Data-driven modeling of core gene regulatory network underlying leukemogenesis in IDH mutant AML

6576 Background: Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations play a significant role in acute myeloid leukemia (AML), yet optimal treatment for AML patients with IDH mutations remains unclear. IDH mutations have been associated with a globally hypermethylated genomic state, as the result of increased levels of 2-HG, a regulator of histone methylation. We sought to retrospectively determine responses of patients with IDH-mutated AML to treatment with hypomethylating agents. Methods: We reviewed clinical data for AML patients treated at The University of Texas M. D. Anderson Cancer Center from June 2001 to December 2011 whose stored leukemia samples from bone marrow aspirateshad been tested retrospectively for IDH1 and IDH2 mutations. We searched three databases that contained records for 407 AML patients. We selected patients who had been treated with the hypomethylating agents 5-azacitidine or decitabine, alone or in conjunction with other therapeutics. We retrospectively reviewed the patients’ medical records to obtain demographic, laboratory, treatment, and clinical data. We evaluated response measured by remission status, reduction in bone marrow blasts and peripheral blood blasts, and time to relapse. Results: We identified 104 patients (25.6%) who had either an IDH1 or IDH2 mutation, and of these, 8 patients had also been treated with hypomethylating agents. A ninth AML patient, who is IDH2-mutant, is currently undergoing treatment with decitabine alone. Of these 9 patients, three had a durable complete remission without recovery of blood counts (CRp), three had a partial response (PR), with or without blood count recovery, and three had no response. All 9 patients did have an initial decrease in the percentage of bone marrow blasts, peripheral blood blasts, or both. Conclusions: This series of patients demonstrates the possibility that AML patients with IDH1 or IDH2 mutations benefit from the use of hypomethylating agents, and suggests it is worthwhile to prospectively investigate treatment of patients with IDH-mutated AML using hypomethylating agents.

Blast clearance dynamics and time to response across IDH1- and IDH2-mutated AML

Representation of Older Adults in FDA and EMA Registrational Trials for Acute Myeloid Leukemia (AML)

Mutations in isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) are found in 15% to 20% of patients with acute myeloid leukemia (AML). IDH inhibitors have been introduced as targeted treatment and are currently under investigation as maintenance therapy after allogeneic transplantation (allo-SCT). Since reports about the outcome of IDH1- and IDH2-mutated (IDHmut) AML after allo-SCT are limited, we retrospectively analyzed 356 IDH-mutated AML patients (IDH1 40%, IDH2 60%). Ten patients (4%) had received an IDH inhibitor prior transplantation, but none had received maintenance with IDH inhibitors. After a median follow-up of 24 months 3-year probabilities of overall (OS) and event-free (EFS) survival, relapse and nonrelapse mortality (NRM) for the entire cohort were 73%, 60%, 27% and 13% respectively. While 3-year OS (78% versus 70%), EFS (56% versus 63%) and NRM (10% versus 14%) rates were similar for IDH1mut and IDH2mut patients, relapse incidence was numerically higher in IDH1mut patients (34% versus 24%) and landmark analysis suggested a continuous rise of relapse incidence preferentially in IDH1mut AML also beyond the first year. Concordantly, IDH2 mutation was associated with superior EFS and by trend with lower relapse incidence. The strongest risk factor for adverse outcomes, however, was AML not in CR. This analysis provides benchmarks for interpretation of results emerging from post-transplant maintenance trials in IDHmut AML and suggest that maintenance strategies may further optimize the promising outcome in this molecularly defined subgroup by reducing relapse risk, especially for patients whose AML is not in remission at time of alloHCT.

DNA Methylation Profiling of AML Reveals Epigenetic Subgroups with Distinct Clinical Outcome

Recurrent mutations in IDH1 or IDH2 in acute myeloid leukemia (AML) are associated with increased DNA methylation, but the genome-wide patterns of this hypermethylation phenotype have not been comprehensively studied in AML samples. We analyzed whole-genome bisulfite sequencing data from 15 primary AML samples with IDH1 or IDH2 mutations, which identified ~4000 focal regions that were uniquely hypermethylated in IDHmut samples vs. normal CD34+ cells and other AMLs. These regions had modest hypermethylation in AMLs with biallelic TET2 mutations, and levels of 5-hydroxymethylation that were diminished in IDH and TET-mutant samples, indicating that this hypermethylation results from inhibition of TET-mediated demethylation. Focal hypermethylation in IDHmut AMLs occurred at regions with low methylation in CD34+ cells, implying that DNA methylation and demethylation are active at these loci. AML samples containing IDH and DNMT3AR882 mutations were significantly less hypermethylated, suggesting that IDHmut-associated hypermethylation is mediated by DNMT3A. IDHmut-specific hypermethylation was highly enriched for enhancers that form direct interactions with genes involved in normal hematopoiesis and AML, including MYC and ETV6. These results suggest that focal hypermethylation in IDH-mutant AML occurs by altering the balance between DNA methylation and demethylation, and that disruption of these pathways at enhancers may contribute to AML pathogenesis.

Acute Myeloid Leukemia (AML) Patients with Myelodysplasia (MDS) Related-Gene Mutations in First Complete Remission (CR1) Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplant (HCT)

LR11 Plays a Critical Role in Leukemogenesis of Acute Myeloid Leukemia by Regulating Adhesion and Homing Ability to Bone Marrow.

Based on current findings, the presence of NPM1 mutations in acute myeloid leukemia (AML) patients is associated with an increased probability of complete remission (CR) and better overall survival (OS). We determined the incidence and prognostic relevance of NPM1 mutations, their association with FLT3 and IDH mutations, and other clinical characteristics in Serbian adult AML patients. Samples from 111 adult de novo AML patients, including 73 AML cases with a normal karyotype (NK-AML), were studied. NPM1, FLT3, and IDH mutations were detected by PCR and direct sequencing. NPM1 mutations were detected in 22.5% of patients. The presence of NPM1 mutations predicted a low CR rate and shorter OS. NPM1 mutations showed an association with both FLT3 and IDH mutations. Survival analysis based on NPM1/FLT3 mutational status revealed a lower OS for NPM1⁺/FLT3^– compared to the NPM1^–/FLT3^– group in NK-AML patients. The lack of impact or unfavorable prognostic effect of NPM1 mutations found in this study can be assigned to a small cohort of analyzed AML patients, as can the presence of FLT3 and IDH mutations or other genetic lesions that cooperate with NPM1 mutations influencing prognosis.

Genomic Characterization of Newly Diagnosed Acute Myeloid Leukemia in Patients Age 60 Years and Older; A Report from the Beat AML Master Trial

Comparative Analysis for Effectiveness of Azacitidine Versus Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes in New Mexico: A Single Institution Experience

Eighty-two de novo AML patients and 18 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the expression and methylation data of CASC15. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of CASC15 expression to identify AML. The patients were divided into CASC15high group and CASC15low group by X-tile method, and the prognostic value of CASC15 was identified by Kaplan-Meier method and univariate and multivariate Cox regression analysis. The expression level of CASC15 was significantly decreased in BM cells of AML patients compared with healthy donors (P<0.001). ROC curve analysis suggested that CASC15 expression might be a potential biomarker to discriminate AML from controls. The expression of CASC15 was high at the early stage of hematopoiesis, and reached a peak at the stage of multipotent progenitors differentiation, then decreased rapidly, and was at a range of low level fluctuations in the subsequent process. Among FAB subtypes, CASC15 expression in M0 was significantly higher than that in M1-M7. Clinically, CASC15low patients were more likely to have NPM1 mutations than CASC15high patients (P=0.048), while CASC15high patients had a significantly higher frequency of IDH1 and RUNX1 mutations (P=0.021 and 0.014, respectively). Moreover, CASC15low group had a shorter overall survival (OS) in patients with NPM1 mutations. Furthermore, multivariate analysis confirmed that CASC15 expression was a significant independent risk factor for OS in NPM1 mutated AML patients. In addition, CASC15 methylation level in BM samples of AML patients was significantly decreased compared with healthy donors. Patients with CASC15 high methylation had poor OS and disease-free survival. The expression of CASC15 is decreased in AML, and low CASC15 expression may predict adverse prognosis in AML patients with NPM1 mutations. Moreover, CASC15 methylation level in AML is significantly decreased, and high CASC15 methylation may predict poor prognosis in AML.

Large-scale multiparameter screening has become increasingly feasible and straightforward to perform thanks to developments in technologies such as high-content microscopy and high-throughput flow cytometry. The automated toolkits for analyzing similarities and differences between large numbers of tested conditions have not kept pace with these technological developments. Thus, effective analysis of multiparameter screening datasets becomes a bottleneck and a limiting factor in unbiased interpretation of results. Here we introduce compaRe, a toolkit for large-scale multiparameter data analysis, which integrates quality control, data bias correction, and data visualization methods with a mass-aware gridding algorithm-based similarity analysis providing a much faster and more robust analyses than existing methods. Using mass and flow cytometry data from acute myeloid leukemia and myelodysplastic syndrome patients, we show that compaRe can reveal interpatient heterogeneity and recognizable phenotypic profiles. By applying compaRe to high-throughput flow cytometry drug response data in AML models, we robustly identified multiple types of both deep and subtle phenotypic response patterns, highlighting how this analysis could be used for therapeutic discoveries. In conclusion, compaRe is a toolkit that uniquely allows for automated, rapid, and precise comparisons of large-scale multiparameter datasets, including high-throughput screens.

Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) occur in about 15% of acute myeloid leukemia (AML) patients, and lead to the production of an oncometabolite, the 2-hydroxyglutarate (2-HG). Importantly, many clinical studies demonstrated that IDH1/2 mutations are systematically conserved at relapse, suggesting that this mutated enzyme could contribute to early leukemogenesis. In an effort to identify therapy specifically targeting IDH1 R132H mutated blasts, we developed an AML cell line model carrying IDH1 R132H mutation. We first stably transduced HL60 cell line to establish clones carrying either empty vector, IDH1 WT gene or IDH1-R132H mutated gene. We then characterized these clones with different features including 2-HG production. Since demethylation of many genes involved in the retinol metabolism is impaired by IDH1-R132H mutation and because all-trans retinoic acid (ATRA), a retinol metabolite, is already used in clinic, we studied its effects on IDH1-R132H mutated AML cells. Here, we showed on our transduced models as well as on primary AML patient samples (n=6), that AML blasts harboring IDH1-R132H mutation were highly sensitive to low doses of ATRA, compared to the IDH1 WT. In fact, IDH1-R132H mutation sensitized to ATRA-induced differentiation, as shown by increased expression of the differentiation marker CD11b and morphological changes evidenced with May-Grünwald-Giemsa staining. This was followed by changes of CD14 and CD15 expression, as well as granulocytic enzymatic activity measured with the nitroblue tetrazolium reduction assay. This terminal granulocytic differentiation came with the reduction of proliferation and colony formation, and then, quickly led to apoptosis of IDH1 R132H mutated AML cells. Moreover, we showed that the level of differentiation of IDH1 R132H mutated models was correlated to the magnitude of IDH1-R132H protein expression and thus to the 2-HG production. We further demonstrated that treatment with a cell-permeable form of 2-HG sensitized empty construct to ATRA-induced differentiation, whereas the inhibition of IDH1-R132H activity reduced its ATRA-sensitivity in IDH1-R132H mutant. Altogether, our results show that IDH1-R132H mutation sensitizes to ATRA-induced granulocytic terminal differentiation in a 2-HG concentration-dependent manner and that it leads to their apoptosis. Since serum 2-HG concentration of IDH mutated patients is approximately 100 fold higher than for non mutated patient, we could expect a high and specific effect of ATRA treatment on IDH1 mutated AML patients. Furthermore, as this mutation could contribute to relapse, this might be a promising therapeutic strategy to achieve a long-term remission in AML with IDH1 mutations. Citation Format: Helena Boutzen, Fabienne De Toni, Estelle Saland, Eric Delabesse, Véronique De mas, Cécile Demur, Florence Castelli, Lara Gales, Virginie Penard-lacronique, Stéphane De Botton, Jean-Charles Portais, Christophe Junot, Stéphane Manenti, Christian Récher, Jean-emmanuel Sarry. All-trans-retinoic acid as a new therapeutic approach to target isocitrate dehydrogenase mutations in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2678. doi:10.1158/1538-7445.AM2014-2678

Bzip in-frame mutations might abrogate the adverse prognosis of therapy-related CEBPA-mutated AML