B3: Molecular and functional characterization of cooperating events in acute myeloid leukemia with NPM1 mutation
Prof. Dr. med. Lars Bullinger
Medizinische Klinik m.S. Hämatologie, Onkologie und Tumorimmunologie
Charité Universitätsmedizin Berlin
Augustenberger Platz 1
Mutations in the Nucleophosmin gene (NPM1mut) are among the most recurrent genetic abnormalities in acute myeloid leukemia (AML). Despite the association with distinct biological and clinical features, NPM1mut AML is characterized by a great heterogeneity with regard to clinical outcome. Emerging data indicate that addi-tional cooperating events are causative for this heterogeneity. Within project B3 we will further elucidate the cooperating events in NPM1mut AML by using an integrated genomic approach.
So far, we have profiled copy number alterations by SNP arrays in 250 AML samples and detected recurrent genomic aberrations in a significant number of the cases such as deletion 9q with a minimally deleted re-gion (MDR) on 9q21. We also performed gene mutation screening in NPM1mut AML by targeted resequenc-ing (n=435) and whole exome sequencing (n=32), which e.g. revealed recurrent mutations in HNRNPK (Het-erogeneous Nuclear Ribonucleoprotein K), a gene located in the MDR on 9q21, as well as recurrent mutations in RAD21, a cohesin factor gene, PTPN11 and MYC. To further evaluate the role of these dele-tions/mutations, gene expression data were generated by microarray technology in 138 NPM1mut cases. These showed a deregulated expression of genes involved in splicing and mRNA processing for 9q deleted cases. First functional analyses of HNRNPK by RNAi-based knock-down experiments in leukemia cell lines suggested a growth advantage for haploinsufficient cells. Ongoing and future functional experiments are also focusing on the interaction of NPM1mut with RAD21 as well as MYC and PTPN11.
With regard to clonal evolution of NPM1mut AML, we analyzed 129 paired (diagnosis/relapse) NPM1mut sam-ples and identified e.g. persisting pre-leukemic DNMT3A mutations as well as relapse-associated copy number changes and mutations. Subsequent analysis by whole exome sequencing of 12 AML with loss of NPM1mut at relapse identified an enrichment of gene mutations associated with anthracycline resistance.
During the next funding period, the major goal of the project remains to better understand the molecular basis of the clinically and biologically heterogeneous NPM1mut AML subgroup. We will continue our integrat-ed genomics approach by targeted resequencing, whole exome sequencing and transcriptomic analyses using RNA-seq to follow up on novel recurrent mutations in larger data sets and to investigate the role of mutations impairing the transcriptomic regulation. In addition, we will advance our analyses of clonal evolu-tion and clonal heterogeneity to get novel insights with regard to treatment resistance/escape mechanisms. A comprehensive evaluation of larger cohorts of NPM1mut diagnostic-relapse pairs will define recurrent muta-tional evolution patterns and provide a basis for improved minimal residual disease (MRD) monitoring. To further study the impact of NPM1mut associated events and to develop models for clonal evolution, selected candidates (e.g. HNRNPK, RAD21, PTPN11 and MYC) will be further investigated functionally in vitro and in vivo using cell line models, primary human AML samples, xenograft and murine leukemia models as well as novel CRISPR/Cas9 gene editing approaches. Finally, novel state-of-the-art proteomics technology, such as stable isotope labelling by amino acid in cell culture (SILAC), will allow us to get insights into the functional role of the NPM1mut protein itself. This will provide important insights into synergistic protein interactions, especially with regard to the development of novel treatment options.
For a current list of project-related publications, please go to this page