IDH2 mutations in patients with normal karyotype AML predict favorable responses to daunorubicin, cytarabine and cladribine regimen

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients’ outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37–0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.

patients had missense IDH1/2 mutations (IDH1/2 + ). Among the IDH1/2 + subgroup, 30 (37.5%) subjects carried IDH1 mutations in the R132 position. Of the 50 IDH2 + patients, 35 (43.75%) and 15 (18.75%) patients carried mutations in the R140 and R172 position, respectively. The median follow-up was 40.8 months and the median survival reached 18.8 months. The estimated 4-year survival for the whole group was 37.5% with standard error ± 3. Demographic and clinical characteristics of the patients are summarized in Table 1.
Hypomethylating activity of cladribine as a possible mechanism leading to improved survival of IDH2 + NK-AML patients. Since our analyses indicated that cladribine was associated with improved outcomes for IDH2 + patients, we further investigated possible biological mechanisms underlying this phenomenon. Mutations in IDH2 endow the enzyme with the neomorphic activity to produce 2-hydroxyglutarate (2HG), which functions as a competitive inhibitor of 2-ketoglutarate-dependent enzymes, such as TET2, a DNA-demethylating enzyme 5 . We therefore investigated, whether cladribine could limit 2HG-dependent DNA hypermethylation in AML cells. To this end, HEL and MOLM14 cell lines were treated with synthetic cell-permeable derivative of 2HG, octyl-2HG, alone or in combination with cladribine for 24 h. For these experiments, we used low cladribine doses (10 nM and 25 nM), which were non-toxic to the cells over the 24 h treatment period (Supplemental Fig. S4). Octyl-2HG significantly increased DNA methylation, measured by 5-methylcytosine abundance, whereas simultaneous addition of cladribine suppressed DNA hypermethylation (Fig. 4A). We next tested the hypomethylating effect of cladribine in HEL cells overexpressing IDH2 R140 and IDH2 R172 mutants.  The overproduction of 2HG in generated IDH2 mutant cell lines was confirmed by liquid chromatographymass spectrometry analyses (Supplemental Fig. S5). As expected, cells with IDH2 R140 and IDH2 R172 mutations induced DNA hypermethylation, comparing to IDH2 wild type (IDH2wt) cells (Fig. 4B). Incubation of cells overexpressing IDH2-mutants with cladribine (10 nM or 25 nM, 24 h) decreased 5-methylcytosine levels comparably to the IDH2-R140-specific inhibitor AGI-6780 (Fig. 4B). Of note, combination of cladribine with AGI-6780 further decreased DNA methylation, as compared to the either compound used alone (Supplemental Fig. S6). Although introduction of IDH1 R132H mutation induced 2-HG production, the global DNA methylation level did not differ between the mutant and wild type cells, and remained unchanged after addition of cladribine or IDH1 R132H-targeting inhibitor (AGI-5198), (Supplemental Fig. S7). At low doses, cladribine inhibits the activity of S-adenosylhomocysteine hydrolase, a key enzyme in the biosynthesis pathway of S-adenosylmethionine (SAM), which constitutes a methyl group donor in DNA methylation reactions [18][19][20]35 . Therefore we determined, whether cladribine compromises DNA methylation by affecting the cellular SAM level. Consistent with our hypothesis, incubation of HEL cells overexpressing IDH2 mutants with cladribine decreased SAM pool without influencing 2HG production, in contrast to AGI-6780, which reduced 2HG without affecting the SAM level (Fig. 4C,D).

Discussion
The prognostic significance of IDH1/2-mutations in patients with NK-AML is controversial, with conflicting reports in the literature 2,8,9,12,36,37 . In the present study, we report that the impact of IDH2 mutations on patient outcomes was related to the specific regimen used: the addition of cladribine to standard daunorubicin and cytarabine (DA) induction was independently associated with longer survival for IDH2 + patients (after censoring www.nature.com/scientificreports/ observations at alloHSCT). Our findings suggest that the mechanism for this beneficial effect is related to cladribine hypomethylating activity. In our study, the IDH2-R140 mutation was associated with superior outcomes in the entire NK-AML, uniquely when accompanied by NPM1 mutations, confirming the previous results 8 . Interestingly, this effect was not only NPM1 mutation-dependent, but also IDH2-specific: we found the favorable effect of NPM1 mutations only in patients with co-occurring IDH2 mutations, suggesting synergy between the two mutations. Neither IDH2-R140 nor R172 impacted outcomes of patients in NPM1 − /FLT3-ITD − subgroup. These data are similar to the findings of Patel et al., but different from other studies reporting a poor or uniquely favorable impact of the IDH2 R172 mutation on prognosis 2,8,37,38 . These discrepancies may be related to study inclusion criteria, type of IDH1/2 mutation, age, disease history as well as cytogenetic background of the analyzed population 2,8,12,14,37,[39][40][41] . In addition, recent high-throughput sequencing studies have shown that de novo IDH1/2 + NK-AML frequently coexist with adverse risk-associated mutations in DNMT3A, ASXL1, RUNX1, SRSF2, PHF6 38,[42][43][44] . Thus, the variable mutational spectra and co-occurring mutations in different patient cohorts may have contributed to the discrepancies in the reported prognostic impact of IDH1/2 + mutations between studies.
The effect of specific treatment has not been evaluated in the previous reports concerning the prognostic significance of IDH1/2 mutations. In our study, two high-intensity induction regimens: daunorubicin + cytarabine (DA) versus daunorubicin + cytarabine + cladribine (DAC) were used to treat NK-AML patients 15,16 . Our analysis showed that the addition of cladribine was associated with significantly improved outcomes in IDH2mutated patients. In the NPM1 − /FLT3-ITD − genotype, both IDH2 R140 and R172 mutations showed favorable  www.nature.com/scientificreports/ impact in the DAC-treated group, suggesting that the effect was IDH2-specific. Neither IDH2 R140 nor IDH2 R172 mutations were prognostic in the DA-treated subgroup, consistent with Patel et al. 8 . Multivariate analysis identified cladribine as an independent prognostic factor for longer survival for IDH2 + patients in both the entire NK-AML cohort and the NPM1 − /FLT3-ITD − subgroup. Thus, cladribine may be beneficial both in IDH2 + and FLT3-ITD + leukemias 22 . Intriguingly, the favorable effect of cladribine in the IDH2-mutated cohort was significant only when censoring for alloHSCT in most of the analyses. Therefore it is possible, that the impact of cladribine in IDH2 + patients is overshadowed in the setting of alloHSCT, e.g. due to improved survival of transplanted IDH2 − patients. Our data may also suggest that early alloHSCT in IDH2 + patients does not offer an advantage over chemotherapy, as has been observed for NPM1 + patients 26 . These possible explanations are further being investigated in an ongoing, prospective randomized clinical trial.
The mechanism of sensitivity of IDH2-mutant cells to cladribine is unknown. Our data show that in cells overexpressing IDH2-mutants, cladribine decreased SAM levels and DNA cytosine methylation, with no impact on 2HG production. Thus, in IDH2-mutant cells, cladribine may deplete the methyl donor pool, impair methylation reactions, and lead to decreased global DNA methylation, despite sustained production of 2HG and ongoing inhibition of 2HG-dependent enzymes, including DNA demethylases. Importantly, as concentrations similar to those used in our in vitro studies are achieved clinically using the standard doses of cladribine, corresponding levels of demethylating activity likely also occur in vivo 45 . Thus, cladribine and IDH2 inhibitors may have different, and potentially synergistic mechanisms of DNA demethylation and our preliminary in vitro data confirmed the synergy between cladribine and IDH2 R140Q-specific AGI-6780. Importantly, in the light of our findings, cladribine could be an interesting treatment alternative in patients with trans or cis resistance to IDH2 inhibition 46 .
Although both IDH2-and IDH1-mutants are reported to overproduce 2HG, in our study cladribine did not improve the survival of patients with IDH1 mutations 5,47 . Despite parallel mechanisms of transformation, IDH1 + and IDH2 + leukemias show differences in both in vitro and clinical studies. This discrepancy might be related to distinct cellular localization of IDH1 and IDH2 molecules (cytoplasmic vs mitochondrial), followed by various downstream metabolic consequences, including differential response to cytotoxic drugs [48][49][50][51] . In our in vitro IDH overexpressing model, global DNA hypermethylation was attributed only to IDH2 mutations, but not to IDH1 R132H cells. To support, although DNA hypermethylation was previously reported in both IDH1 and IDH2-mutants overexpressing HEK293T cells, 5-methylcytosine level was considerably lower in IDH1 than IDH2-mutants 5 . Furthermore, IDH1 + and IDH2 + leukemias differ in their mutational profiles, with high incidence of DNA (cytosine-5)-methyltransferase 3A (DNMT3A) mutations reported in IDH1 + , but not IDH2-R140 + AML 38,43 . As DNMT3A mutations impact DNA methylation profile, it is very likely, that their co-segregation with IDH1 mutations might change the response to cladribine 52 .
In summary, our data show that the addition of cladribine to standard AML induction therapy resulted in improved outcomes in patients with IDH2 mutation. The mechanism of this synthetic effect likely involves cladribine's demethylating activity in a molecular background of the mutation-induced DNA hypermethylation. Given the limitations of this study (retrospective nature, lack of comprehensive mutational profile at diagnosis, and relatively small IDH1/2 + subgroups), further investigations on cladribine as a treatment option for IDH1/2 + patients are warranted. Of note, a randomized, international study comparing DA versus DAC regimens has been already launched, with complete remission, overall survival and multimodality assessments of measurable residual disease as the study endpoints.

Patients and methods
Patients characteristics, material collection and molecular tests. A total of 398 de novo NK-AML patients treated in 9 PALG centers between 1999 and 2014 were either prospectively randomized to 1 of the 3 treatment groups (in the years 2000-2006): daunorubicin + cytarabine (DA; n = 18), daunorubicine + cytarabine + cladribine (DAC; n = 24), daunorubicine + cytarabine + fludarabine (DAF; n = 20), or treated outside the trial (2006-2014), according to DA (n = 173), DAC (n = 152) or DAF (n = 11) induction protocols, at the discretion of the treating physician (Table 1, Supplemental Table S3). Of note, fewer patients were included from years 2000-2006 due to limited access to molecular genetic data. Analysis of the prognostic significance of IDH1/2 mutations was performed for the entire population (DA-, DAC-and DAF-treated; Supplemental Table S2), while the impact of cladribine on outcomes of the IDH2 + NK-AML population was evaluated in the DAC-vs DA-treated groups (Table 3 and Supplemental Table S3). All patients included in the study were eligible for intensive induction treatment with the age range from 18 to 76 years and median age of 50 years. All samples were obtained with written informed consent, in accordance with the Declaration of Helsinki. The study was approved by the local Bioethics Committees of Warsaw Medical University for all participating institutions. The mutation status of IDH1/2 was determined as previously described 7,53,54 . Details of the material collection and molecular tests are described in Supplementary Figures  www.nature.com/scientificreports/ Statistical analysis. The study end points were rate of complete remission (CR), median overall survival (OS), and relapse-free survival (RFS). Complete remission rate was defined according to previously published criteria 16 . Overall survival was defined as time from diagnosis to either death or last observation alive. Data analyses were performed with and without censoring the observations at the time of allogeneic hematopoietic stem cell transplantation (alloHSCT) if performed in 1st CR. Log-rank test was used to compare OS in univariate analysis. For comparison of CR rates or frequency distribution of other characteristics between subgroups, we used Chi-square or Fisher exact test (when the number of patients per subgroup was < 5). In multivariate analyses logistic regression and Cox proportional model were used to compare CR rates and OS, respectively. The statistical analyses were performed using STATISTICA 12 (StatSoft Inc. Tulsa, OK, USA).
Chemicals, antibodies and cell culture reagents. Details on chemicals, antibodies, cell culture reagents and generation of AML HEL cells overexpressing IDH2-mutants are available in the Supplementary Figures