Functional and Biological Implications of CUX1 Mutations and Deletions in Myeloid Neoplasms

'," authors equally contribute

The CUX1 gene, located on 7q22.1, has been characterized genetically as a haplo-insufficient tumor suppressor gene. Somatic CUX1 mutations (CUX1^MT) and deletions (CUX1^DEL) have been reported in myeloid neoplasms.The p200CUX1 is the most abundant isoform, containing 4 DNA-binding domains (CR1, CR2, CR3, and HD) with rapid "on" and "off" DNA-binding kinetics. Depending on promoter context, the p110 isoform can be either a repressor or an activator. We aim to define the clinical and biological/functional relevance of CUX1 lesions in myeloid neoplasms.

We first established the clinical prevalence and phenotypic associations of CUX1 . In a cohort of 1480 patients with myeloid neoplasms including, lower-risk MDS (24%, n=359), higher-risk MDS (17%, n=249), pAML (22%, n=322), sAML (14%, n=205), MDS/MPN (15%, n=217) and MPN (9%, n=128), we correlated CUX1 lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and concomitant somatic mutations. We found that somatic CUX1^MT and CUX1^DEL were detected in 4% and 6% of our cohort, respectively. All CUX1^MT were heterozygous except in 6% were homozygous (uniparental disomy; UPD) and 7%, compounded heterozygous fashion. CUX1 is under expressed in 60% of MDS with -7/del(7q) and 70% of AML with -7/del(7q) vs. 18% and 8% of diploid controls (P=.004 and P <.0001,respectively). In addition, 15% of MDS and 5% of AML cases with cytogenetic abnormality other than -7/del(7q) showed low expression of CUX1 . Truncations and missense mutations were present in 25% and 75% of mutant cases, respectively. CUX1^MT were more common in MDS (52%) than AML(16%) and MDS/MPN ( 32%; P=.02), while CUX1^DEL more frequently exist in RAEB1/2 and sAML (64%) compared to lower-risk MDS (12%), pAML (12%) and MDS/MPN (14%; P <.0001). Regards concomitant genetic events, CUX1 lesions were significantly associated with TET2, ASXL1 and BCOR (P=.02, P=.004, P=.0009 respectively) compared to CUX1 wild type (WT). In contrast to WT which was mutually exclusive with FLT3 (3%). Clonal architecture analyses showed that 36% of CUX1 mutations were founder events with secondary BCOR (25%) and ASXL1 (17%) , while CUX1^MT were subclonal in 64% in which TET2 (22%), BCOR (9%), and SF3B1 ( 9%) served as ancestral events. Prognostic analyses showed that both CUX1^MT and CUX1^DEL were significantly associated with shorter overall survival compared WT (CUX1^MT: 56 vs. 94 mo.; P=.02; CUX1^DEL: 46 vs. 94 mo., P=.004) where truncating mutations had a significant impact on worse prognosis than WT (39 v s. 94 mo.; P=.01).

CUX1 functions as an auxiliary factor in base excision repair. Specifically, the CUT domains within CUX1 stimulate the enzymatic activities of the 8-oxoguanine DNA Glycosylase-1 (OGG1). We therefore verified whether genetic lesions of CUX1, either CUX1^MTor CUX1^DEL, delay oxidative DNA damage repair and thereby contribute to leukemogenesis by establishing a clonal mutator phenotype. First, we compared 8-oxoG cleavage activity in extracts from HCC1419 cells harboring CUX1 loss-of-heterozygosity (CUX1^LOH), Hs578T cells diploid for CUX1 and Hs578T cells expressing CUX1 shRNA. We observed decreased 8-oxoG cleavage in HCC1419 cells and Hs578T-shCUX1 cells in which CUX1 expression was reduced. Secondly, we obtained bone marrow cells from healthy donors and patients with frameshift CUX1^MTor del7q, and performed single cell gel electrophoresis (comet assays) to monitor DNA repair following exposure to H₂O₂. We found that repair of oxidized bases in genomic DNA was delayed in both samples with heterozygous and homozygous CUX1 inactivating mutations and in samples with CUX1^DEL. Having noted that CUX1 lesions cause a defect in BER, we performed whole exome sequencing of CUX1^MT (n=3) and WT (n=117). Strikingly, samples with either CUX1^MT or low CUX1 expression (n=23) harbored a significantly higher number of mutations compared to WT and high CUX1 expression samples (n=156; P=.03, P=.04).

Altogether, our results show that reduced CUX1 expression in myeloid cancer cells is associated both with a decrease in DNA repair efficiency and an increase in somatic mutations. We propose that genetic and epigenetic changes that reduce CUX1 expression cause a DNA repair defect that leads to the accumulation of somatic mutations and ultimately, shorter patient survival.