In acute myeloid leukemia (AML), between 15-20% of all patients harbor a somatic mutation in the isocitrate dehydrogenase 1 or 2 gene (IDH1 and IDH2). Therefore, these mutations are among the common ones in AML. However, the prognostic significance of mutated IDH in AML patients remains controversial. Several research groups reported distinct outcomes within specific patient subsets depending on the biological and clinical context. Additionally, the prognostic impact seems to be depended by the co-mutations, the specific location of the mutation (i.e., regarding the hotspot locations IDH1 R132, IDH2 R140, and IDH2 R172) as well as the applied treatment. Today, allogeneic hematopoietic stem cell transplantation (HSCT) remains the consolidation therapy with the highest chance of sustained remission for most younger and older AML patients. Even though many IDH mutated AML patients are consolidated by HSCT and several trials testing IDH inhibitors in a maintenance setting are active, very little data are available on the influence of IDH mutations at diagnosis and as measurable residual disease (MRD) marker in the HSCT context. The first aim of our study was to study the frequency of IDH mutations and assess their associations with other biological and pretreatment markers. In our cohort of 292 AML patients, who all received an HSCT for consolidation, we identified somatic IDH mutations in 70 (24%) patients. IDH1 mutations were found in 11.4% of the patients, all of which were R132 substitutions. Regarding mutations in the IDH2 gene, we identified 8.9% and 5.1% patients harboring a R140 or a R172 substitution, respectively. Generally, IDH mutated patients did not differ significantly from IDH wild type patients in our set regarding their biological characteristics with the exception that IDH mutated patients had significant higher bone marrow blasts at diagnosis. When we analyzed the mutational landscape of our cohort, we found that IDH mutated patients more frequently also harbored DNMT3A mutations, while RUNX1 mutations and TP53 mutations were found in lower frequencies. For the diagnostic bone marrow variant allele fractions (VAFs) associated with the three IDH mutation types, we observed a mutation specific pattern. While IDH2 R140 mutations clustered around a median VAF of about 50%, the VAFs for IDH1 R132 and IDH2 R172 mutations at diagnosis were significantly lower by median but showed a wider distribution. Next, we aimed to examine the prognostic value of different IDH1 and IDH2 mutations in AML patients that receive a consolidating allogeneic HSCT. Here, we observed no differences in the cumulative incidence of relapse (CIR), event-free survival (EFS), and overall survival (OS) according to the IDH mutational status at diagnosis. This also held up when we analyzed the three mutations, i.e., IDH1 R132, IDH2 R140, and IDH2 R172 mutations separately. Also in multivariable analyses, the diagnostic IDH mutation status did not significantly associate with outcomes after HSCT. However, we also analyzed the prognostic impact in the context of the ELN2017 classification and observed a distinct prognostic impact of the IDH mutations. With respect to outcome following HSCT consolidation in the ELN2017 Favorable-risk group, IDH mutation status did not influence outcomes. In the ELN2017 Intermediate-risk group, IDH mutated patients had an increased relapse rate compared to IDH wild-type patients. Nevertheless, this did not translate into significant shorter EFS or OS. Within the ELN2017 Adverse-risk group, IDH mutated patients had a lower CIR and by trend longer OS and EFS. The final major objective of our study was to analyze the role of IDH mutations as MRD marker in AML patients in CR prior to an allogeneic HSCT. In our cohort, 44 mutated patients had material for IDH mutation status detection on MRD level using digital droplet PCR (ddPCR) at HSCT available. DdPCr is a novel method that allows absolute quantification of gene mutations and/or expression without the necessity of standard curves. We established an assay that allows the quantification of IDH1 and IDH2 mutations with very high sensitivity and specificity. Of the 44 patients, 33 (75%) had detectable IDH MRD. The ddPCR based IDH mutation MRD positive patients were differently distributed (IDH1 R132: 65%; IDH2 R140: 94%; IDH2 R172: 44%). Interestingly, the VAFs at HSCT were much lower in IDH1 R132 (median 0.17%) and IDH2 R172 (median 0.20%) compared to IDH2 R140 (median 11.6%). Looking for association between IDH MRD status at HSCT and outcome, we observed a strong relapse association of IDH1 R132 positivity or IDH2 R172 positivity. Patients that were MRD positive for IDH1 R132 or IDH2 R172 mutations also had a shorter - though not significant - EFS and OS. Thus, clinically, the elimination of persisting IDH mutations – especially of IDH1 R132 and IDH2 R172 – before HSCT could be an important milestone towards a cure for these patients. On the other hand, IDH2 R140 MRD positivity at HSCT did not associate significantly with the CIR, EFS, and OS. Together with the previous mentioned finding of a higher VAF at diagnosis, we speculated that in our cohort, IDH2 R140 mutations behaved more like a clonal hematopoiesis-related aberrations.:Inhalt/Content Bibliographische Beschreibung/Bibliographic Description 1 Referat/Abstract 2 Einleitung/Introduction 3 Acute myeloid leukemia 3 Epidemiology 3 Clinical presentation and pathogenesis 3 Diagnostic workup and classification 3 Risk stratification and treatment 6 2022 European LeukemiaNet genetic risk classification 6 Standard Treatment 7 Measurable Residual Disease 10 Isocitrate Dehydrogenase 1 and 2 mutations 11 Definition 11 Pathology and Epidemiology 12 IDH mutations as prognostic markers 12 IDH inhibitors 13 Publikation/Publication 15 Zusammenfassung/Summary 22 Perspektive/Outlook 26 Literaturverzeichnis/References 27 Anlage/Supplemental Material 36 Referenz der Publikation/Reference of the Publication 55 Erklärung über den wissenschaftlichen Anteil des Promovenden 56 Erklärung über eigenständige Abfassung der Arbeit 58 Lebenslauf/Curriculum Vitae 59 Publikationen/Publications 61 Erst- und Letztautorschaften/First and Last authorship 60 Ko-Autorschaften/Co authorship 61 Reviews 66 Danksagung/Acknowledgments 67

Background The role of allogeneic hematopoietic cell transplantation (alloHCT) in acute myeloid leukemia (AML) with mutated IDH1/2 has not been defined. Therefore, we analyzed a large cohort of 3234 AML patients in first complete remission (CR1) undergoing alloHCT or conventional chemo-consolidation and investigated outcome in respect to IDH1/2 mutational subgroups (IDH1 R132C, R132H and IDH2 R140Q, R172K). Methods Genomic DNA was extracted from bone marrow or peripheral blood samples at diagnosis and analyzed for IDH mutations with denaturing high-performance liquid chromatography, Sanger sequencing and targeted myeloid panel next-generation sequencing, respectively. Statistical as-treated analyses were performed using R and standard statistical methods (Kruskal–Wallis test for continuous variables, Chi-square test for categorical variables, Cox regression for univariate and multivariable models), incorporating alloHCT as a time-dependent covariate. Results Among 3234 patients achieving CR1, 7.8% harbored IDH1 mutations (36% R132C and 47% R132H) and 10.9% carried IDH2 mutations (77% R140Q and 19% R172K). 852 patients underwent alloHCT in CR1. Within the alloHCT group, 6.2% had an IDH1 mutation (43.4% R132C and 41.4% R132H) and 10% were characterized by an IDH2 mutation (71.8% R140Q and 24.7% R172K). Variants IDH1 R132C and IDH2 R172K showed a significant benefit from alloHCT for OS (p = .017 and p = .049) and RFS (HR = 0.42, p = .048 and p = .009) compared with chemotherapy only. AlloHCT in IDH2 R140Q mutated AML resulted in longer RFS (HR = 0.4, p = .002). Conclusion In this large as-treated analysis, we showed that alloHCT is able to overcome the negative prognostic impact of certain IDH mutational subclasses in first-line consolidation treatment and could pending prognostic validation, provide prognostic value for AML risk stratification and therapeutic decision making.

Les gènes de l'isocitrate déshydrogénase (IDH) sont mutés dans 70 à 80 % des gliomes de bas grade. Les enzymes mutantes IDH qui en résultent présentent une activité de gain de fonction, produisant du R-2-hydroxyglutarate (R-2-HG), appelé oncométabolite en raison de son accumulation anormale dans les tumeurs et de ses activités oncogéniques potentielles. Parmi les caractéristiques du cancer telles que la reprogrammation métabolique et épigénétique, le stress réplicatif et la stabilité du génome ont été peu caractérisés dans les cancers IDH-mutants. Par conséquent, cette étude vise à étudier l'impact de l'accumulation de R-2-HG sur la réplication de l'ADN et sa contribution au stress réplicatif dans les cancers IDH-mutants. Nous avons étudié la dynamique de la fourche de réplication dans des astrocytes humains normaux et confirmé les résultats dans d'autres lignées cellulaires normales et cancéreuses. Nous avons constaté que le traitement exogène par l'octyl-R-2-HG entravait la progression de la fourche de réplication et retardait par conséquent l'achèvement de la phase S. L'évaluation des niveaux de phosphorylation des protéines RPA, CHK1 et H2AX a révélé que la réponse classique au stress réplicatif (RSR) n'était pas activée. Un état cellulaire dans lequel la réplication de l'ADN est altérée sans activation de la RSR a notamment été décrit dans la littérature comme résultant de l'activation de la réponse au stress intégré (ISR). Cependant, l'activation de la RSI dans les cancers mutants IDH n'est pas bien étudiée. En évaluant les marqueurs d'activation de la RSI, tels que la phosphorylation de l'eIF2α et les niveaux de protéines ATF4, nous avons montré que l'octyl-R-2-HG activait la RSI. De plus, le blocage de l'ISR a partiellement sauvé la fourche de réplication et la progression de la phase S. Nous avons répliqué cette étude oncométrique. Nous avons reproduit ce défaut de réplication de l'ADN lié à l'oncométabolite ainsi que l'effet de sauvetage partiel de l'ISRIB lors de l'induction de la surexpression du gène IDH mutant. Nos résultats indiquent que la production de R-2-HG associée à la mIDH peut inhiber la dynamique normale de réplication de l'ADN via la signalisation ISR. / The isocitrate dehydrogenase (IDH) genes are mutated in 70-80% of low-grade gliomas. The resulting IDH mutant enzymes exhibit gain-of-function activity, producing R-2-hydroxyglutarate (R-2-HG), which is referred to as an oncometabolite due to its abnormal accumulation in tumours and potential oncogenic activities. Among the hallmarks of cancer such as metabolic and epigenetic reprogramming, replicative stress and genome stability have been poorly characterized in IDH-mutant cancer. Therefore, this study aims to investigate the impact of R-2-HG accumulation on DNA replication and its contribution to replicative stress in IDH-mutant cancers. We investigated replication fork dynamics in normal human astrocytes and confirmed the results in other normal and cancer cell lines. We found that exogenous treatment with octyl-R-2-HG impaired replication fork progression and consequently delayed S-phase completion. Assessment of RPA, CHK1 and H2AX protein phosphorylation levels revealed that the classical Replicative Stress Response (RSR) was not activated. Among others, a cell state in which DNA replication was impaired without activation of the RSR has been described in the literature as a result of activation of the Integrated Stress Response (ISR). However, ISR activation in IDH-mutant cancers is not well studied. Hence, by assessing ISR activation markers such as eIF2α phosphorylation and ATF4 protein levels, we showed that octyl-R-2-HG activated ISR. Moreover, blocking ISR partially rescued the replication fork and S-phase progression. We replicated this oncometabolite-related DNA replication defect as well as ISRIB’s partial rescue effect upon induction of mutant IDH gene overexpression. Our results indicate that mIDH-associated R-2-HG production possibly inhibits normal DNA replication dynamics via ISR signalling.