U2AF1 Mutations in S34 and Q157 Create Distinct Molecular and Clinical Contexts
Authors: Adema Vera , Hirsch Cassandra M. , Przychodzen Bartlomiej P , Nazha Aziz , Kuzmanovic Teodora , Negoro Eiju , You Dewen , Makishima Hideki , Clemente Michael J. , Carraway Hetty E. , Sekeres Mikkael A. , Visconte Valeria , Maciejewski Jaroslaw P. 
Published: October 2019

Abstract
Background: U2AF1 forms a heterodimer for the recognition of the 3' splice site during pre-mRNA splicing. Somatic U2AF1 mutations are present in approximately 10% of MDS patients. Most U2AF1 mutations are recurrent at 2 highly conserved hotspots, while non-canonical mutations are rare. U2AF1S34 and U2AF1Q157 mutations map within the zinc finger domains of the protein, resulting in distinct downstream effects. We have previously shown that U2AF1Q157 mutant patients have distinct splicing patterns compared to U2AF1WT with a set of misspliced targeted genes, including ARID2 and EZH2. In contrast, recent work focusing on S34 suggests a distinct subset of misspliced genes, including ATG7 (Park SM, Molecular Cell, 2016). The biological and clinical implications of these 2 distinct mutations are unknown. We investigated the differences between these mutations with respect to clinical outcomes and molecular background, including their impact on clonal architecture.
Methods: We first collected molecular and clinical data on a cohort of 1700 patients with myeloid neoplasms (median follow up 1.0 year, range 1-5 years), median age was 65 years (range, 11-93). Targeted deep sequencing of a panel of frequently mutated genes (64) was applied. Our analyses included somatic mutational patterns, clonal hierarchy, and mutational correlation of the cohort of patients with U2AF1S34 and U2AF1Q157 and those without mutations in this gene. U2AF1 mutations were found in 5% (78/1700) of patients, all of them were missense and in a heterozygous configuration.
Results: Both mutations were equally distributed in the cohort: U2AF1S34 (45%, 35/78), and U2AF1Q157 (46%, 36/78). Other mutations (Q84, E124, E152, and R156) were detected at a lower frequency (9%). We then dissected the clonal hierarchy of both U2AF1 mutations and found that 44% (34/77) were ancestral while 56% (43/77) were secondary. In MDS, most U2AF1 mutations (77%, P=.002) were dominant, while subclonal U2AF1 mutations were evenly distributed between the subentities. U2AF1S34 or U2AF1Q157 were equally likely to be dominant (21% vs. 27%; ancestral events; P=.09, respectively). Similarly, S34 and Q157 mutant clones had similar median variant allele frequencies (3-52% vs. 8-64%).
U2AF1 S34 mutant cases had similar OS to patients carrying U2AF1Q157 (N=35 vs. N=36; 10 vs. 15 months; P=.209; LogR=.65). When we compared the impact of ancestral vs. secondary U2AF1S34 and U2AF1Q157 we found that MDS patients carrying ancestral U2AF1 mutations had a shorter OS compared to MDS patients carrying secondary U2AF1 patients (N=26 vs. N=18; 13 vs. 34 months; LogR=.04). Of note, ancestral U2AF1S34 patients had shorter OS compared to ancestral U2AF1Q157 patients (13 vs.11; 10 vs.15 months; P=.03; LogR=.86).
Given these differences, we also investigated the mutational spectrum of U2AF1MUT patients. Cross sectional analysis identified that the top genes mutated in the U2AF1 mutant cohort were: ASXL1 (26%), BCOR/L1 (15%), TET2 (13%), DNMT3A and PHF6 (12%), ETV6 (10%), RUNX1 and STAG2 (9%), and SETBP1 (8%). Transcriptional factor and DNA-methylation genes were predominantly mutated in U2AF1MUT patients (35% and 24%, respectively). Exploring the association between S34/Q157 vs. other gene mutations, S34 co-occurred with BCOR/L1 mutations (P=.007, 24%), while Q157 mutations co-occurred with ASXL1 (P=.003, 44%) irrespective of their rank in the clonal hierarchy. When S34 was the dominant mutation, secondary mutations included ETV6, BCOR, and CUX1. In contrast, when Q157 was the ancestral event, secondary mutations included ASXL1 and DNMT3A. Subclonal S34 occurred in the context of ancestral RUNX1, BCOR/L1, CUX1 and DNMT3A, while subclonal Q157 followed ancestral ASXL1, EZH2, PHF6 and TET2.
Conclusion: In sum, U2AF1S34 and U2AF1Q157, consistent with their differential missplicing consequences, create a distinct molecular milieu leading to differences in clinical outcomes.

Disclosures
Makishima: The Yasuda Medical Foundation: Research Funding. Carraway:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Celgene: Consultancy, Honoraria, Speakers Bureau; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.