Genome-wide DNA methylation analysis pre- and post-lenalidomide treatment in patients with myelodysplastic syndrome with isolated deletion (5q)

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a biologically and phenotypically distinct subtype of MDS. Clinically, it is often characterized by severe macrocytic anemia and normal, or elevated, platelet counts. Patients with MDS del5q show a good overall survival and lower risk of evolution to acute myeloid leukemia (AML) compared to most MDS subtypes [1]. Furthermore, several large studies demonstrated an excellent response to the immunomodulatory drug lenalidomide for patients with MDS del5q [2‐4].

The German MDS study group conducted a prospective, open-label, non-randomized, single-arm, multicenter, phase II trial to evaluate the safety of lenalidomide and identify patterns of disease progression [5]. Due to the strict inclusion criteria and rigid screening process, the study population was very homogenous. As expected, two-thirds of patients achieved sustained transfusion independence. However, progression to AML was observed in 15% of patients. This was not predictable by any clinical marker and raises the question of feasibility for potential combination therapies in addition to lenalidomide alone [5].

The mechanism of action of lenalidomide in MDS del5q has just recently been better understood [6]. As MDS del5q is hypothesized to be a disease of haploinsufficiency [7, 8], the specific sensitivity to lenalidomide is partially explained through its suppression of haplodeficient proteins by a cereblon-dependent increase of ubiquitination and degradation [9]. Additionally, lenalidomide has direct antiproliferative effects by inhibiting CDC25C and PP2A phosphatase levels which leads to G2/M-phase cell cycle arrest. It also induces T-cell and NK-cell proliferation and cytotoxic activity, resulting in immunomodulation [10‐12].

Altered methylation of the genome is a known pathogenetic mechanism of MDS and has been shown to contribute to progression to AML [13‐15]. Hypomethylating agents (HMA) are therefore widely used in the therapy of high-risk MDS and AML with myelodysplasia-related changes. HMAs are potential candidates for combination with lenalidomide in MDS del5q and are undergoing clinical evaluation. Whether lenalidomide itself has an effect on the methylation patterns of MDS patients is unknown. One recent study in multiple myeloma cell lines showed that DNA hypermethylation might be a mechanism of resistance to lenalidomide treatment [16]. This prompted us to evaluate the methylation status of patients with MDS del5q before and after uniform treatment with lenalidomide. We sought to answer the questions whether lenalidomide treatment alters methylation patterns in MDS, and whether methylation status can predict response to lenalidomide treatment and outcome of patients with MDS del5q.

We present a comprehensive genome-wide DNA methylation analysis on 51 MDS del5q patients. This represents the largest cohort of MDS del5q patients analyzed for methylation so far. In an earlier analysis by Zhou et al. [15] on 20 MDS patients, only one patient with del5q was included and, interestingly, this patient clustered with the healthy controls rather than the other MDS subtypes. In a larger, more recent study, Reilly et al. [21] analyzed 10 patients with del5q in their cohort of 141 MDS patients and found genes in the respective cluster were mostly hypermethylated; especially, the genes WT1, NBEA (Neurobeachin), and AP1M2 (Adaptor Related Protein Complex 1 Subunit Mu 2) were significantly differentially methylated. All three genes were also found to be significantly differentially methylated in our cohort. Of note, WT1 hypermethylation was associated with inferior survival in their study.

The main focus of the present study was the comparison of global methylation prior to and after treatment with lenalidomide. For this, we were able to obtain paired samples from 17 patients. As expected in this very homogenous patient population, we found a very homogenous methylation pattern across patients before the start of therapy. Comparing pre- and post-treatment samples, we found no relevant changes in methylation. This observation was independent of whether patients had achieved a cytogenetic remission. On one hand, this highlights the consistency of our sample and data processing. On the other hand, it shows that methylation patterns seemed to be mostly independent of the size of the del5q clone and lenalidomide treatment had little effect on the genome-wide methylation status of the patients’ bone marrow cells in this study. Lenalidomide does not seem to exert its observed effect by inducing DNA methylation changes.

Despite the fact that the study population of the LE-MON-5 study was very homogenous due to the rigid screening process, there was still one third of patients who did not respond to lenalidomide treatment. Therefore, we next wanted to explore whether differences in the baseline methylation patterns between patients could help predict the response to lenalidomide treatment. Unsupervised hierarchical clustering showed a clear distinction of patients into two main clusters. Even though the percentage of responders in cluster B was higher than in cluster A, this difference was not significant and the methylation status not predictive of response. We additionally performed supervised clustering between responders and non-responders to lenalidomide treatment. The resulting heatmap showed no differences in methylation patterns. This was further confirmed by multiple t-testing on the sample M-values, which resulted in zero significantly differently methylated CpGs between responders and non-responders (data not shown). In contrast, there was a clear trend towards inferior survival for patients in cluster A. One potential explanation could be the higher number of patients harboring a mutation in TP53, which has previously been associated with inferior outcome in MDS del5q [22]. However, even after excluding patients with TP53 mutations from the analysis, the remaining patients still clustered into the same two clusters and there was still a trend towards inferior survival for patients in cluster A independent of TP53 mutations. Only half of the deaths in cluster A were due to progressive disease (only one of them in a TP53 mutant patient; the other three deaths were due to cardiac disease or unknown reasons) and PFS was in fact not significantly different between the two clusters.

To evaluate whether the methylation differences between the clusters were influenced by the significantly higher number of male patients in cluster A, we compared our data to previously published data on the influence of gender on the methylation of autosomes [23]. In contrast to the reported finding that male gender is associated with less methylation, cluster A was predominantly composed of hypermethylated CpGs. Additionally, none of the genes reported to be significantly altered by gender and only a very small fraction (around 5%) of the genes considered as potentially altered by gender in the study by Liu et al. [23] were among our most differentially methylated CpGs. Therefore, we conclude that the methylation differences observed between the clusters cannot be simply explained by gender distribution.

Analyzing the significantly differentially methylated genes leading to the clusters, we found that the majority of genes were hypermethylated in cluster A compared to cluster B. Among those genes, there were some interesting candidates whose aberrant methylation had been associated with inferior outcome in MDS in prior studies, e.g., WT1 [21], DLX6 (Distal-Less Homeobox 6) [24], members of the PI3K-Akt-mTOR and MAPK pathways [14] and, importantly, members of the Wnt signaling pathway. There have been several reports of a role of Wnt signaling in the pathogenesis of MDS del5q and its potential as a therapeutic target [25, 26]. Initially, it was observed by Liu et al. [27] that CTNNA1 (Catenin Alpha 1), a tumor suppressor gene encoded on chromosome 5q31, was expressed at lower levels in MDS del5q due to suppressed expression of the second copy by methylation of the gene promotor. Since then, several studies have reported aberrant methylation of Wnt family members in MDS including del5q underlining the potentially important role of the regulation of this pathway [14, 28‐30].