Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy

Abstract

Background: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated.

Methods: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression.

Results: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression.

Conclusions: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Figure 1.. Increased SALL4 Expression in Patients with Myelodysplastic Syndrome after Treatment with a Hypomethylating Agent.

Panel A shows a waterfall plot of the log₂ factor change in SALL4 expression in 25 patients after four cycles of azacytidine treatment in cohort 1. Panel B shows a waterfall plot of the log₂ factor change in SALL4 expression in 43 patients after three to five cycles of treatment with a hypomethylating agent in cohort 2. The term t0 denotes before any cycles of treatment, and t4 after four cycles of treatment. Panel C shows overall survival among patients with SALL4 up-regulation and those with SALL4 down-regulation in cohort 1, and Panel D shows the corresponding data in cohort 2. In Panels C and D, tick marks indicate censored data. A log-rank test was used to compare survival curves for statistical significance in Panels C and D.

Figure 2.. Demethylation of a Critical CpG Island and SALL4 Expression in Leukemic Cells Treated with CRISPR-DiR.

Panel A shows the CpG region within SALL4 5′ untranslated region (5′UTR)–Exon 1–Intron 1. Depicted at the top of the diagram are the 30 CpG residues; depicted below are the four SALL4 exons (in blue) and introns (in purple), with the location of the 30 CpG residues in the Exon 1–Intron 1 region. The location of the target of the guide RNA for CRISPR-DiR (clustered regularly interspaced short palindromic repeats–DNA methyltransferase 1 [DNMT1]–interacting RNA) is shown below, targeting CpG number 11. The term dCas9 denotes nuclease-dead Cas9, and sgDiR single-guide DNMT1-interacting RNA. Panel B shows bisulfite sequencing after CRISPR-DiR of HL-60 cells transduced with either a nontargeting negative control guide RNA (DiR_NT) or a targeting guide RNA (DiR_SALL4), leading to demethylation, with higher methylation indicated by redder shading. Panel C shows up-regulation of SALL4 transcript (copies per cell assessed by droplet digital polymerase-chain-reaction [ddPCR] assay) in HL-60 cells treated with CRISPR-DiR 8 days after transduction of the CRISPR-DiR. A Wilcoxon rank-sum test was used to evaluate the statistical difference in copies per cell between NT (in three patients) and sgSALL4 (in three patients). Data are shown as means, and error bars indicate the standard deviation.

Figure 3.. Demethylation of a Critical CpG Island and SALL4 Expression in Leukemic Cells and Patients with Myelodysplastic Syndrome Treated with a Hypomethylating Agent.

Panel A shows SALL4 copies per cell assessed by ddPCR assay in HL-60 cells treated with decitabine. A Wilcoxon rank-sum test was used to evaluate the statistical difference in copies per cell between dimethylsulfoxide (DMSO) and decitabine treatments. Data are shown as means, and error bars indicate standard deviations of three biologic replicates. Panel B shows Western blot analysis of HL-60 cells treated with decitabine. Panel C shows methylation profiling in HL-60 cells treated with vehicle alone as compared with those treated with decitabine at a dose of 250 nmol per liter. Treatment with a hypomethylating agent leads to demethylation of SALL4 in patients with myelodysplastic syndrome. Panel D shows average methylation across SALL4 5′UTR CpG loci (1 through 11) in patients with myelodysplastic syndrome before any cycles of azacytidine treatment (t0) and after four cycles of such treatment (t4). Red dots indicate patients with up-regulation of SALL4 expression after azacytidine treatments, and blue dots indicate patients with down-regulation of SALL4 after azacytidine treatments. Methylation values at t0 were compared with methylation values at t4 in each up- and down-regulated group separately. The average difference in methylation between the two time points was calculated with the use of a Wilcoxon matched-pairs signed-rank test (one-tailed) for each up- and down-regulated group separately. Panel E shows methylation changes in six patients with SALL4 up-regulation and nine patients with SALL4 down-regulation at t0 and t4.