We have recently shown that lower CAT expression identifies CLL patients with an indolent clinical course while higher CAT levels are associated with an aggressive disease . In this study, we show that the rs1001179 SNP T allele in the CAT promoter is associated with higher CAT levels in CLL cells and provides binding sequences for ETS-1 and GR-β transcription factors. Moreover, methylation of the CpG Island II in the CAT promoter, likely driven by the DNMT1 enzyme, is a further crucial element in the regulation of CAT expression in CLL. Remarkably, statistical linear models suggest that the rs1001179 T allele and CAT promoter methylation cooperate in regulating CAT expression. The key advance of this study is to identify genetic and epigenetic mechanisms at the basis of the differential expression of CAT in CLL subsets.
Herein, we show that CLL cells express higher CAT mRNA and protein levels than normal B cells, thus confirming and extending previous data . Moreover, we document that higher mRNA and protein CAT expression identifies a subset of treatment-naïve patients with a faster disease progression, thus validating our previous findings in an independent set of patients and extending the results also at the protein level. Differential CAT expression in CLL supports the existence of two main disease subtypes characterized by a disparity in clinical outcome, probably as a consequence of differences not only in underlying genetic lesions, epigenetic changes, activated signaling pathways, and interactions with the microenvironment, but also in the redox machinery. Therefore, the elucidation of mechanisms regulating CAT expression in CLL is of preeminent importance to unveil mechanisms of disease and to develop strategies for improving its clinical management. In this study, we focus on the rs1001179 SNP in the CAT promoter, since it is associated with altered CAT expression [22–24]. In-silico alignment sequence analysis of the region in close proximity to the rs1001179 SNP shows several conserved sequences among phylogenetically related species, with a higher percentage identity among primates, suggesting that this region plays a fundamental role in the CAT gene expression regulation. In line with the putative functional role, CLL cells harboring the rs1001179 SNP T allele exhibit higher average CAT mRNA levels compared with cells bearing the wild-type C allele. This finding is in accordance with previous studies showing an association between the rs1001179 SNP T allele and higher CAT levels in normal peripheral blood cells [22–24]. Moreover, a possible correlation between the rs1001179 SNP in the CAT promoter and susceptibility to disease has been suggested in prostate cancer and hepatocellular carcinoma [33–35]. In contrast, the rs1001179 SNP is not a risk factor for non-Hodgkin lymphoma development . Taken together, these data point to genetic polymorphism as a possible mechanism underlying the heterogeneous expression of CAT associated with variable CLL clinical behavior. However, we do not document an association between the rs1001179 SNP and clinical progression, measured as TTFT (data not shown). This finding could be explained by the multifactorial pattern of CAT expression regulation in cancer, which include not only genetic but also epigenetic changes and transcriptional regulation [15, 37].
The in-silico prediction of TF-binding sites indicates that the rs1001179 SNP in the CAT promoter lies on a putative consensus sequence for specific TFs involved in the regulation of CAT expression. This analysis predicts a putative binding sequence for TFII-1 and GATA-1 in presence of the C allele, and for STAT4, ETS1 and GR-β in presence of the T allele. While previous in-silico analyses have already predicted the binding of GATA-1 and TFII-1 to the rs101179 SNP C allele [22, 38], and of STAT4 to the rs101179 SNP T allele , the putative binding of GR-β and ETS1 to rs101179 SNP has never been predicted so far. In this study, we validate the binding of GR-β and ETS-1 to the CAT promoter harboring the T -but not the C- allele. GRs can either directly bind canonical GC response elements (GREs) or act through indirect "tethered" interaction with other TFs, mediating transactivation or transrepression . Moreover, several ChIP-seq studies also showed that GR can bind sequences that differ from canonical binding sequences, directly or indirectly, via other TFs [39–41]. Taken together, these data suggest that GR-β could directly bind the CAT promoter bearing the T allele, thus competing with ETS-1 or, alternatively, it can indirectly bind the promoter through a "tethered" interaction with ETS-1. GR-transcriptional programs exert effects on apoptosis, metabolism, and inflammation, often in collaboration with other TFs [42–44]. ETS1 is the major extracellular signal-regulated kinase 1/2 (ERK1/2) downstream effector [45, 46]. Interestingly, higher ERK1/2 activation identifies CLL patients with a faster disease progression [47, 48]. The findings that CLL patients with a more aggressive disease are characterized by higher CAT levels  and ERK1/2 activation [19, 49], together with data on the function of rs1001179 T as a binding sequence for ETS1, could be suggestive of a possible role of the ERK1/2-ETS1 pathway in the transcriptional regulation of CAT that deserves to be further investigated.
This study also shows that CLL cells exhibit lower CAT promoter methylation compared with normal B cells, which could reflect the massive DNA hypomethylation that characterize CLL cells . Moreover, while in normal B cells the methylation degree of CpG sites positively correlated with each other, in CLL cells we show an overall lower or even negative correlation of methylation levels among the CpG analyzed sites. Overall, methylation has been described as a well-regulated, non-random process throughout the genome and, based on this regulated process, closer neighboring CpG sites are more likely to share the same methylation status . Thus, this leukemia-specific methylation pattern suggests that the co-methylation process between nearby CpG sites may be dysregulated in the CAT promoter of CLL cells. Moreover, methylation of the CpG Island II of the human CAT gene promoter negatively correlates with CAT mRNA levels. Remarkably, inhibition of DNA methyltransferase in CLL cells induces augment of CAT mRNA levels, thus functionally validating the role of methylation in regulating CAT gene expression in CLL.
The expression of DNMT1 resulted significantly reduced in CLL cells compared with HD B cells, reflecting the lower methylation levels within the CAT promoter shown by CLL versus HD B cells. In addition, DNMT1 expression level inversely correlated with CAT expression in CLL, highlighting its role in modulating methylation of the CpG Island II in CAT promoter. Therefore, these results identify DNMT1 as driver of differences in methylation levels underlying catalase expression.
Using statistical linear models, we show that CLL cells carrying the rs1001179 SNP T allele also exhibit a lower CpG Island II methylation in the CAT promoter and a higher CAT expression. This finding suggests that methylation of the promoter region encompassing the rs1001179 SNP could modify the effects of this SNP on CAT expression in leukemia cells, for example influencing the binding affinity of TFs to DNA sites, as reported for other genes [51–53]. Indeed, some transcription factors preferably bind hypermethylated DNA while others are inhibited by hypermethylated CpG sites . Herein, we also show that ETS-1 can bind the CAT promoter in presence of rs1001179 SNP T allele, which in turn results associated with higher CAT levels in CLL cells but not in HD B cells. Interestingly, DNA binding of ETS-1 is known to preferably bind hypomethylated DNA . Taken together, these data could account for the finding that rs1001179 SNP does not influence CAT expression in HD B cells, which are indeed characterized by higher CAT promoter methylation levels, compared with leukemic cells. Remarkably, SNPs can also influence the methylation status of surrounding CpG sites operating as a cis-acting factor for methylation of adjacent CpG sites [30, 55]. Therefore, the potential interactions of these regulatory mechanisms can alter the binding of TFs to DNA in an allele-specific manner, thus playing a role in disease risk and cancer progression. However, further investigations are required to address the mechanisms of this interaction and their effects on leukemia progression.
In conclusion, our data advance the knowledge on the role of genetic and epigenetic mechanisms controlling CAT expression in leukemia. Future challenges are to design therapeutics strategies targeting CAT regulatory pathways that could implement the effectiveness of current therapies and overcome drug resistance in CLL.