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The hydroxymethylome of multiple myeloma
cohort of patients at relapse treated by bortezomib mono- therapy (Mulligan cohort, Online Supplementary Figure S10A). Conversely, no significant correlation was identi- fied between FAM72 expression and survival in a cohort of patients at relapse treated by dexamethasone mono- therapy (Online Supplementary Figure S10B) and between FAM72 expression and in vitro HMCL response to melpha- lan, lenalidomide or dexamethasone (not shown). Of partic- ular interest, high FAM72 expression tended to be signifi- cantly correlated with a better response to panobinostat (n=10, P=0.061; Online Supplementary Figure S10C), sug- gesting that HDACi could have a therapeutic interest in FAM72high myeloma patients associated with poor sur- vival. Since a combination of both HDACi and DNMTi has been shown to reprogram HMCL cells,45 we next investigated the ability of the combined DNMTi and HDACi decitabine (5aza-dC) and trichostatin A (TSA) to regulate FAM72 and FOXM1 expression in HMCL. Data indicated that the combined treatment reduced, although to different extent, the expression of both genes in a majority of investigated cell lines (Figure 5B). Finally, pri- mary MM cells were treated with a similar combination (5aza-C and the HDACi SAHA) and their resistance to these drugs was inversely correlated to the expression of FAM72 and FOXM1 (Figure 5C and Online Supplementary Figure S10D). When considering FAM72high/FAM72low and FOXM1high/FOXM1low expressing cells, only FAM72high samples remained significantly correlated with higher sen- sitivity to the HDCAi/DNMTi combination (Figure 5C). These results suggest that high-risk patients overexpress- ing FAM72 could benefit from treatment by a HDACi/DNMTi combination.
Discussion
Collectively, our data point to a prominent role of DNA demethylation events occurring at 1q21.1, and specially at the FAM72D locus, in MM biology and malignancy. One of the most common genetic features in MM linked to high-risk prognosis is the gain (three copies) or amplifica- tion (four and more copies) of part or all of the q arm of chromosome 1.46 Among the partial gains of 1q, 1q21 is particularly detected both in newly diagnosed (30%) and relapse (70%) cases. However, there is still no unifying picture explaining the functional role of the 1q arm ampli- fication and its wide occurrence among high-risk patients. Interestingly, MM-like 1q alterations can be experimental- ly triggered by inhibiting DNA methylation. Indeed, 5aza- dC treatment of activated B cells leads to a decondensa- tion of the 1q12 pericentromeric chromatin, a process that might enable local rearrangements of the 1q arm.47 Although DNA methylation levels are generally low in MM cells, residual methylation accumulates at specific sites17 and can still be erased by active demethylation through TET enzymes. Such mechanisms could play a role in disease progression provided that TET are targeted to the pericentromeric region of chromosome 1 and its adjacent region 1q21. Consistent with this idea, we show that one fourth of the genes associated with MM-specific hydroxymethylated regions lie within the 1q21.1 region. Among them, FAM72D, a gene encoding for a protein of unknown function, was shown here to enhance prolifera- tion of MM cells. Whereas most mammals have only one copy of the FAM72 gene, due to recent duplication events, four genes encode the human FAM72 proteins A to D.39 The FAM72 genes are known to be overexpressed in can-
Figure 6. 5-methylcytosine (5mC) oxidation at FAM72D enhancer and GAS2, DEPTOR and MYC super enhancers associate with multiple myeloma. 5mC oxidation in multiple myeloma (MM) cells occurs at super-enhancers (SE) and normal enhancers and as such participates in the establishment of an MM-specific transcription program and the maintenance of plasma cell identity. Several scenarios may lead to FAM72D overexpression in MM, including 1q21 gain/amplification, FOXM1 upregulation and DNA demethylation, and might combine in high risk MM to enhance survival and proliferation.
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