Determination of featured signatures in clonal drift based on Dx_Rel pairwise analysis of transcriptomic dynamics within a persistent TCR clone. (A) Stacked bar plots comparing cell cycling composition dynamics in persisted Dx-Rel major TCR clonotypes (M clones) from T593 and T856 Dx_Rel paired samples. Clonotypes are derived and ordered according to scTCR clonotyping. (B) Venn diagram showing intersection of commonly upregulated mRNA counts at relapse from drifted TCR M clones across T856 (T856Rel-M, blue) and T593 (T593Rel-M, orange). (C) Heat map showing hierarchy of the topmost significantly differently expressed genes (adjusted P < .05, average log2FC > 1) in drifted TCR clonotypes obtained by FindAllMarkers. Relative gene-expression levels are normalized and visualized by color intensity. Detailed information is provided in supplemental Table 8. (D) Bar plot displaying chromatin immunoprecipitation (ChIP) and qPCR analyses of trimethylation of histone H3 lysine 4 (H3K4me3; left panel) and acetylation of histone H3 lysine 27 (H3K27ac; right panel) levels on endogenous MSI2 transcriptional start site (TSS) for T593 and T856 from diagnosis to relapse. Data is presented after normalizing to nonspecific binding control as fold enrichment of immunoprecipitated DNA relative to input DNA. Data are presented as mean values ± standard error of the mean (SEM). P values were estimated using the 2-tailed Student t test; ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. (E) MSI2 RNA immunoprecipitation and high-throughput mRNA sequencing tracks surrounding the MYC locus of T-ALL cell lines, Molt4 (blue), Jurkat (orange), and CEM (green), with data shown as fold enrichment over input. Pink area indicates MSI2-binding region on MYC mRNA. Number of replicates: Molt4 MSI2, n = 2; Jurkat MSI2, n = 2; and CEM MSI2, n = 2. (F) MYC mRNA half-lives (t_1/2) evaluation in T-ALL cell lines, Jurkat and Molt4, after treating with MSI2 RNA-binding inhibitor Ro at the final concentration of 20 μM in presence or absence of actinomycin D (ActD), in comparison with those in ActD-treated T-ALL cell lines. (G) Western blot assay validation of MYC expression in MSI2-knockout (MSI2^KO) T-ALL cell lines, Jurkat and Molt4. sgRNAs MSI2-sg1, MSI2-sg2 and MSI2-sg3 were used in CRISPR–associated protein 9 (Cas9)-expressed parental cells in the 2-vector CRISPR/Cas9 system. Experiments were performed in triplicate. Representative chemiluminescence images are shown. (H) MSI2 RNA immunoprecipitation and MYC quantitative PCR of reverse-transcribed complementary DNA (cDNA) after treatment of T-ALL cell lines, Jurkat and Molt4, with 10 μM of Ro for 6 and 12 hours compared with the no-treatment vehicle subgroup. Data are presented as mean values ± SEM. P values were estimated using the 2-tailed Student t test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (I) Western blot assay examination of MYC and cleaved-PARP protein levels after treatment of T-ALL cell lines, Jurkat and Molt4, with 5, 10, or 20 μM Ro for 12 and 24 hours compared with the no-treatment vehicle subgroup. Experiments were performed in triplicate. Representative chemiluminescence images are shown. (J) Comparison of apoptosis in T-ALL cell lines, Jurkat, and Molt4, after treatment with Ro (5 or 10 μM) for 24 and 48 hours compared with the no-treatment vehicle subgroup by annexin V and DAPI staining. Experiments were performed in triplicate. Data are presented as mean values ± SEM. Representative scatterplots are shown. (K) DNA damage assessment by phosphorylated H2AX (γH2AX) staining of T-ALL cell lines, Jurkat and Molt4, after treatment with Ro (5 or 10 μM) for 12 hours compared with the no-treatment vehicle subgroup. Experiments were performed in triplicate. Representative overlaid histograms are shown.