Downregulation Of The Wnt Inhibitor CXXC5 Predicts a Better Prognosis In Acute Myeloid Leukemia

Different CXXC zinc finger proteins like MLL, DNMT1 and TET1 have been identified to play a role in leukemia development. The CXXC family member CXXC5 is located on 5q31, a region recurrently deleted in acute myeloid leukemia (AML), suggesting that inactivation of CXXC5 might be involved in leukemogenesis. CXXC5 has previously been shown to exert tumor suppressor functions in solid tumor cells and to negatively regulate Wnt signaling, a known pro-survival pathway in leukemia. Here, we investigated prognostic and biological implications of CXXC5expression in AML.

CXXC5 mRNA expression was determined by real time quantitative PCR in pretreatment bone marrow samples of 186 AML patients enrolled on the Medical Research Council (MRC) AML15 and UK National Cancer Research Institute (NCRI) AML17 trials. We found downregulation of CXXC5 in AML compared to healthy controls, particularly in cases with del(5q)/-5, MLL rearrangements and t(8;21). To investigate CXXC5-associated gene expression, microarray data from 351 normal karyotype AML derived from the Microarray Innovations in LEukemia study and 529 unselected AML patients treated in Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group (HOVON/SAKK) trials were analyzed. CXXC5 was associated with expression of several genes implicated in leukemogenesis (WT1, GATA2, MLL, DNMT3B, RUNX1), indicating that alteration of CXXC5 expression is a downstream effect of different leukemic pathways. Data from the HOVON/SAKK cohort (median age 46 years (range 15-77) was further used to perform outcome analyses according to microarray-based CXXC5 expression. Patients with CXXC5 expression below the median level had a lower relapse rate (45% vs 59%; P=0.007) and a better overall survival (OS: 46% vs 28%; P<0.001) and event-free survival (EFS: 36% vs 21%; P<0.001) at 5 years. This was of independent significance in multivariate analyses after adjustment for age, white blood cell count, cytogenetic risk group, FLT3-ITD status, biallelic CEBPA mutations, as well as mutations of NPM1, DNMT3A and ASXL1.

DNA sequencing of CXXC5 exons 1-3 on 84 AML cases revealed no mutations, suggesting that CXXC5 is predominantly inactivated by gene regulatory mechanisms in AML. Pyrosequencing analysis showed that lower expression of CXXC5 in AML samples was correlated with hypermethylation within the promoter region. Treatment of NB4 and U937 cell lines with 5-Aza-2´-deoxycytidine led to demethylation of this region and induction of CXXC5 at the mRNA and protein level.

Functional analyses confirmed Wnt inhibitor function of CXXC5 in leukemic cells. K562 cells overexpressing pEGFP-C2-CXXC5 showed impaired upregulation of active β-catenin protein, decreased upregulation of Wnt target genes Axin2 and Survivin, as well as decreased Wnt reporter activity after Wnt3a stimulation. In addition, we found evidence for involvement of CXXC5 in the DNA damage-induced p53 response in leukemia. Knockdown of CXXC5in OCI-AML3 cells harboring wildtype p53 led to attenuated p53 upregulation and cell cycle arrest after γ-irradiation.

In summary, we have identified CXXC5 as a novel independent prognostic factor in AML. Our data suggest a tumor suppressor function of CXXC5 in AML and association of the gene with different leukemic pathways. These functional and prognostic implications build the framework for further investigating the role of CXXC5 in leukemia. AML patients with downregulation of CXXC5 might be candidates for evaluation of Wnt inhibition and cases with epigenetically silenced CXXC5 expression may particularly benefit from demethylating agents.