaying enrichment in the CoMMpass dataset of indicated gene ontology terms; “LOW” and “HIGH” rectangles indicate groups of patients with a low and high expression of oxidative stress response, respectively. (B) Scatter-bar plot showing CD38 RNA levels in LOW and HIGH groups from panel E. (C) Kaplan–Meyer curves of the progression-free survival probability of the LOW and HIGH groups from panel A, p-value is indicated. Data are presented as mean ± S.D (n = 3). (** p ≤ 0.01; unpaired t-test).

Figure 10

Graphic of proposed model: CD38 upregulation by genetic or pharmacologic (ATRA, LEN, or POM) approaches result in energetic remodeling with mitochondria dynamic shifts and oxidative stress priming MM cells for low doses of NAD⁺-depleting agents.