Abstract The HDAC-inhibitor sodium-butyrate, promotes differentiation of colon cancer cells as evidenced by induction of alkaline phosphatase (ALP) enzyme activity. Screening of a panel of 34 colon cancer cell lines identified cell lines responsive (44%) and refractory (56%) to butyrate-induced differentiation. Refractory cell lines were significantly enriched for those harbouring the CpG island methylator phenotype (CIMP) (P=0.016). The ALP gene family comprises 4 members - ALPi, ALPL, ALPP and ALPPL2 - each of which was induced by butyrate in responsive cell lines, demonstrating co-ordinate regulation of this gene family in response to HDACi treatment. Deletion analyses of the ALPi promoter identified a KLF/Sp1 cis element essential for butyrate-induction of ALPi promoter activity. Remarkably, we observed that a CpG dinucleotide within this site was preferentially methylated in refractory cell lines. Furthermore, the majority of cell lines with methylation of this site in the ALPi promoter also harboured methylation of similar sites within the ALPL and ALPP promoters, indicating coordinate methylation of this gene family in refractory cell lines. Frequent methylation and reduced expression of ALPi and ALPL were also observed in primary human colon cancers. Targeted inactivation of the DNA methyltransferases Dnmt1 and Dnmt3b (HCT116-DKO) and co-treatment with decitabine restored butyrate-inducibility of ALP in the methylated HCT116 cell line. However, butyrate-induction of an exogenous ALPi promoter-reporter paralleled induction of endogenous ALPi mRNA across the cell lines, suggesting ALPi promoter methylation may not be the primary determinant of ALP inducibility. We therefore explored whether the differential induction of the ALP gene family in colon cancer cells was due to selective induction of a common transcriptional regulator. To address this, responsive and refractory cell lines were treated with butyrate for 72h and transcription factors selectively induced in responsive cell lines identified by microarray. By focussing on factors which bind KLF/Sp1 regulatory elements, we identified KLF5 as selectively induced in responsive cell lines. KLF5 was also preferentially induced by butyrate in HCT116 DKO cells. Importantly, knockdown of KLF5 markedly attenuated butyrate-induction of ALP mRNA expression and enzyme activity. These findings demonstrate that the ALP gene family is co-ordinately inducible by butyrate in select colon cancer cell lines, in a KLF5-dependent manner. Cells lines refractory to butyrate-induction of the ALP gene family were enriched for CIMP high lines, and displayed coordinate promoter methylation. We propose that the coordinate methylation of this gene family in CIMP high colon cancers is likely linked to their coordinate regulation. Citation Format: John Mariadason, Azadeh Carr, Georgia A. Corner, Sheren Al-Obaidi, Anderly Chueh, Fiona Chionh, Lars Tögel, Naseem Ahmed, Diego Arango, Daniel Buchanan, Joanne Young, Leonard H. Augenlicht, Madhu S. Malo, Richard Hodin, Oliver M. Sieber, Thomas Weber, Joongho Shin. Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2013-2991