Acquired Gene Mutation of MAP4K2 Identified from Myelodysplastic Syndromes Patients Confers Azacitidine Resistance

Myelodysplastic syndromes (MDS) is clinically characterized by ineffective hematopoiesis and frequent development of acute myeloid leukemia (AML). Accumulating literature has addressed a variety of abnormalities in MDS hematopoietic stem cells, including chromosomal abnormalities, genetic mutations and epigenetic changes. At present, however, these recent discoveries have hardly led to improvement in patient outcomes. Except for allogeneic stem cell transplantation as a potentially curative treatment, few treatment options are available. Among them, DNA methyltransferases inhibitor azacitidine has been widely used against MDS and AML with proven efficacy in overall survival improvement, but subsequent recurrence is inevitable even after marked response. In this study, we searched genomic mutations associated with azacitidine resistance in high risk MDS patients by next generation sequencing, using longitudinally collected samples from patients undergoing azacitidine treatment in our institution. Mutational analysis was performed by whole exome sequencing in 4 consecutive cases, with patients' BM samples taken at the point of diagnosis, during treatment, after relapse. Control sequencing was performed using T cell fraction from during-treatment samples. We verified variant allele frequency of the detected mutations by targeted deep sequencing, to identify gene mutations strongly related to clinical azacitidine resistance.

We identified an acquired gene mutation of MAP4K2, which is a member of the serine/threonine protein kinase family and activated by TNF-α/TRAF2 signal. MAP4K2 is required for the efficient activation of JNKs by stress-induced pathway. The mutation was exclusively found in the region encoding the protein-binding domain, which is required for signal transduction of JNK and p38 pathway by binding MAP3Ks such as MEKK1. We introduced the mutated form of MAP4K2 retrovirally into the human AML cell line Kasumi-1, and assessed its impact on azacitidine resistance. We found that the MAP4K2 mutation recaptured azacitidine resistance without affecting proliferation in the absence of azacitidine. In addition, another AML cell line HL60 with MAP4K2 mutation demonstrated higher growth ratio compared with control vector or MAP4K2 wild type after azacitidine treatment. Moreover, Kasumi-1 cells harboring MAP4K2 mutation showed low apoptosis rate and increased colony forming capacity after azacitidine treatment. We knocked in the mutations into MAP4K2 alleles Kasumi-1 cells using CRISPR/Cas9 system and found that Kasumi-1 cells with heterozygous MAP4K2 mutation similarly showed azacitidine resistance compared with control cells.

We examined whether associated with stress-induced cytokines are associated with MAP4K2 mutation-induced azacitidine resistance. We revealed that MAP4K2 mutation increased TNF-α expression after azacitidine treatment. In addition, the mutant-form of MAP4K2 exhibited enhanced stability compared with the wild type. These results imply that MAP4K2 mutation we detected in this study is gain of function mutation that confers altered cytokine signaling and growth advantage on MDS cells by stabilizing MAP4K2 protein.

In summary, we found that MAP4K2 mutation in MDS patients who have lost azacitidine responsiveness. Mutant MAP4K2 played a causative role in azacitidine resistance. Targeting MAP4K2 has become an attractive therapeutic strategy to conquer azacitidine resistance in MDS.