Two Novel Distinct Subtypes of Myeloid Neoplasms Molecularly Associated with Histone H3K36 Methylations

Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia.  A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia.

In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets.

SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2-mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004).

ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36.

We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies.