UTX mutations in Myeloid Neoplasms
Authors: Nagata Yasunobu , Radivoyevitch Tomas , Makishima Hideki , Hirsch Cassandra M. , Przychodzen Bartlomiej P , Kuzmanovic Teodora , Negoro Eiju , Clemente Michael J. , Mukherjee Sudipto , Carraway Hetty E. , Sekeres Mikkael A. , Maciejewski Jaroslaw P 
Published: October 2019

Genetic studies in myeloid neoplasms have revealed that somatic mutations and deletions frequently affect epigenetic regulation via DNA methylation and histone modification. One frequently affected epigenetic complex is polycomb repressive complex 2 (PRC2), which trimethylate Histone3Lysine27 (H3K27) to create a repression mark for the expression of a variety of genes that includes essential homeobox genes. Members of this complex include EZH2, EED and SUZ12, which exert methyltransferase activity, and perhaps indirectly also DNTM3A and ASXL1. The histone demethylase ubiquitously transcribed X (UTX) counters the enzymatic activity of PRC2 by removing di- and trimethyl groups from H3K27. It thus removes repressive marks. We were the first to report UTX mutations in a small portion of CMML and MDS cases. In another cohort, frequent somatic loss-of-function mutations in UTX were found in ALL. The goal of the present study was to understand the functional role of UTX and the consequences of its mutations on H3K27 methylation status, specifically with respect to their relevance to myeloid neoplasia.

A total of 1,979 patients with various myeloid neoplasms (n = 1,686) and other hematologic disorders (n = 293) were screened for gene mutations in UTXand other reported driver genes relevant to myeloid neoplasms using whole exome sequencing and/or targeted deep-sequencing. Expression array analyses were performed on 200 samples. In addition, we also used sequencing and expression data from the de novo AML TCGA repository.

UTXwas mutated in 55 (2.8%) cases out of 1,979 cases. The mutations were found in 2.5% of MDS, 3.1% de novo AML (including 8.3% CBF AML), 4.8% MDS/MPN, and 1.4% secondary AML (sAML). Of those, 77% were missense mutations and 23% nonsense, splice site and frameshift mutations, without an evident hot spot. The male-to-female ratio was 1.2 (55% vs. 45%). UTX gene expression was significantly higher in females than in males (p<.001). After gender adjustment and dichotomized using a threshold of expression of 2 standard deviations from the mean, low UTX expression levels were identified in 13/183 (7%) individuals. When we focused on clonal burden using variant allele frequencies (VAFs) to investigate clonal architecture within the tumor population, in 37 cases UTX constituted subclonal events and in 18 they were dominant.

We then examined the molecular context of UTX lesions. Average mutation burden in patients with UTX mutations was higher than in WT UTX carriers (4 vs. 1.5, p<.001). UTX mutations co-occurred with other driver genetic mutations such as ASXL1, ZRSR2, CUX1, NF1. Surprisingly, EZH2 mutations are also enriched in UTX mutated cases although they function in the opposite direction of H3K27 trimethylation. Focusing on dominant clone and subclonal events in cases with these two mutations, EZH2mutations were enriched in cases with dominant UTX clone. This suggests that they play important roles in the cancer's pathogenesis.

To clarify the role of UTX in the maintenance of leukemia, genomic knockouts of UTX were developed in human leukemic cell lines using the CRISPR-Cas9 system. RNA sequencing revealed that knockout cell enrichment for developmental regulators such as Hox genes. In addition, we made knockdowns of human CD34+ cells using short hairpin RNAs against UTX. The cells showed enhanced colony formation and increased replating efficiency consistent with retained clonogenicity.

The truncating pattern of UTX mutations in myeloid neoplasms suggests that they are loss-of-function hits. Missense mutations thus need to be confirmed. Functional analysis in vitro shows that low expression of UTX may have functionally equivalent consequences. If so, an additional 7% of patients may have low UTX expression, and may thus phenocopy patients with UTX mutations. Combined, a total of ~10% out of myeloid neoplasm patients may harbor UTX deficiency. Epigenetic modifying drugs related to H3K27 such as inhibitors of EZH2and histone deacetylases are in development. It is controversial to which patients they should be applied. Our findings could be key to a deeper understanding of epigenetic alterations, drug function, and response.

Disclosures

Makishima: The Yasuda Medical Foundation: Research Funding. Mukherjee:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.