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M-M. Ji et al.
mutations (P=0.045 and P=0.224) (Figure 2C,D). Overall, these data expand the prognostic role of histone modifica- tion in disease progression in PTCL-NOS.
Histone modifier gene mutations sensitized T-lym- phoma cells to the histone deacetylase inhibitor chi- damide and/or the hypomethylating agent decitabine
A possible structure-function relationship of the mutants was addressed using the crystal structure of the proteins encoded by KMT2D (PDB4Z4P) and EP300 (PDB4PZR), the two most frequently mutated histone modifier genes. As shown in Figure 3A, KMT2D R5389W, E5444K and V5486M might destabilize the SET domain and reduce histone methylation activity, EP300 E1377R, W1466_ and E1515V might disrupt the acetyl-CoA bind- ing pocket, destabilize the HAT domain and reduce his- tone acetylation activity. Next, representative missense mutants KMT2D (V5486M) and EP300 (H1377R), as well as WT KMT2D and EP300, were established and trans-
fected into Jurkat cells. Compared with WT protein, while KMT2D mutant reduced the level of H3K4me3, this reduction was restored by the HDAC inhibitors romidepsin and chidamide (Figure 3B and Online Supplementary Figure S2). On the other hand, the level of H3K18ac was reduced in EP300 mutant, but this effect was restored by the HDAC inhibitors valproic acid, suberoylanilide hydroxamic acid, or romidepsin and chi- damide (Figure 3C). These results were observed by west- ern blot and by immunofluorescence assay (Figure 3B,C).
In tumor samples from PTCL-NOS patients, a signifi- cantly lower fraction of nuclear H3K4me3 positivity (+++~++++, 30%) was observed in cases with the KMT2D mutation than in those without mutations. Similarly, a lower fraction of nuclear H3K18ac positivity (+++~++++, 17%) was present in cases with EP300/CREBBP mutations than in those without muta- tions (Figure 3D). Interestingly, upon treatment with chi- damide (administered orally at a dose of 30 mg twice per
Table 1. Multivariate analysis of predictors of progression-free survival in patients with PTCL-NOS controlled by International Prognostic Index.
Variable
International Prognostic Index
Low & low/intermediate vs. intermediate/high & high risk
Histone modifying gene mutations
positive vs. negative
RR. relative risk; 95% CI: confidence interval.
Training Cohort Validation Cohort
RR 95%CI Pvalue RR 95%CI Pvalue
4.209 2.148-8.250
3.633 1.885-7.005
<0.001 2.013
<0.001 2.017
1.178-4.213
1.062-4.414
0.033
0.049
AB
CD
Figure 2. Progression-free survival and overall survival curves of patients with peripheral T-cell lymphoma not otherwise specified according to histone modifi- er gene mutations. (A) Progression-free survival and (B) overall survival curves of the training cohort. (C) Progression-free survival and (D) overall survival curves of the validation cohort.
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