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T. Braun et al.
files of T-PLL cells with those of TCR-activated T cells. By integrating T-PLL’s miR profiles with transciptome data via GSEA (based on differentially expressed mRNA and on mRNA ordered by their correlation to the respective miR), we uncovered prominent regulatory networks around DNA damage response and prosurvival pathways in T-PLL.
Increased miR-223-3p expression is linked to activated T-cell phenotypes and associates with signatures
of altered DNA damage response and cell cycle deregulation
We next focused on phenotypic and clinical associations of miR (i) differentially expressed in our cohort and (ii) already linked to B- and / or T-cell leukemogenesis. In our sequencing analysis of small-RNA, miR-223-3p was signifi- cantly upregulated in T-PLL over CD3+ pan-T cells from age-matched healthy donors (fc=9.85, P=0.0002, Figure 3B). GSEA based on mRNA ranked by their correlation to miR- 223-3p expression revealed an association of miR-223-3p with signatures of altered DNA damage responses (e.g., P53 PATHWAY: NES=2.59, q<0.0001) and deregulated cell-cycle mediators (e.g., G2M CHECKPOINT: NES=-2.34, q=0.003, Figure 3C). We further evaluated potential miR-223-3p tar- get mRNA, by combining target prediction databases and miR-ome – transcriptome correlations. Defining criteria of mRNA as putative miR targets in T-PLL are outlined in the Methods section. In total, we identified eight putative tar- gets of miR-223-3p in T-PLL (Figure 3D). Notably, FOXO1 (rho=-0.41, P=0.005) was identified as one of them and was significantly downregulated in T-PLL (n=32 of 48 cases with fc<0.5 compared to normal T-cell controls). Tumor suppres- sive FOXO1 is a prominent regulator of redox balances and DNA insult-mediated cell death.32
When T-PLL cases were divided into three subgroups based on miR-223-3p expression (high, medium, low), we found that miR-223-3p expression correlated significantly with surface expression levels of the TCR activation mark- ers CD38 (mean surface expression: 65.5% vs. 3.6%, P=0.006, MWW) and CD69 (mean 8.5% vs. 1.0%, P=0.1, MWW, Figure 3E, Online Supplementary Table S8 with sum- mary of clinical data).
Increased levels of miR-200c and miR-141 species are associated with deregulated cell cycle molecules, activated phenotypes, and more aggressive presentations
Another miR family, miR-200c/-141, was significantly upregulated in a subset of 23 T-PLL cases (Figures 4A and 1C). Upregulation for miR-141-3p was 43.2-fold (P<0.0001), for miR-141-5p 29.0-fold (P=0.0001), for miR- 200c-3p 38.2-fold (P<0.0001), and for miR-200c-5p 56.6- fold (P=0.003) over all cases. MiR-141-3p showed the high- est absolute CPM values among all deregulated miR in the entire cohort of T-PLL (mean CPMmiR-141-3p =26,561; mean CPM of all significantly deregulated miR in T-PLL =1,440, fc=18.4; Online Supplementary Figure S2). GSEA based on mRNA ranked by their correlation to miR-200c/-141 family members revealed significant enrichments of the HALL- MARK E2F TARGET (NES=3.64, q<0.0001) and HALL- MARK G2M CHECKPOINT gene sets (NES=3.05, q<0.0001, both based on miR-141-3p correlated mRNA, Figure 4B). Additionally, we identified 93 mRNA as poten- tial targets of miR-200c/-141 (Figure 4C) in T-PLL. While nine mRNA showed an overlap between miR-141-3p and
miR-200c-3p target mRNA, we found only a small set of putative targets which were shared between either miR- 141-3p or miR-200c-3p and miR-141-5p. Exemplarily, KAT2B, a known tumor suppressor affecting DNA damage and cell cycle regulation,33 emerged as a potential target of miR-200c-3p (rho=-0.41, P=0.005, Spearman). Surface expression of the T-cell activation marker CD40L was ele- vated in cases with high miR-141-3p (mean 16.5% vs. 0.05%, P=0.03, MWW), high miR-200c-3p (mean 16.5% vs. 0.05%, P=0.03, MWW), and high miR-200c-5p expression (mean 20.0% vs. 0.0%, P=0.009, MWW, Figure 4D). Serum LDH levels at the time of sample correlated with increased miR-141-3p (mean 898 U/L vs. 509 U/l, P=0.03, MWW, Figure 4E) and elevated miR-200c-3p expression (mean 917 U/L vs. 509 U/L, P=0.02, MWW, Online Supplementary Table S9 with summary of clinical data).
Reduced miR-21 expression is linked to features of advanced or aggressive disease
The small-RNA sequencing analysis also revealed a 3.7- fold reduction of miR-21-3p expression (fc=0.27, P<0.0001) and a 3.2-fold reduction of miR-21-5p expression (fc=0.31, P<0.0001) in T-PLL (Online Supplementary Figure S9A). Interestingly, absolute expression (CPM) values of miR-21- 5p were the second most altered among all deregulated miR in T-PLL (mean CPM of all significantly deregulated miR in T-PLL of 1440 vs. mean CPMmiR-21-5p of 15,526, fc=10.8, Online Supplementary Figure S2), suggesting a highly T-PLL-specific loss of expression. GSEA of miR-21-associated mRNA implicated relevance of this miR in apoptosis and cell cycle regulation, as gene sets like HALLMARK P53 PATHWAY and G2M CHECKPOINT were significantly deregulated (NES of APOPTOSIS PATHWAY considering miR-21-5p-associat- ed mRNA=3.89, q<0.0001; NES of G2M CHECKPOINT considering miR-21-3p-associated mRNA=2.39, q=0.001, Online Supplementary Figure S9B). Furthermore, we assessed potential target mRNA of miR-21-3p and miR-21-5p as described above (n=42, e.g., MAP3K1 as a putative target of both miR-21-3p and miR-21-5p, Online Supplementary Figure S9C). In contrast to the current concept of miR-21 being a potent suppressor of cell cycle arrest and apoptosis induc- tion, we did not find negative correlations with mRNA mediating these published effects (e.g., PDCD4: rho=0.04, P=0.79; BTG2: rho=0.28, P=0.05; correlations based on miR-21-5p expression, Spearman).34 Dichotomized by mean miR-21-5p expression, T-PLL with low miR-21-5p levels revealed higher white blood cell (WBC) counts (mean 154 G/L vs. 90.0 G/l, P=0.02, MWW) and lower platelet counts (mean 110 G/L vs. 153 G/L, P=0.03, MWW, Online Supplementary Figure S9D) at the time of sampling, indicat- ing a more active growth behavior of T-PLL with low miR- 21 expression. Fittingly, serum levels of LDH (mean 933 U/L vs. 522 U/L, P=0.02, MWW, Online Supplementary Figure S9E) were elevated in patients with low cellular miR-21-5p expression (Online Supplementary Table S10 with summary of clinical data).
Reduced expression of miR-29 clusters is associated with alterations of survival signaling and cell cycle regulators reflected in features of a more active disease
As analyzed by small-RNA sequencing, the miR-29 fam- ily members miR-29a-3p (fc=0.29, P<0.0001), miR-29b-1- 5p (fc=0.47, P=0.001), and miR-29c-3p (fc=0.29, P<0.0001) showed a homogenous downregulation in T-PLL over
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