Cladribine Induction Therapy Improves Prognosis in IDH2 Mutant NK-AML Patients

2.1 Prognostic Value of IDH1/2 Mutations in Overall NK-AML Population and Different NPM1/FLT3 Mutation Status Subgroups

This study included 398 newly diagnosed NK-AML patients, of which 80 (20.1%) had IDH1/2 mutations (IDH1/2+). The overall complete remission rate (CR) was 75.4% (300/398); univariate and multivariate analyses showed that IDH1 and IDH2 gene mutations did not affect the complete remission rate. Stratifying the overall NK-AML population based on IDH1 mutation status revealed that IDH1+ patients had a declining trend in overall survival (OS), but no statistically significant difference; in contrast, univariate and multivariate analyses after excluding allogeneic hematopoietic stem cell transplantation (alloHSCT) showed that IDH2+ patients significantly benefited in terms of OS (Figure 1-A).

Subsequent analyses compared low-risk (LR: NPM1⁺/FLT3-ITD^–) and high-risk (HR: FLT3-ITD⁺ , NPM1^–/FLT3-ITD^–) genotypic populations to determine the impact of NPM1/FLT3 genotype on the prognostic value of IDH1/2. Univariate analyses showed that IDH1 had an adverse impact on HR NK-AML patients’ OS (IDH1+ vs IDH1− OS rates were 15% vs 36%; P=0.03; Figure 1-B), and the impact was particularly significant for NPM1^–/FLT3-ITD^– (IDH1+ vs IDH1− OS rates were 15% vs 43%; P=0.026); multivariate analysis after excluding alloHSCT showed that IDH1 mutations were independently associated with increased mortality risk in HR NK-AML and NPM1^–/FLT3-ITD^– subgroup patients. Unlike IDH1, when assessed alongside NPM1 and FLT3 mutations, IDH2 mutations had a positive impact on the prognosis of both HR and LR subgroups (Figure 1-B,C); moreover, IDH2 R140 and IDH2 R172 mutations did not affect the survival of NPM1^–/FLT3-ITD^– subgroup. Notably, except for the NPM1−/FLT3-ITD− genotype, all other IDH2 R140 mutations were usually accompanied by NPM1 mutations (with or without FLT3-ITD); thus, the OS benefit for IDH2 mutation patients in the overall cohort was primarily driven by the NPM1+/IDH2-R140+ genotypic subgroup.

Figure 1 Overall NK-AML Population (A), HR NK-AML(B) and LR NK-AML(C) Patients’ OS

2.2 Prognostic Significance of IDH2 Mutations in Patients Receiving DA vs DAC Treatment

IDH2 mutations positively impacted the survival rate of NK AML patients receiving DAC treatment (IDH2+ vs IDH2-: 54% vs 33%, P=0.0087; Figure 2-A), but had no effect on the survival rate of patients receiving DA treatment (IDH2+ vs IDH2-: 21% vs 23%, P=0.22; Figure 2-B). In the DA treatment group, IDH2 R140 and IDH2 R172 mutations had no effect on OS.

2.3 Impact of Cladribine Combined with Standard DA Induction Therapy on Prognosis of IDH1/2+ NK-AML Patients

Further comparisons of clinical outcomes in IDH1/2+ patients receiving DAC vs DA treatment show that, after excluding HSCT, DAC induction therapy was associated with improved 4-year OS in high-risk IDH2+ patients compared to standard DA regimen (OS: 50% vs 13%; P=0.04; Figure 3-A/B); among which, the improvement in OS was particularly significant in the NPM1⁻/FLT3-ITD⁻ subgroup (HR: 0.3; 95%CI 0.08-0.95; P=0.04); however, IDH2- or IDH1 patients’ OS did not improve. In the IDH2 + subgroup, the good benefit of cladribine was limited to younger patients (< 50 years). However, multivariate analysis for IDH2 + patients showed that after excluding alloHSCT, DAC induction therapy was independently associated with reduced mortality risk (HR: 0.21; 95%CI 0.056 – 0.8; P = 0.02).

Figure 3: DAC vs DA Treatment of IDH2+ Overall NK-AML Patients (A) and HR NK-AML Patients (B) OS

IDH2 mutations induce the production of enzymes with novel variant activity that generate 2-hydroxyglutarate (2HG), which is a competitive inhibitor of 2-ketoglutarate-dependent enzymes, such as TET2 (an enzyme involved in DNA demethylation). Therefore, this study aims to evaluate whether cladribine can inhibit 2HG-dependent DNA hypermethylation in AML cells. HEL and MOLM14 cell lines were treated with synthetic cell-permeable derivatives of 2HG and octyl-2HG alone or in combination with cladribine for 24 hours, and the results showed that only the octyl-2HG treatment group exhibited significantly increased DNA methylation levels.

Furthermore, compared to IDH2 wild-type (IDH2wt) cells, IDH2 R140 and IDH2 R172 mutant cells induced DNA hypermethylation (Figure 4). Compared to cells treated with the specific IDH2-R140 inhibitor AGI-6780, cells treated with cladribine (10 nM or 25 nM, 24 h) showed reduced levels of 5-methylcytosine, and cladribine combined with AGI-6780 further reduced DNA methylation. Further studies found that cladribine inhibited S-adenosylhomocysteine hydrolase, which is involved in the biosynthesis of S-adenosylmethionine (SAM), and can serve as a methyl donor in DNA methylation reactions; thus, cladribine primarily inhibits DNA methylation by affecting SAM levels.

Figure 4: Cladribine Inhibits IDH2 R140Q and R172K Mutation-Induced DNA Hypermethylation