Mutation Profiling Reveals Novel Epigenetics and Splicing Mutations in Chemorefractory Pediatric AML

Despite substantial improvement of treatment outcome in pediatric acute myeloid leukemia (AML), chemotherapy resistance remains to be a major concern. While molecular basis behind therapeutic failure was intensely interrogated, genomic alteration of chemotherapy resistance remains poorly characterized in pediatric AML. Here, we performed whole exon sequencing (WES) to identify gene mutations associated with primary and secondary chemo-resistance in pediatric AML.

Taking advantage of the available WES dataset from pediatric AML patients in two hospital centers, we compared mutation frequency of total 45 pediatric AML cases, including 24 cases of induction remission and 21 cases of chemo-resistance, to that of a cohort of 200 adult AML in TCGA dataset. We observed mutation frequency of signaling molecules were much higher in pediatric AML than in adult AML. The most frequent mutated genes are NRAS (27%, 12/45) and KIT (20%, 9/45) in pediatric AML, which has significant higher mutation frequency than in adult AML (NRAS, P=0.0002; KIT, P=0.0001). In consistent with previous findings, epigenetic regulators such as DNMT3A (0%, 0/45), TET2 (0%, 0/45), IDH1/2 (2%, 1/45) and ASXL1/2 (2%,1/45) are rarely mutated in pediatric AML patients, while they are commonly found mutated in adult AML. It's worth noting that we uncovered several novel epigenetic mutations which were not found in TCGA dataset, such as KDM5C, SIRT6, CHD4, SS18 and SETD8.

We assessed whether any mutated genes are associated with primary chemo-resistance group as comparing to primary chemo-sensitive group in studies cohorts. The chemo-sensitive group contained 24 primary induction remission samples, and 3 primary chemo-sensitive diagnosis samples of secondary chemo-resistance patients. Besides high recurrence of FLT3-ITD, we observed none of signaling molecule mutations are significantly enriched in primary chemo-resistance group. It's been reported that KIT mutations are frequently mutated in pediatric AML, and associated with adverse outcome. In our studied cohort, KIT mutations are more frequently found in relapsed AML (57%, 4/7), than primary chemo-resistance (28%, 2/7). Thus, poor clinical outcome of KIT mutations is more likely attributed to disease recurrence but not primary chemo-resistance.

Furthermore, epigenetic mutations were enriched at primary chemo-resistance group (7/18 vs 2/27, P=0.009), including histone modified enzyme KDM5C, SIRT6, SETD8, splisomal factors PRPF6, PRPF8 and other epigenetic regulators CHD4, and IDH2. We found high allele frequency of KDM5C point mutation (35%) and SIRT6 frameshift mutation (42%) in one chemorefractory t (8:21) translocated AML patient. In addition, we also identified splicing mutation PRPF6 and PRPF8 in chemorefractory AML patients. In light of recent discovery of epigenetic alteration mediating chemo-resistance in adult AML, we thought these epigenetic mutations found in our cohort may also play a critical role in chemo-refractory pediatric AML.

The development of new treatment strategies for pediatric AML must be based on an improved understanding of the molecular mechanisms underlying chemotherapy resistance. Previously, Fiona et al, shown that SETBP1 and ASXL1 and RELN mutations were associated with primary chemo-resistance in adult and pediatric AML patients by utilizing targeted sequencing of 670 genes. We have, for the first time, performed whole exon sequencing into 21 pediatric chemo-resistance AML. We identified novel epigenetic and splicing alterations such as KDM5C, SIRT6, SS18, CHD4 and PRPF8, PRPF6, which may play critical role in leukemogenesis and chemo-resistance in pediatric AML patients. Taking together, our study shed light into genetic lesions and evolution patterns underlying chemotherapy resistance and failure in pediatric AML. Larger cohort of patients and functional studies are needed to further understand molecular spectrum underlying chemo-resistance in pediatric AML.