Thalidomide and its derivatives, lenalidomide and pomalidomide (also known as IMiDs), have significantly changed the treatment landscape of multiple myeloma, and the recent discovery of cereblon ...(CRBN) as their direct biological target has led to a deeper understanding of their complex mechanism of action. In an effort to comprehend the precise mechanisms behind the development of IMiD resistance and examine whether it is potentially reversible, we established lenalidomide‐resistant (‐LR) and pomalidomide‐resistant (‐PR) human myeloma cell lines from two IMiD‐sensitive cell lines, OPM2 and NCI‐H929, by continuous culture in the presence of lenalidomide or pomalidomide for 4–6 months, until acquirement of stable resistance. By assessing genome‐wide DNA methylation and chromatin accessibility in these cell lines, we found that acquired IMiD resistance is associated with an increase in genome‐wide DNA methylation and an even greater reduction in chromatin accessibility. Transcriptome analysis confirmed that resistant cell lines are mainly characterized by a reduction in gene expression, identifying SMAD3 as a commonly downregulated gene in IMiD‐resistant cell lines. Moreover, we show that these changes are potentially reversible, as combination of 5‐azacytidine and EPZ‐6438 not only restored the observed accessibility changes and the expression of SMAD3, but also resensitized the resistant cells to both lenalidomide and pomalidomide. Interestingly, the resensitization process was independent of CRBN. Our data suggest that simultaneous inhibition of DNA methyl transferases and EZH2 leads to an extensive epigenetic reprogramming which allows myeloma cells to (re)gain sensitivity to IMiDs.
Using a cell line model for IMiD resistance, this study shows that epigenetic resensitization with simultaneous inhibition of DNA methyl transferases and EZH2 can restore sensitivity to IMiDs. In addition to acquired IMiD resistance, this combined epigenetic therapy was also effective in myeloma cells with primary resistance to IMiDs, and thus deserves further testing in a preclinical and clinical setting.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known ...about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders. MDS is frequently associated with deletions on chromosome 5q as well as aberrant DNA methylation patterns ...including hypermethylation of key tumor suppressors. We have previously shown that hypermethylation and silencing of the non-coding RNA VTRNA2-1 are correlated with poor outcomes in acute myeloid leukemia patients. In this study, we find that VTRNA1-2 and VTRNA1-3, both located on chromosome 5q, can be regulated and silenced by promoter DNA methylation, and that the hypomethylating agent 5-aza-2-deoxycytidine causes reactivation these genes. In normal hematopoiesis, we find that vault RNAs (vtRNAs) show differential methylation between various hematopoietic cell populations, indicating that allele-specific methylation events may occur during hematopoiesis. In addition, we show that VTRNA1-3 promoter hypermethylation is frequent in lower risk MDS patients and is associated with a decreased overall survival.
Spliceosome mutations are frequently observed in patients with myelodysplastic syndromes (MDS). However, it is largely unknown how these mutations contribute to the disease. MicroRNAs (miRNAs) are ...small noncoding RNAs, which have been implicated in most human cancers due to their role in post transcriptional gene regulation. The aim of this study was to analyze the impact of spliceosome mutations on the expression of miRNAs in a cohort of 34 MDS patients. In total, the expression of 76 miRNAs, including mirtrons and splice site overlapping miRNAs, was accurately quantified using reverse transcriptase quantitative PCR. The majority of the studied miRNAs have previously been implicated in MDS. Stably expressed miRNA genes for normalization of the data were identified using GeNorm and NormFinder algorithms. High-resolution melting assays covering all mutational hotspots within SF3B1, SRSF2, and U2AF1 (U2AF35) were developed, and all detected mutations were confirmed by Sanger sequencing. Overall, canonical miRNAs were downregulated in spliceosome mutated samples compared to wild-type (P = 0.002), and samples from spliceosome mutated patients clustered together in hierarchical cluster analyses. Among the most downregulated miRNAs were several tumor-suppressor miRNAs, including several let-7 family members, miR-423, and miR-103a. Finally, we observed that the predicted targets of the most downregulated miRNAs were involved in apoptosis, hematopoiesis, and acute myeloid leukemia among other cancer- and metabolic pathways. Our data indicate that spliceosome mutations may play an important role in MDS pathophysiology by affecting the expression of tumor suppressor miRNA genes involved in the development and progression of MDS.
•EMCN is a novel marker of human HSCs.•EMCN is a more specific marker of HSCs than CD34 as it can discriminate HSCs from lineage-committed HPCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The immunomodulatory drugs (also known as IMiDs) thalidomide, lenalidomide and pomalidomide, play a pivotal role in the treatment of multiple myeloma (MM). Recent studies have deciphered the ...mechanism of action of these drugs and shown that they directly interact with cereblon (CRBN), which is part of an E3-ubiquitin ligase complex. The IMiD-CRBN interaction selectively alters the affinity of CRBN to two important transcriptional factors, Ikaros (IKZF1) and Aiolos (IKZF3), leading to their ubiquitination and subsequent proteasomal degradation, representing a novel mechanism of therapy through ubiquitin-mediated alteration of gene expression.
Several studies have underlined the importance of CRBN for the biologic activity of IMiDs, as both human cell lines and patient samples with acquired IMiD-resistance, exhibit a significant reduction of CRBN expression. However, it still remains unknown how CRBN expression is regulated, and subsequently, whether the acquired resistance to IMiDs is potentially druggable and reversible.
In this study, we have used the IMiD-sensitive human myeloma cell lines OPM2 and NCI-H929, which were cultured with subtoxic doses of Lenalidomide and Pomalidomide for 4-6 months for acquisition of stable resistance. The lenalidomide- and pomalidomide-resistant cell lines (OPM2-LR, NCI-H929-LR and OPM2-PR, NCI-H929-PR subsequently) were then used as a model of acquired IMiD-resistance. In line with previous studies, we confirmed a significant reduction of CRBN expression, but not it downstream targets, IKZF1 and IKZF3, in the resistant cell lines, both at mRNA and protein level. Using methylation-specific melting curve analysis, we next showed that the downregulation of CRBN is not due to promoter DNA methylation. We therefore proceeded with the assessment of chromatin accessibility through nucleosome positioning using AcceSsIble. Our data show that acquired IMiD-resistance is accompanied by increased nucleosome occupancy (and thus decreased chromatin accessibility - figure 1A) rather than DNA methylation, an event that is also more prominent in promoters and enhancers (figure 1B). Among the top 100 genes whose promoters are nucleosome-occupied was CRBN, an event that could explain the reduced expression observed upon acquired IMiD-resistance.
Since both DNA methylation and nucleosome positioning are two potentially reversible epigenetic modifications, we next treated the resistant cell lines with a combination of decitabine (a DNA-methyl-transferase inhibitor) and GSK-126 (an EZH2-inhibitor) for 3 days and then exposed them again to IMiDs. Even though the combination treatment by itself was mildly toxic for the cells, cell viability was even more reduced upon treatment with IMiDs three days after exposure to this combination of epidrugs, suggesting that epigenetic reprogramming and resensitization of IMiD-resistant cells could be feasible (figure 1C).
Taken together, our data could potentially explain the mechanism behind CRBN downregulation observed upon IMiD-resistance and provide evidence for possible epigenetic resensitising myeloma cells to IMiDs.
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No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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