Abstract
Background
Isocitrate dehydrogenase (IDH)–mutant tumors exhibit an altered metabolic state and are critically dependent upon nicotinamide adenine dinucleotide (NAD+) for cellular survival. NAD+ steady-state levels can be influenced by both biosynthetic and consumptive processes. Here, we investigated activation of sirtuin (SIRT) enzymes, which consume NAD+ as a coenzyme, as a potential mechanism to reduce cellular NAD+ levels in these tumors.
Methods
The effect of inhibition or activation of sirtuin activity, using (i) small molecules, (ii) clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 gene editing, and (iii) inducible overexpression, was investigated in IDH-mutant tumor lines, including patient-derived IDH-mutant glioma lines.
Results
We found that Sirt1 activation led to marked augmentation of NAD+ depletion and accentuation of cytotoxicity when combined with inhibition of nicotinamide phosphoribosyltransferase (NAMPT), consistent with the enzymatic activity of SIRT1 as a primary cellular NAD+ consumer in IDH-mutant cells. Activation of Sirt1 through either genetic overexpression or pharmacologic Sirt1-activating compounds (STACs), an existing class of well-tolerated drugs, led to inhibition of IDH1-mutant tumor cell growth.
Conclusions
Activation of Sirt1 can selectively target IDH-mutant tumors. These findings indicate that relatively nontoxic STACs, administered either alone or in combination with NAMPT inhibition, could alter the growth trajectory of IDH-mutant gliomas while minimizing toxicity associated with cytotoxic chemotherapeutic regimens.
Keywords: glioma, IDH, NAD+, NAMPT, sirtuin
Key Points.
Blockade of Sirt1 activity can rescue cytotoxicity from NAD+ depletion in IDH-mutant cell lines.
A combination drug strategy using NAMPT inhibition with augmentation of NAD+ consumption by sirtuin 1 activation significantly inhibits IDH-mutant cell growth.
Importance of the Study.
IDH-mutant tumors exhibit low basal levels of the critical metabolite NAD+, which renders them sensitive to drugs that inhibit NAD+ biosynthesis through the enzyme NAMPT. Translation of this pharmacologic strategy clinically has been stalled because of concerns related to NAMPT inhibitor toxicity, particularly at high doses. We therefore sought to explore alternative ways to disrupt NAD+ equilibrium and find that the enzyme sirtuin 1, for which NAD+ serves as a cofactor, is a major consumer of NAD+ in IDH-mutant cells. Sirtuin 1 activation by genetic or pharmacologic means can drive NAD+ consumption and potentiate the effects of NAMPT inhibition, leading to effective targeting of IDH-mutant cancers.
The metabolic enzymes isocitrate dehydrogenase 1 and 2 (IDH1/2) are frequently mutated in a variety of tumor types, including in adult diffuse gliomas,1–5 chondrosarcoma,6 leukemia,7 and cholangiocarcinoma.8,9 While the wildtype IDH1/2 enzymes promote conversion of isocitrate to alpha-ketoglutarate (α-KG), the tumor-associated mutant enzymes acquire a novel enzymatic function that mediates conversion of α-KG to R-2-hydroxyglutarate (2-HG).10 The resulting excess quantities of 2-HG, which cannot be cleared by normal cellular mechanisms, then promote tumorigenesis through inhibition of various α-KG–dependent enzymes involved in controlling the epigenetic state of the cell.11–14 Mutation of IDH is an early and persistent event in the lifespan of a tumor,15,16 and much effort has been put forth to reduce excess 2-HG levels with direct IDH inhibitors, in the hope of reversing these associated tumorigenic phenotypes. These compounds have been shown to be effective in acute myeloid leukemia,17,18 although the efficacy of this approach in gliomas remains unclear at this time.19,20 The clinical course of IDH-mutant glioma is relatively prolonged and can span several years in the indolent lower-grade phase of disease natural history,21 offering a time window for intervention with relatively nontoxic therapeutics to deflect the growth trajectory.
To this end, it is notable that mutant IDH has been associated with a variety of other changes in the metabolic program of the cell, leading to alterations in numerous metabolites22–27 and altering flux through the citric acid cycle.28 Along these lines, we previously discovered that IDH-mutant cancers exhibit low basal levels of the ubiquitous cellular coenzyme NAD+. While IDH-wildtype gliomas retain the ability to maintain adequate NAD+ levels by upregulation of the salvage NAD synthesis pathway29 or utilization of NAD+ pools in the microenvironment via quinolinic acid phosphoribosyltransferase,30 excess 2-HG produced in the context of IDH mutation leads to epigenetic changes that make this class of tumor less able to compensate for perturbations in NAD+ stores. This results in a metabolic vulnerability that renders IDH-mutant tumors highly sensitive to inhibitors of the salvage NAD synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT).31 Despite the marked efficacy of NAMPT inhibitors both in vitro and in vivo in IDH1-mutant tumor models, opportunities for translation of this finding to clinical therapy have been hampered by concerns about toxicities observed in animal studies.32,33
NAD+ and its reduced form NADH serve an important role in cellular energy generation by facilitating redox reactions to produce ATP. In addition, NAD+ itself is a substrate for the key signaling enzymes, poly(ADP)ribose polymerases (PARPs) and sirtuins (SIRTs), which are involved in DNA repair, gene expression, and cell fate. Indeed, PARP and SIRT pathways are the primary mediators of intracellular NAD consumption.34 Recognizing the current limitations to using NAMPT inhibitors as monotherapy, we sought to identify alternative means to modulate cellular NAD+ pools as a strategy for targeting IDH-mutant tumors. In a recent study, we showed that PARP activation and the resulting burst of NAD+ consumption, triggered by exposure to the cytotoxic alkylating chemotherapy temozolomide, induces a transient metabolic stress that potentiates NAMPT inhibition.35 In this study, we extend this inquiry to the SIRT family of deacetylases, to investigate whether the comparatively nontoxic enhancement of SIRT activity can accelerate NAD+ consumption to target IDH-mutant tumors.
Materials and Methods
Human Cell Lines
HT1080, U251, and HEK293T cell lines were obtained from American Type Culture Collection (male) and grown in EMEM or DMEM supplemented with 10% FBS (Thermo Fisher Scientific). The patient-derived glioma lines MGG152, MGG119, MGG18, and MGG123 have been previously described and were maintained following the published protocol.36 The MGG123 cell line overexpressing IDH1-R132H was generated by lentivirus infection using pLenti6.3/TO/V5 containing IDH1-R132H, pCMV-psPAX2 (Addgene), and pCMV-VSVG (Addgene), followed by selection with blasticidin (0.5 μg/mL). TS603 was a kind gift from T. Chan at the Memorial Sloan-Kettering Cancer Center. Cell lines were authenticated by short tandem repeat fingerprinting, as well as assessment of IDH1-R132H mutation by immunoblot where applicable. Screening for mycoplasma contamination was performed every 3 months. Genotyping data are provided in Supplementary Table 1.
Compounds and Chemicals
The following compounds were added to cell culture media where indicated: FK866 (Cayman Chemical), EX-527 (Selleckchem), dimethyl sulfoxide (Sigma-Aldrich), doxycycline hyclate (Sigma-Aldrich), SRT1720, SRT2183, SRT2104, or SRT3025 (Selleckchem), nicotinamide mononucleotide (Sigma-Aldrich), puromycin dihydrochloride (Sigma-Aldrich), G418 (InvivoGen).
Cell Viability Assay
Cell viability was assessed by measuring luminescence at the indicated timepoints using the Cell Titer-Glo Assay Kit (Promega) according to the manufacturer’s protocol with a microplate reader (BioTek).
Clonogenic Assay
HT1080 cells were seeded in a 6-well plate at a density of 50–100 cells per well, treated with indicated drug after 24 hours. At 8–14 days, cells were washed 1x in phosphate buffered saline, fixed with 6% glutaraldehyde, then stained with 0.05% crystal violet diluted. Images were captured with the EVOS FL Auto 2 Imaging System.
Sphere Formation Assay
Glioma cell lines were seeded at a density of 100 cells per well in 96-well plates, exposed to drug as indicated. Sphere number was counted 14–21 days later.
NAD+/NADH Assay
NAD+ and NADH were measured using the NAD/NADH-Glo Assay (Promega) according to manufacturer’s recommendations at indicated timepoints. Luminescence values were obtained using a microplate reader (BioTek).
CRISPR/Cas9 Plasmids
Small guide (sg)RNAs targeting SIRT1, SIRT2, and SIRT3 and nontargeting control were obtained from GenScript’s GenCRISPR Plasmid Collection containing sgRNAs pre-validated by the Broad Institute and inserted into pLentiCRISPRv2 all-in-one plasmid containing CRISPR (clustered regularly interspaced short palindromic repeat) associated protein 9 (Cas9).
Expression Plasmids of cDNA
SIRT1 cDNA was custom ordered from GenScript and inserted into pDONR/Zeo vector (Thermo Fisher Scientific). pINDUCER20 and pINDUCER21 were obtained from Addgene. SIRT1 was inserted into pINDUCER20 and pINDUCER21 using the Gateway Cloning System (Thermo Fisher Scientific) and authenticated by DNA sequencing.
Lentiviral Transduction of Cell Lines
Lentiviral particles were made by co-transfection of HEK293T cells with expression vectors, packaging plasmid psPAX2 (Addgene), and VSV.G (Addgene) in a ratio of 2:1:1 using FuGENE HD (Promega) and transduced into IDH-mutant cell lines using previously published protocols.31 Selection was performed using puromycin (pLentiCRISPRv2), G418 (pINDUCER20), or flow cytometry (pINDUCER21).
Immunoblot Analysis of Protein Expression
Harvested cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) supplemented with protease inhibitor, phosphatase inhibitor cocktail (Roche), and trichostatin A (Sigma). Membranes were probed with the following antibodies: Sirt1, Sirt2, Sirt3, acetyl-p53 Lys382, acetyl-histone H3 Lys9, cyclophilin B, beta-tubulin, beta-actin horseradish peroxidase (HRP) conjugate (Cell Signaling Technology), IDH1-R132H (Dianova), vinculin (Thermo Fisher Scientific), anti-rabbit immunoglobulin (Ig)G HRP and anti-mouse IgG HRP (GE Healthcare). Immunoblots were visualized on Gel Doc XR+ and images were saved and cropped in Image Lab (Bio-Rad) and exported as TIFF files.
Quantification and Statistical Analysis
Details of statistical analyses for individual experiments are provided in the Figure legends. All experiments were performed in triplicate. Data are presented as mean ± SD. Statistical analyses were performed with GraphPad Prism software. Differences were compared using unpaired Student’s t-test for 2 groups or ANOVA for multiple groups, using a cutoff of significance of P < 0.05. Correction for multiple comparisons was performed as indicated according to the statistical test being used.
Results
Blockade of Sirtuin Activity Can Rescue IDH-Mutant Cells from Toxicity Induced by NAMPT Inhibition
To investigate the extent to which activity of the SIRT family of NAD-dependent enzymes influences NAD+ equilibrium in IDH-mutant tumor cells, we assessed the effect of a small-molecule SIRT inhibitor, EX-527. HT1080 cells, which have an endogenous IDH1-R132C mutation, exhibited decreased cell viability when exposed to the NAMPT inhibitor FK866; however, co-treatment with EX-527 resulted in rescue of cell viability (Fig. 1A). We found that cellular NAD+ levels, which are significantly lower following FK866 treatment, are rescued in part by the addition of EX-527 (Fig. 1B). We observed a comparable rescue of cell viability and NAD+ levels by EX-527 in patient-derived IDH1-R132H mutant glioma stemlike cell lines,36 MGG152 (Fig. 1C, D) and MGG119 (Supplementary Figure 1A, B). Together, these experiments suggest that inhibition of SIRT activity decreases IDH-mutant cell sensitivity to NAD+ depletion.
Fig. 1.
Sirt1 small-molecule inhibitor rescues IDH-mutant tumors from effects of NAMPT inhibition. (A and B) HT1080 cell line treated with indicated doses of the FK866 in combination with dimethyl sulfoxide (DMSO) or EX-527 for 48 hours and then assessed for cell viability (A) and NAD level (B). (C and D) The MGG152 glioma line treated with indicated doses of FK866 in combination with DMSO or EX-527 and assessed for cell viability at 48 hours (C) and NAD level at 24 hours (D). Experiments were performed in triplicate and are representative of multiple experiments. Data are presented as means ± SD. P-values are derived from ANOVA using Dunnett’s multiple comparisons test. NS, non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005.
Sirt1 Deletion Mimics the Effect of Sirtuin Inhibitor in IDH-Mutant Cell Lines
While EX-527 can have pleiotropic effects, it has been reported to have >200-fold more selectivity for inhibition of Sirt1 compared with Sirt2 and Sirt3.37,38 To determine if the rescue we observed with EX-527 was directly related to Sirt1 activity, we engineered IDH-mutant cell lines with deletion of SIRT1 using CRISPR/Cas9 gene editing. Two independent sgRNAs (of 3 tested) effectively deleted Sirt1 in HT1080 (Fig. 2A) and MGG152 (Fig. 2E). As expected, consistent with loss of Sirt1 function as an NAD-dependent deacetylase,39,40 we observed an increase in acetylation of p53 in SIRT1 null lines (Fig. 2A, E). Inactivation of Sirt1 did not impact cell growth rate or viability (Fig. 2B), suggesting that the activity of Sirt1 is dispensable for cell proliferation in IDH-mutant cancer lines. However, SIRT1 null cells created from both cell lines were dramatically less sensitive to NAMPT inhibition, as detected by cell viability assay (Fig. 2C, F). Further, the decrease in NAD+ level induced by NAMPT inhibition was significantly smaller in SIRT1 null cells (Fig. 2D, G; Supplementary Figure 2A, B), evidence supporting our hypothesis that the resistance to NAMPT inhibition is related to lack of Sirt1-dependent NAD+ consumption.
Fig. 2.
Sirt1 deletion mimics the effect of Sirt inhibitor EX-527 in IDH-mutant cell lines. (A) Immunoblot of HT1080 stable cells expressing the indicated SIRT1 sgRNAs or nontargeting (NT) sgRNA together with Cas9, probed for Sirt1, acetylated p53 (ac-p53) as a marker of Sirt1 activity or vinculin as a loading control. (B) HT1080 SIRT1 null stable cells and control cells exhibit similar growth kinetics. (C) Cell viability in nontargeting (NT) or SIRT1 null cell lines 48 hours after exposure to DMSO or FK866 at indicated doses. (D) NAD level in NT or SIRT1 null cell lines 24 hours after exposure to FK866 (10 nM). (E) Immunoblot of MGG152 stable cells expressing the indicated SIRT1 sgRNAs or NT sgRNA together with Cas9, probed for Sirt1, acetylated p53 (ac-p53) or vinculin as a loading control. (F) Cell viability in NT or SIRT1 null cell lines 48 hours after exposure to DMSO or FK866 at indicated doses. (G) NAD level in NT or SIRT1 null cell lines 24 hours after exposure to FK866 (10 nM). Experiments were performed in triplicate and are representative of multiple experiments. Data are presented as means ± SD. P-values are derived from ANOVA using Dunnett’s multiple comparisons test. NS, non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005.
Because EX-527 also has some inhibitory effect on Sirt2 or Sirt3 activity, particularly at higher doses, we additionally used CRISPR/Cas9 gene editing to delete SIRT2 or SIRT3 from IDH-mutant cells to determine if loss of these Sirt family members influences sensitivity to NAMPT inhibitor‒mediated NAD+ depletion. SIRT2 was stably deleted from HT1080 cells with 3 distinct sgRNAs (Supplementary Figure 3A). Despite testing several sgRNAs, we were able to achieve only partial deletion of SIRT3 (Supplementary Figure 3B). Sirt1 expression was not affected by loss of Sirt2 or Sirt3 (Supplementary Figure 3A, B). Notably, complete deletion of SIRT2 and partial loss of SIRT3 did not significantly change the sensitivity of HT1080 to NAMPT inhibition, as we observed a similar decrement in cell viability following NAMPT inhibition in all stable cell lines (Supplementary Figure 3C, D). In the SIRT2 deleted lines, NAD+ levels measured slightly higher following NAMPT inhibition compared with the nontargeting line (Supplementary Figure 3E), but this change was not large enough to preserve cell viability. NAD+ did not change significantly in the absence of SIRT3 (Supplementary Figure 3F). These data indicate that sensitivity to NAMPT inhibitor in IDH-mutant cancer cells is primarily influenced by activity of Sirt1, but not Sirt2 or Sirt3.
Sirt1 Overexpression Increases NAD+ Consumption and Enhances the Cytotoxicity of NAMPT Inhibitor in IDH-Mutant Cells
Since the cytotoxic effect of inhibition of NAD biosynthesis in IDH-mutant cancer cells can be reversed by inhibiting Sirt1 activity, we next assessed whether Sirt1 activation could potentiate the effects of NAMPT inhibition in these cells. To this end, we engineered an IDH-mutant cell line to express Sirt1 downstream of a tetracycline-inducible promoter.41 We observed strong overexpression of Sirt1 following the addition of doxycycline to the growth media (Fig. 3A). Sirt1 overexpression was associated with an increase in Sirt1 enzymatic activity, as detected by an increase in Sirt1-deacetylating activity resulting in decreased level of acetyl-lysine 9 of histone H3 (acetyl-H3K9) (Supplementary Figure 4A). In addition, we observed a decrease in overall NAD+ level in the timeframe shortly after doxycycline induction of Sirt1 expression, although at steady state there was recovery in baseline NAD+ level in Sirt1 overexpressing cells compared with control cells not exposed to doxycycline (Supplementary Figure 4B–D). Notably, Sirt1 overexpression did lead to a significant decrease in colony formation (mean, 76.1 colonies for control compared with 58.5 colonies for Sirt1 overexpressing cells, P = 0.0003; Fig. 3B).
Fig. 3.
Excess Sirt1 activity leads to increased NAD consumption in IDH-mutant cell lines. (A) Immunoblot of HT1080 line engineered to allow for inducible Sirt1 overexpression (HT1080-Sirt1) exposed to the indicated doses of doxycycline for 48 hours. Lysates were probed for Sirt1 and tubulin, as loading control. (B) Clonogenic survival of HT1080-Sirt1 line following exposure to 0.5 µg/mL dox (+ Dox) compared with control (− Dox). (C) Cell viability of HT1080-Sirt1 cell line exposed to 0 (No Dox) or 0.5 µg/mL doxycycline (+ Dox) and treated with varying doses of FK866, assessed at 24 hours. (D) NAD levels in HT1080-Sirt1 cell line exposed to 0 (No Dox) or 0.5 µg/mL doxycycline (+ Dox) and treated with varying doses of FK866 for 24 hours. (E) Cell viability of HT1080-Sirt1 cell line exposed to 0 (No Dox) or 0.5 µg/mL doxycycline (+ Dox) and treated with indicated doses of FK866 ± 100 µM NMN, assessed at 24 hours. (F) NAD level of HT1080-Sirt1 cell line exposed to 0 (No Dox) or 0.5 µg/mL doxycycline (+ Dox) and treated with indicated doses of FK866 ± 100 µM NMN, assessed at 24 hours. Experiments were performed in triplicate and are representative of multiple experiments. Data are presented as means ± SD. P-values are derived from two-tailed unpaired t-test. NS, non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005.
Importantly, Sirt1 overexpression also led to increased sensitivity to NAMPT inhibition. Cell viability was significantly lower after FK866 treatment in the presence of Sirt1 overexpression (Fig. 3C). We observed a dramatic decrease in NAD+ level following the combination of NAMPT inhibition and Sirt1 overexpression (Fig. 3D), consistent with the hypothesis that the decrement in cell viability is driven by NAD+ consumption. Further supporting this proposal, addition of nicotinamide mononucleotide (NMN), an immediate precursor of NAD+ that bypasses the need for NAMPT, led to a rescue in the effects observed following NAMPT inhibition and Sirt1 combination (Fig. 3E, F).
Sirt1 Activator Increases NAD+ Consumption and Leads to Cytotoxicity in IDH-Mutant Cells
We then tested whether we can observe effects similar to genetic overexpression by deploying small-molecule Sirt1-activating compounds (STACs).42 We confirmed Sirt1 activation with SRT3025 by examination of ac-p53 following drug exposure and observed a decrease of ac-p53 level to 43% of control at the maximum dose tested (Fig. 4A). Using a panel of endogenous IDH-mutant cell lines, we observed a decrease in IDH-mutant line proliferation and cell viability following treatment with STACs. There was a significant decrease in cell viability using SRT3025 in MGG152, TS603, HT1080, and MGG119 (Fig. 4B). The decrease in cell viability coincided with a significant decrease in NAD+ level, as shown for the MGG119 glioma line (Fig. 4C). We observed a similar decrease in proliferative capacity in IDH-mutant lines using additional STACs, including SRT1720 (Supplementary Figure 5A, B), SRT2183 (Supplementary Figure 5C), and SRT2104 (Supplementary Figure 5D). In contrast to the consistent STAC sensitivity of IDH-mutant lines, we observed variability in STAC sensitivity in IDH-wildtype lines (Fig. 4B). To therefore strictly isolate the effect of the IDH mutation, we engineered an isogenic model of IDH mutation using an IDH-wildtype glioma (MGG123) background (Fig. 4D). As evidence of the causal role of IDH-mutant function in mediating this effect, expression of the IDH mutation (IDH1 R132H) in MGG123 induced increased sensitivity to SRT3025 (Fig. 4E, Supplementary Figure 5E, F). Furthermore, this sensitivity correlated with the degree of NAD+ depletion observed upon SRT3025 treatment (Fig. 4E).
Fig. 4.
Sirt1 activator increases NAD+ consumption and leads to cytotoxicity in IDH-mutant cells. (A) Immunoblot of MGG119 lysates treated with SRT3025 and probed for Sirt1, acetylated-p53, and beta-actin. The intensity of acetylated p53 band compared with control (0 µM) is indicated below. (B) Cell viability of a panel of IDH-mutant (MGG119, MGG152, TS603, HT1080) and IDH-wildtype cell lines (MGG123, MGG18, U251) exposed to varying doses of SRT3025. (C) NAD level in the MGG119 glioma line exposed to SRT3025 at 48 hours. (D) Immunoblot of MGG123 parental (IDH wildtype) and MGG123-IDH1-R132H expressing line (IDH-mutant) probed for IDH1-R132H and vinculin. (E) Cell viability (left panel) and NAD level (right panel) in MGG123 parental (IDH-wildtype, black) or engineered IDH mutant expression line (gray) after treatment with SRT3025 (10 μM). (F) Cell viability in MGG119 glioma line following treatment with combination of SRT3025 and FK866. (G) Model figure showing NAD+ equilibrium in balance between biosynthetic and consumptive processes. IDH-mutant tumors are dependent primarily on NAMPT for NAD+ synthesis. NAD+ consumption is driven by PARP activity and sirtuin activity. Augmentation of Sirt1 activity enhances NAD+ consumption leading to critically low intracellular levels of NAD+ and results in IDH-mutant cell death. FK866: NAMPT inhibitor, ac-p53: acetylated p53; PARP: poly(ADP)ribose polymerase, STAC: sirtuin activating compound. Experiments were performed in triplicate and are representative of multiple experiments. Data are presented as means ± SD. P-values are derived from ANOVA using Dunnett’s multiple comparisons test. NS, non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005.
Consistent with Sirt1 overexpression experiments, SRT3025 increased sensitivity to NAMPT inhibitor, with the most striking augmentation observed at low dose of FK866 (Fig. 4F). Taken together, Sirt1 activation by a STAC leads to increased NAD+ consumption and resulting cytotoxicity in IDH-mutant cells.
Discussion
IDH mutations lead to an altered metabolic landscape that may be exploited for therapeutic use. NAD+ is an essential metabolite that has recently become the focus of targeting strategies for a wide variety of cancer types. Cancer cell vulnerability to NAD+ depletion is dependent on which NAD biosynthetic pathways are used by the cell of origin.43 In IDH-mutant tumors, including gliomas, baseline NAD+ levels are low compared with IDH-wildtype cells, secondary to an exclusive dependence on the NAD+ salvage pathway, utilizing NAMPT, for biosynthesis.31 We have observed in a variety of IDH-mutant models that small differences in NAD+ levels following NAMPT inhibitor treatment can translate into large differences in cell viability. We hypothesize that there is a critical NAD+ depletion threshold, beyond which cell viability is markedly affected. Therefore, mechanisms to deplete cellular NAD+ pools offer an attractive therapeutic approach in IDH-mutant tumors.
NAD+ steady state is maintained by a balance of biosynthetic and consumptive processes. We demonstrate here that Sirt1 is responsible for a substantial proportion of NAD+ consumption in IDH-mutant cells. As such, pharmacologic Sirt1 activation disrupts NAD+ equilibrium by increasing NAD+ consumption, thereby depleting cellular NAD+ pools (for model, see Fig. 4G). It is striking that genetic modulation of Sirt1 has a minimal effect on cell viability in equilibrium. Interestingly, we observed only a modest decrease in equilibrium NAD+ levels with genetic Sirt1 activation alone, which we speculate may be secondary to the ability of Sirt1 to promote NAMPT transcription to maintain NAD+ availability in a positive feedback loop.44–46 Despite the small effect of genetic Sirt1 activation alone, we find that the combination of Sirt1 activation and NAMPT inhibition results in a striking augmentation of tumor cell killing. Further, the dual strategy resulted in a significant decrease in cell survival at concentrations of FK866 that were at least an order of magnitude lower than FK866 alone. While Sirt1 has important roles in cellular stress response and metabolism,47,48 the effects of the combination strategy were rescued by exogenous NAD+, demonstrating that depletion of this critical metabolite underlies the efficacy of this strategy.
To strengthen this observation beyond our engineered model line, we used pharmacologic activation of Sirt1. STACs have been developed and studied for their purported ability to promote longevity and prevent aging-related diseases.42,48 There exists some controversy surrounding whether these agents activate Sirt1 directly or indirectly,49 potentially explaining the range of responses we observed between the effect of genetic Sirt1 activation and drug-mediated Sirt1 activation in IDH-mutant models. Nevertheless, there is much enthusiasm for this class of drug and, thus far, a STAC tested in human clinical trials has demonstrated easy tolerability with minimal toxicities.50,51 This raises the exciting possibility of an IDH-mutant-targeted treatment involving a STAC alone, or in combination with a low-dose NAMPT inhibitor, which would potentiate the effectiveness of NAMPT inhibition while minimizing toxicity. Further studies using in vivo models of IDH-mutant glioma are needed to characterize the efficacy. For IDH-mutant glioma, in particular, there is a potential window of opportunity in the long, indolent phase of disease prior to first progression,21 during which an effective treatment with a low side effect profile could prove useful for slowing tumor growth trajectory. In conclusion, Sirt1-mediated enhancement of NAD+ consumption in combination with inhibition of NAMPT offers a potential targeting strategy for IDH-mutant tumors.
Funding
This work was supported by the Tawingo Fund (D.P.C.), National Institutes of Health R01CA227821 (D.P.C., H.W.), P50CA165962 (D.P.C.), Paul Calabresi Career Development Award in Clinical Oncology for Nervous System Tumors K12CA090354 (J.J.M.); Richard B. Simches Scholars Award (J.J.M.), Seeman Family Massachusetts General Hospital Scholar in Neuro-Oncology Award (J.J.M.).
Supplementary Material
Acknowledgments
We thank members of the Cahill and Brastianos laboratories, William G. Kaelin, Matthew Vander Heiden, A. John Iafrate, and Tracy T. Batchelor for helpful discussions and the MGH Pathology Flow Cytometry Core at Charlestown Navy Yard for assistance with cell sorting.
Conflict of interest statement. D.P.C. has received honoraria and travel reimbursement from Merck and has served as a consultant for Lilly. All other authors declare no competing interests.
Authorship statement. Conception and design: J.J.M., K.T., H.W., D.P.C. Acquisition of data: J.J.M., A.F., J.A.B., H.N., M.S., C.K.L., S.S.T., L.M. Analysis and interpretation of data: J.J.M., H.N., M.S., C.K.L., H.W., D.P.C. Writing, review, and/or revision of manuscript: J.J.M., M.S., C.K.L., H.W., D.P.C. Study supervision: H.W., D.P.C.
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