Page 156 - 2022_01-Haematologica-web

S.J. Erkeland et al.
T-cell prolymphocytic leukemia show a relatively homogeneous gene expression profile with TCL1A amongst the highest upregulated genes
We then performed gene expression array analysis of T-PLL samples and normal T-cell subsets. A correlation plot and unsupervised clustering analysis of gene expres- sion showed that most T-PLL samples, with the exception of T-PLL21 and T-PLL24, and to a lesser extent T-PLL3, T- PLL22, T-PLL-25, clustered together; at the other end, nor- mal T-cell subsets showed a high correlation to each other (Figure 1A and B).
Next, we focused on CD4+ T-PLL, which is the major group within T-PLL, and compared their gene expression to that of normal CD4+ T-cell subsets. We found 2,282 probe sets differentially expressed (at least 2-fold up- or downregulated) in CD4+ T-PLL compared to normal CD4 subsets. Of the top 100 differentially-expressed probe sets in CD4+ T-PLL, the average expression of most probe sets was upregulated, including the T-PLL charac- teristic T-cell leukemia driving oncogene TCL1A (Figure 1C; Online Supplementary Table S5). Collectively these data showed that the protein encoding gene expression profile of T-PLL is clearly different from normal T-cell subsets.
A subset of microRNA is differentially expressed in T-cell prolymphocytic leukemia of which miR-141 and miR-200c show prognostic impact
In order to evaluate the role of small non-coding RNA in the T-PLL cohort, we then performed genome-wide miRNA expression profiling by small RNA sequencing of 21 T-PLL cases. Based on miRNA expression, all T-PLL cases clustered together in a principal component analysis (PCA) plot and were most similar to healthy effector CD4 cells (Figure 2A). Therefore, we then compared the expres- sion of individual miRNA in T-PLL with healthy effector CD4 cells. Strikingly, we did not find any miRNA signifi- cantly downregulated in T-PLL compared to normal effec- tor CD4 cells (false discovery rate [FDR]<0.05). However, we did identify a set of 35 miRNA that were abundantly expressed and significantly upregulated (FDR<0.05; fold change [log2FC] >1.5) in the T-PLL cohort compared to healthy effector CD4 cells (Figure 2B; Online Supplementary Table S6). These included miR-17-5p, miR-19b-3p, miR- 20a, miR-106b, miR-92a of the oncogenic miR-17~92 clus- ters and other well-known oncogenic miRNA, including miR-125a, that were also expressed at higher levels in T- PLL cases (Online Supplementary Table S6). miRNA of the miR-200c/141 cluster (FDR=0.011; average overexpression FC >55; range, 0.95-1,211) and the miR-181a/b cluster (FDR=0.025; average overexpression FC >20; range, 0.2-451), which were the highest overexpressed miRNA in T-PLL (Figure 2B; Online Supplementary Table S6). Notably, miR-141, miR-200c, miR-181a and miR-181b were signif- icantly overexpressed compared to other normal T-cell fractions (Figure 2C). Subgroups of T-PLL with pro- nounced overexpression of miR-200c/141 and/or miR-181a/b (T-PLL-1-5,10, 11, 16, 17, 21-25, 28) (range, 10-1,200-fold) significantly correlated with higher white blood cell counts (Figure 2D). Furthermore, after splitting the patients in two groups (>5-fold overexpression com- pared to effector CD4 cells and <5-fold overexpression compared to effector CD4 cells) and performing a survival analysis, we found that the ten patients with the higher miR-141 and miR-200c levels (T-PLL-1-4, 11, 21, 22, 23, 25
and 28, had a shorter survival compared with the eleven patients with lower levels of miR-141 and miR-200c (T-PLL-5, 9, 10, 16-20, 24, 26, 29) (P=0.042, Mantel-Cox log rank test) (Figure 2E). In contrast, no difference in sur- vival was found based on miR-181 expression (data not shown). A multiple regression analysis with risk factors including age, sex, white blood cell count and immunophenotype did not determine a confounding effect on the survival. Together, these data present the first evidence that miRNA are deregulated but heterogeneous- ly expressed in T-PLL and that miR-141 and miR-200c expression levels may have prognostic value to define a subgroup of T-PLL with relatively poor survival.
The miR-200c/141-ZEB2-TGFb axis is aberrant in T-cell prolymphocytic leukemia
Next, we asked whether aberrant expression of miR- 200c/141 and miR-181a/b results in downregulation of their TargetScan-predicted well-conserved mRNA targets (Online Supplementary Tables S2 to S4) in T-PLL. In order to directly correlate miRNA levels to mRNA expression pro- files, we generated transcriptomics data of CD4+ T-PLL samples with high expression levels (>10-fold overex- pressed compared to effector CD4 cells) of these miRNA (high miR-200c/141 samples: T-PLL-1, 3, 4, 22, 23, 25; high miR-181a/b samples: T-PLL 3, 4, 10, 16, 17, 23 and 25) (Figure 2B). Effector CD4 T cells from healthy individuals were used as controls, as the miRNA profiles of T-PLL cells appeared most similar to this T-cell subset.
We first analyzed several genes that may be correlated with the upregulation of miR-200c/141 and miR-181a/b. For instance, involvement of enhanced miRNA expression was postulated based on elevated Argonaute-2 (AGO2) expression as a result of chromosome 8 gains in a subset of T-PLL patients.2 However, AGO2 mRNA expression was not significantly increased in our T-PLL cohort (data not shown). The miR-200c/141 cluster is frequently co- expressed with protein tyrosine phosphatase, non-recep- tor type 6 (PTPN6) through transcriptional read-through and complex 3D chromatin interactions between PTPN6 and miR-200c/141 promoters.28 Accordingly, we found a significant upregulation of PTPN6 in T-PLL samples with enhanced miR-200c/141 expression (FC=3.7; FDR=0.0003; Online Supplementary Figure S2A). Furthermore, MIR200C/141 is known to be transcription- ally upregulated by MYC,41,42 an oncogenic transcription factor that is frequently overexpressed in T-PLL.2,43 In agreement, we noted a significant overexpression of MYC in T-PLL (FC=4.9; FDR=0.0019; Online Supplementary Figure S2B). In normal T-cell development miR-181 con- trols expression of various phosphatases, such as SHP2, PTPN22, DUSP5, DUSP6 and NRARP, thereby enhancing NOTCH and T-cell receptor signaling.29 In agreement, we found a significant downregulation of DUSP5 mRNA expression in T-PLL cells (FC=5.7; FDR=1.43x10-6; Online Supplementary Figure S2C).
Gene set enrichment analysis (GSEA) showed a signifi- cant enrichment of miR-200c/141 and miR-181a/b targets in the downregulated fraction of genes in T-PLL compared to normal effector CD4 T cells (P<0.05) (Figure 3A and B). Ingenuity pathway analysis (IPA) of significantly deregu- lated genes in T-PLL revealed that the TGFb network is affected in T-PLL, including transforming growth factor b1 (TGFb1) (FC=0.276; P<7.68x10-10), TGFb2 (FC=0.339, FDR=1.85x10-5 and TGFb receptor 3 (TGFbR3)
148
haematologica | 2022; 107(1)