Nishi et al a b have developed a

Nishi et al. (2014a, 2014b) have developed a method of inducing cancer stem-like the following site (CSCs) through the reprogramming and partial differentiation of the immortalized but otherwise normal MCF10A human mammary epithelial cell line. To experimentally test our computational model of oncometabolic nuclear reprogramming, we similarly employed the MCF10A cell line and an isogenic derivative endogenously heterozygous for the R132H mutation of isocitrate dehydrogenase 1 (IDH1) gene, generating 2HG (Grassian et al., 2012). Quantification of intracellular 2HG showed that the levels of the oncometabolite were more than 30-fold higher in cell lysates from the knockin MCF10A IDH1 cells, confirming neomorphic IDH1 enzymatic activity (Figure 2A). To corroborate that the sole accumulation of 2HG in an otherwise isogenic background was sufficient to significantly impair histone demethylation, we examined the pattern of histone lysine methylation in IDH1 knockin and parental cells. Western blot analysis showed that the global levels of H3K4me3, H3K9me3, and H3K27me3 were increased in 2HG-overproducing MCF10A knockin cells compared with IDH1 parental cells (Figure 2A). These results were consistent with 2HG-induced broad inhibition of histone demethylation, showing agreement with previous models overexpressing IDH mutants (Duncan et al., 2012; Lu et al., 2012). We then employed a commercially available ELISA-based global DNA methylation able to indirectly provide a global measurement of 5-methtylcytosine (5-mC) levels in genomic DNA obtained from IDH1 and IDH1 knockin cells. Measurement of 5-mC levels of long interspersed nucleotide element 1 (LINE-1) confirmed that LINE-1 methylation significantly increased in 2HG-overproducing IDH1 cells when compared with IDH1 parental cells. Remarkably, a 2-day treatment with the selective R132H-IDH1 inhibitor AGI-5198, which fully suppressed 2HG to background levels (Figure 2A), partially reverted LINE-1 hypermethylation in IDH1 cells. Because it could be argued that 2HG-induced chromatin reorganization might promote the pluripotency-associated genes transition from an inactive to an active stage, we assessed whether the overproduction of 2HG promoted the expression of pluripotency regulators in normal breast epithelial cells. Flow cytometry analyses confirmed that the baseline expression of the core transcription factors OCT4 and SOX2 remained essentially unaltered in IDH1 knockin cells compared with parental IDH1 cells (Figure 2B). A preliminary evaluation of the top ten most significant Gene Ontology (GO) “molecular function” and “biological process” term annotations overrepresented in the 290 differentially hypermethylated CpG sites that were identified in IDH1 knockin cells (Figure 2C) strongly suggested that 2HG had a significant impact on the transcriptional repression of differentiation programs. Intracellular accumulation of the oncometabolite 2HG due to the heterozygous expression of the IDH1 allele is therefore sufficient to notably alter global histone lysine methylation without varying the baseline expression of the most critical reprogramming factors (i.e., OCT4 and SOX2) but altering expression of differentiation genes, thus providing an idoneous experimental model to validate the stochastic predictions of our biomathematical model in vitro. MCF10A IDH1 and MCF10A IDH1 cells were then transduced with OCT4 and SOX2 (hereafter called OS) to examine whether endogenously produced 2HG could substitute for combinations of stemness factors (i.e., KLF4 and c-MYC) to reprogram MCF10A mammary cells into iPS-like (iPSL-10A) cells. At day 15 after infection, MCF10A IDH1 cells growing on feeder layers showed a >10-fold increase in reprogramming efficiency relative to MCF10A IDH1 cells, as assessed by counting the number of alkaline phosphatase (AP)-positive (AP+) colonies (Figure 3A). We found that the colonies identified by the highly AP+ criterion were also positive for strong endogenous expression of NANOG (data not shown), which was considered a characteristic of bona fide iPSL-10A cells.