Figure 2. NAPRT targeting makes MM cells... | American Society of Hematology

Figure 2.

$NAPRT targeting makes MM cells more sensitive to NAD+-lowering agents. (A) Quantification of indicated extracellular NAD+ metabolites in BM plasma derived from 15 patients with NDMM and 10 healthy donors, using LC-MS/MS. Data are mean ± standard deviation; ∗P < .04 (Welch test), ∗∗P = .007 (t test). (B) Western blot (WB) showing indicated protein expression across a panel of MM cell lines with different genetic backgrounds (black and white squares refer to presence or absence of indicated abnormalities, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (bottom blot). One representative experiment is shown. (C) NAPRT, NADSYN, and NAMPT mRNA levels were evaluated in a panel of HMCLs by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the 2-ΔΔCt method, with normalization to GAPDH. MM cells are divided into low- and high-NAPRT–expressing cells (top); the bottom panel shows the ratio (FC) of FK866 50% inhibitory concentration value for all the tested MM cell lines in the presence vs absence of NA (0.5 μM) supplementation. (D) Heat map showing FK866 activity signature expression in patients with MM derived from the CoMMpass data set grouped by GSVA method as FK866-sensitive patients (a group of patients with gene expression in accordance with FK866 treatment). “Other” includes patients with nonoverlapping profiles (top). Kaplan-Meyer curves of the PFS probability of FK866-sensitive patients, divided into quartiles for their expression of NAPRT. Log-rank test is used to compute the P value (log-rank test). First and fourth quartiles of NAPRT expression are represented in red and blue, respectively (bottom). (E) CD138+ primary cells from patients with MM (3 NDMM and 1 RRMM) and PBMCs from healthy donor (HD; n = 2), were treated with the indicated dose of FK866 in the presence or absence of NA (1 μM) for 96 hours and assessed for cell viability using CTG. (F) MM tumor (CD138+) and normal (PBMCs) cells were treated as in panel E, alone and in combination with the NAPRT inhibitor 2HNA (1 mM), and assessed for cell viability using CTG. (G) RPMI 8226 and MM1S NAPRT–expressing cells were treated with different concentrations of FK866 in the presence or absence of NA (2 μM), 2HNA (1 mM), and their combination for 72 hours. Cell viability was assessed using an MTS-based assay. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; unpaired t test. CTG, CellTiter-Glo; HMCLs, human myeloma cell lines; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay; ns, not significant; PBMCs, peripheral blood mononuclear cells; RRMM, relapsed refractory MM.$

NAPRT targeting makes MM cells more sensitive to NAD⁺-lowering agents. (A) Quantification of indicated extracellular NAD⁺ metabolites in BM plasma derived from 15 patients with NDMM and 10 healthy donors, using LC-MS/MS. Data are mean ± standard deviation; ∗P < .04 (Welch test), ∗∗P = .007 (t test). (B) Western blot (WB) showing indicated protein expression across a panel of MM cell lines with different genetic backgrounds (black and white squares refer to presence or absence of indicated abnormalities, respectively). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control (bottom blot). One representative experiment is shown. (C) NAPRT, NADSYN, and NAMPT mRNA levels were evaluated in a panel of HMCLs by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the 2-ΔΔCt method, with normalization to GAPDH. MM cells are divided into low- and high-NAPRT–expressing cells (top); the bottom panel shows the ratio (FC) of FK866 50% inhibitory concentration value for all the tested MM cell lines in the presence vs absence of NA (0.5 μM) supplementation. (D) Heat map showing FK866 activity signature expression in patients with MM derived from the CoMMpass data set grouped by GSVA method as FK866-sensitive patients (a group of patients with gene expression in accordance with FK866 treatment). “Other” includes patients with nonoverlapping profiles (top). Kaplan-Meyer curves of the PFS probability of FK866-sensitive patients, divided into quartiles for their expression of NAPRT. Log-rank test is used to compute the P value (log-rank test). First and fourth quartiles of NAPRT expression are represented in red and blue, respectively (bottom). (E) CD138⁺ primary cells from patients with MM (3 NDMM and 1 RRMM) and PBMCs from healthy donor (HD; n = 2), were treated with the indicated dose of FK866 in the presence or absence of NA (1 μM) for 96 hours and assessed for cell viability using CTG. (F) MM tumor (CD138⁺) and normal (PBMCs) cells were treated as in panel E, alone and in combination with the NAPRT inhibitor 2HNA (1 mM), and assessed for cell viability using CTG. (G) RPMI 8226 and MM1S NAPRT–expressing cells were treated with different concentrations of FK866 in the presence or absence of NA (2 μM), 2HNA (1 mM), and their combination for 72 hours. Cell viability was assessed using an MTS-based assay. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; unpaired t test. CTG, CellTiter-Glo; HMCLs, human myeloma cell lines; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay; ns, not significant; PBMCs, peripheral blood mononuclear cells; RRMM, relapsed refractory MM.

This Feature Is Available To Subscribers Only