Summary:

Tumors mutated in IDH1 tend to have lower levels of the essential substrate NAD+. In this issue of Cancer Discovery, Nagashima and colleagues exploit this metabolic sensitivity by devising a combinatorial therapy that both further reduces the pools as well as sequesters the remaining substrate in PAR chains, sensitizing the cells to temozolomide and PARG inhibition.

See related article by Nagashima et al., p. 1672.

Gliomas represent the most common primary malignant tumors of the central nervous system, with more than 80% of low-grade gliomas being mutated in isocitrate dehydrogenase I or II (IDH1 and IDH2). Although patients with mutations in IDH1 display longer survival compared with their wild-type counterparts, these tumors do invariably recur as more aggressive lesions despite standard-of-care radiochemotherapy and surgical resection. This is likely due to the diffuse and infiltrative nature of these tumors as well as the metabolic ramifications the mutation has in response to therapy. Considering the high frequency of IDH1 mutations among patients with low-grade gliomas as well as the invariable rate of recurrence, identification of mutant IDH1–induced sensitivities is of great interest and has the potential for significant impact on survival.

Under normal biological conditions, IDH1 functions in the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (α-KG) while reducing NADP+ to NADPH. The mutant form confers neomorphic enzymatic activity with the conversion of α-KG to the oncometabolite D-2-hydroxyglutarate (D-2HG) in a process that consumes rather than produces NADPH while generating NADP+ (1, 2). This metabolic reprogramming has far-reaching impacts, as nicotinamide adenine dinucleotide (NAD) plays a critical role as a coenzyme fueling redox reactions of metabolism as well as a cosubstrate in nonredox reactions important in the regulation of gene expression, DNA repair, inflammation, intracellular trafficking, aging, and cell death.

One class of NAD+-consuming enzymes are the PARPs implicated in DNA-repair mechanisms. Under conditions of DNA damage, PARPs utilize NAD+ to catalyze the transfer of ADP-ribose moieties, leading to repeating units of PAR that could exceed hundreds of units in number. This post-translational modification, referred to as PARylation, functions as a scaffold for assembly of multiprotein complexes involved in a number of biological processes, including DNA damage repair. Upon repair of the damage, the PARylated regions can be enzymatically hydrolyzed through PAR glycohydrolase (PARG). Whereas PARylation requires and sequesters NAD+, the breakdown of PAR chains releases NAD+.

In a series of manuscripts over the past five years, Daniel Cahill's group, among others, has established that mutant IDH1 has profound impacts on canonical metabolic pathways. Specifically, they found that IDH1-mutant tumors have lower basal intracellular NAD+ levels, as was observed in their panel of IDH1 wild-type and mutant cell lines (3). This was found to be due to a reduced expression of NAPRT1 in a mutant IDH1–mediated manner and was suspected of promoting a sensitivity that could be induced through further NAD+ depletion via NAMPT inhibitors, which block NAD+ biosynthesis. In fact, in vivo models harboring flank or intracranial tumors expressing mutant IDH1 that were subjected to NAD+ depletion via administration of NAMPT inhibitors displayed reduced tumor proliferation and prolonged survival, suggesting that the metabolic crisis that ensues upon NAD+ depletion has therapeutic applications in the context of mutant IDH1 tumors. Subsequent studies investigated whether the vulnerability to NAD+ depletion could be enhanced in combination with the alkylating chemotherapeutic agent temozolomide (4). As temozolomide induces a DNA damage response, it leads to activation of PARP and polymerization of PAR, a reaction that utilizes and is dependent on NAD+. The rationale was that in mutant IDH1 tumors that already present with a deficit of NAD+, treatment with temozolomide will lead to the expenditure of the remaining NAD+ and, in combination with NAMPT inhibitors, will lead to an additive effect with enhanced antitumor efficacy. In vitro, the combined effects led to significant reduction in proliferation exclusively among IDH1-mutant lines, with minimal effects in wild-type lines or normal human astrocytes. Similar observations showing antitumor activity against IDH1-mutant tumors, but not wild-type tumors, were observed in vivo.

In their most recent publication in this issue of Cancer Discovery, Nagashima and colleagues expand on the aforementioned studies and explore another route to exploit the sensitivities imparted by NAD+ depletion in IDH1-mutant tumors, specifically, that of PARG inhibition (Fig. 1; ref. 5). Here, it is suggested that PARG inhibition will lead to NAD+ sequestration in nonhydrolyzed PAR chains that result from DNA damage, compounding the NAD+-depleted state and enhancing the sensitivity.

Utilizing a valuable cohort of patient-derived IDH1 wild-type and mutant glioma lines, the group reports variability in the response to treatment with both PARP inhibitor monotherapy and its combination with temozolomide, a variation that was unable to be explained through genomic profiling. Consistently, however, the NAD+ levels were reduced among the IDH1-mutant lines following temozolomide treatment, which was not observed in the wild-type lines. In contrast to their previous publication utilizing NAD+ depletion via NAMPT inhibitors in combination with temozolomide, they investigated further depletion of NAD+ pools through the inhibition of PAR hydrolysis that would otherwise release and replenish NAD+ stores. To this end, the PARG inhibitor PDD00017273 (PDD) was used in combination with temozolomide, and effects on NAD+ levels, viability, and colony formation were determined. Although an impressive combinatorial effect on colony formation was observed in the IDH1-mutant line (HT1080), the control line (U251) used in this case was already sensitive to temozolomide. In the combined PDD with temozolomide treatment, a sustained decrease in NAD+ levels, even after 24 hours, was observed among IDH1-mutant cell lines, but not in the majority of the wild-type lines, suggesting that PARG inhibition and temozolomide is capable of inducing a mutant IDH1–specific NAD+-depleted sensitivity. Proving that this effect was due to NAD+ levels, they supplemented with NAD+ precursors and showed a partial rescue to viability. Subsequent treatment with the PARP inhibitor olaparib displayed a rescue to the viability phenotype; in this case, PAR accumulation and NAD+ was not affected, suggesting that the sensitivity induced by PDD and temozolomide is conferred by PAR accumulation and depleted NAD+ stores.

To address the observed variation in the patient-derived lines, Tet-inducible mutant IDH1 cell lines were generated. It was only under prolonged culturing in the presence of mutant IDH1 (3 months) that the sensitivity to temozolomide and PARG inhibition was able to be phenocopied. This is consistent with previous data showing that it takes several passages for mutant IDH1 to elicit its effects on epigenetic regulation of gene expression (6). In addition, using an IDH1-mutant patient-derived cell line that had undergone prolonged culture in the presence of the mutant IDH1 inhibitor AGI5198 showed that there was no longer a differential effect when treating with temozolomide versus temozolomide with PDD, and there was a restoration to basal levels of NAD+ and NADH. This may argue that although IDH inhibitors have shown efficacy in acute myeloid leukemia (7) and to an extent in gliomas (8), its effects when combined with temozolomide may not be as robust. As the mutant IDH1 inhibitor could elicit beneficial effects, for example, by reducing the production of immunosuppressive D-2HG and illuminating other sensitivities or liabilities, the data reported by Nagashima and colleagues suggest that timing and prolonged use of the mutant IDH inhibitor may make tumor cells less amenable to combined PDD and temozolomide therapy. Further studies investigating the short-term impact of the inhibitor in combination with this therapy and other therapies is warranted.

A main issue surrounding patients with low-grade glioma is recurrence and subsequent resistance to temozolomide, which is commonly associated with treatment-induced mutations in mismatch-repair pathways (9). Here, Nagashima and colleagues knocked out MSH6, inducing a mismatch-repair deficiency, modeling temozolomide resistance and, arguably, recurrence. Excitingly, the sensitivity to temozolomide and PDD persisted, suggesting this therapy could be efficacious in the frequently occurring temozolomide-resistant context that many patients find themselves in. Although current models do not necessarily permit it, further investigations into temozolomide-resistant lines and efficacy of PDD in orthotopic models of recurrence would be exciting to pursue and can offer significant impact on the field.

In the culminating set of experiments, Nagashima and colleagues tested the combined therapy in an in vivo context. In a well-controlled experiment, the IDH1-mutant HT1080 line with either nontargeting sgRNA or PARG-targeting sgRNA was injected into either flank of an individual mouse treated with temozolomide. The in vivo data showed promising results, with a significant reduction in tumor growth when PARG was knocked out in combination with temozolomide. Exploring this effect utilizing the wealth of patient-derived glioma lines while understanding any in vivo variation will be insightful.

IDH mutations are early hits in tumorigenesis, and studies investigating the molecular evolution of recurrence find that they usually persist in recurrent clones (10). This coupled with the high frequency for which patients with low-grade gliomas harbor the IDH1 mutation stresses its importance as a therapeutic target. Given that direct targeting of D-2HG in gliomas has not yet achieved promising clinical outcomes, studies like this one by Nagashima and colleagues, which describe a metabolic liability and an induced sensitivity spurred by the mutation, present a novel therapeutic approach and are of significant importance in moving the field closer to treating this most devastating disease.

H. Yan reports personal fees and other from Genetron Holdings (owner interest); personal fees from Agios, and personal fees from PGDX outside the submitted work; in addition, H. Yan has a patent for “Genetic Alterations in Isocitrate Dehydrogenase and Other Genes In Malignant Glioma” issued, licensed, and with royalties paid from Agios and PGDX; for “Methods for Rapid And Sensitive Detection of Hotspot Mutations” issued, licensed, and with royalties paid from Genetron Holdings; for “Homozygous and Heterozygous IDH1 Gene-Defective Human Astrocytoma Cell Lines” pending, and a patent for “Homozygous and Heterozygous IDH1 Gene-Defective Cell Lines Derived From Human Colorectal Cells” pending. No potential conflicts of interest were disclosed by the other author.

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