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Figure S1 shows the design of regulating the expression of miRNA-363.(A) The construction of FUA based miRNA-363 lentiviral vector; (B) The scheme of miRNA-363 knockout using CRISPR/Cas9 system.

Table S1. Patient Charactertistics and their mi-EP platform type

Purpose:Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non–Hodgkin lymphomas with aggressive clinical behavior. We performed comprehensive miRNA profiling in PTCLs and corresponding normal CD4+ Th1/2 and TFH-like polarized subsets to elucidate the role of miRNAs in T-cell lymphomagenesis.Experimental Design:We used nCounter (NanoString Inc) for miRNA profiling and validated using Taqman qRT-PCR (Applied Biosystems, Inc). Normal CD4+ T cells were polarized into effector Th subsets using signature cytokines, and miRNA significance was revealed using functional experiments.Results:Effector Th subsets showed distinct miRNA expression with corresponding transcription factor expression (e.g., BCL6/miR-19b, -106, -30d, -26b, in IL21-polarized; GATA3/miR-155, miR-337 in Th2-polarized; and TBX21/miR-181a, -331-3p in Th1-polarized cells). Integration of miRNA signatures suggested activation of TCR and PI3K signaling in IL21-polarized cells, ERK signaling in Th1-polarized cells, and AKT–mTOR signaling in Th2-polarized cells, validated at protein level. In neoplastic counterparts, distinctive miRNAs were identified and confirmed in an independent cohort. Integrative miRNA–mRNA analysis identified a decrease in target transcript abundance leading to deregulation of sphingolipid and Wnt signaling and epigenetic dysregulation in angioimmunoblastic T-cell lymphoma (AITL), while ERK, MAPK, and cell cycle were identified in PTCL subsets, and decreased target transcript abundance was validated in an independent cohort. Elevated expression of miRNAs (miR-126-3p, miR-145-5p) in AITL was associated with poor clinical outcome. In silico and experimental validation suggest two targets (miR-126→ SIPR2 and miR-145 → ROCK1) resulting in reduced RhoA-GTPase activity and T–B-cell interaction.Conclusions:Unique miRNAs and deregulated oncogenic pathways are associated with PTCL subtypes. Upregulated miRNA-126-3p and miR-145-5p expression regulate RhoA-GTPase and inhibit T-cell migration, crucial for AITL pathobiology.

Figure S6: Overall Survival in PTCL by miRNA expression. Kaplan-Meier curve representing overall survival of PTCL-NOS cases (n=24) by higher vs lower expression of miR-29c (A) and miR-106 (B) expression. (C) Correlation of n-Counter values between miR-126 and miR-145 expression (r=0.1)

Figure S7: Functional Validation of miR-126 and miR-145: A. Western blot of the miR-126 target S1PR2 in CD4+ T cells and Hut78 T cells transduced with empty vector or miR-126. B. Relative mRNA expression of S1PR2, BCL6, and PRDM1 in CD4+ T cells transduced with miR-126 compared to cells transduced with vector control. C. Flow cytometry histogram of CXCR5 expression in the Raji and DHL16 B-cell lines.D Flow cytometry assessment of the number of migrated CD4+ Jurkat cells expressing empty vector or miR-126, with S1PR2 inhibitor (JTE-013) E. Western blot of S1PR2 and RhoA-GTP in Jurkat cells transduced with empty vector or miR-126-vector treated with S1PR2 inhibitor (JTE-013) F. Cell viability in T cell lines after induction of miR-126 expression. G. Cell viability in Jurkat cells transduced with empty vector or miR-126 in combination with the drug JTE-013. H. Percentage of apoptotic cells in Jurkat cell cultures expressing empty vector or vector containing miR-126 I. Percentage of cells in phases of the cell cycle in Jurkat cell cultures expressing empty vector or vector containing miR-126. J. Expression of miR-145 in normal B cell subsets, CD3+ T cells and Stroma cells profiled using the qRT-PCR based ABI platform. K. Expression of miR-145 in PTCL subsets profiled using nCounter platform. L. miR-145 expression in Jurkat cells after ectopic expression of miR-145 or vector control. M. Cell viability in Jurkat cells transduced with vector control or miR-145.

Figure S6 shows the knockout of CACNA1C using CRISPR/Cas9 system.The expression of CACNA1C was knockout using its sgRNA1 and 5 CRISPR/Cas9 system, which resulted in the obvious apoptosis of these edited cells.

Figure S3 shows the survival curve of patients treated by CHOP regimen.There was survival indifference between patients with distinct CACNA1C expression in the setting of CHOP treatment (p>0.05).

Table S2: List of Antibodies used for the Western Blot Table S3: List of flow cytometry Antibodies used for the expression of different markers Table S4: List of genes and their Forward and Reverse Sequence for the evaluation of mRNA expression by performing Real time RT PCR Table S5: Mature Sequence of miR-126-3p and miR-145-5p taqman Probe used for Taqman base Real time qPCR assay

Figure S2: Expression of lineage specific markers in Polarized CD4+ T cells from different biological donors A: Flow cytometry profiles displaying CXCR5 and PD-1 expression in CD4 T cells cultured with IL-21. B. Flow cytometry profiles displaying GATA3 and TBX21 expression in CD4 T cells cultured in TH1 or TH2 differentiation media.

Figure S1: Clinical outcome of PTCL entities and molecular characterization (A) Heatmap of AITL, PTCL-TBX21 and PTCL-GATA3 classification signatures profiled with the HG-U133 Plus2 platform (n=42); X-axis genes, Y-axis cases (B) Kaplan-Meier curves comparing OS between the PTCL subgroups.

Figure S3: Identification of unique miRNA signatures for TH subsets: Heatmap of differentially expressed miRNA (p2 fold) in polarized CD4+ T-cell subsets. Signatures were derived from the directly polarized TH cells (Donor B/C) and evaluated in the pre-IL2 stimulated TH effector cells (Donor A)

Figure S5 shows the influence of L-type calcium channel modulators on rituximab-mediated calcium influx and CD20 expression.(A) Nimodipine weakened rituximab-induced increase of intracellular calcium ions, whereas Bay K8644 enhanced it in OCI-ly3 and OCI-ly7 cell lines (p

Table S7. Significant signaling pathways associated with the differentially expressed miRNA between PTCL entities and their corresponding normal counterpart

Table S6.Differentially expressed miRNA between naïve CD4+T-cells and different Polarized TH subsets (Red=Upregulated); (Black=Downregulated)

Figure S2 shows probe sets for CACNA1c on HG-U133plus2 platform.

Figure S4: Principal component analysis and hierarchical clustering (HC) of PTCL subgroups. A. PCA of PTCL subgroups and CD3+ T cells based on miRNA expression quantified using with the qRT-PCR platform (ABI, Inc).B. HC based on expression of miRNA in the nCounter signatures in PTCL subtypes and CD3+ T cells profiled with the qRT-PCR platform (ABI, Inc). C. Scatter plot of microRNA expression between n-Counter and ABI qRT-PCR platforms in 4 PTCL cases profiled on both platforms. miRNA-126 and miRNA-145 are colored in red and blue, respectively. miRNA that showed skewed expression (were in the upper quartile in a case on 1 platform, but in the lower quartile for that case when measured by other platform) are noted by the other colored dots. D. Supervised clustering on ABI platform using the differentially expressed genes of PTCL subtypes obtained from n-Counter

Figure S4 shows CACNA1C and CD20 expression and their association with rituximab response in DLBCL cell lines.(A and B) CACNA1C and CD20 mRNA expression were different among OCI-ly3, OCI-ly7, and OCI-ly8 cell lines (p

Figure S5: Validation of nCounter signature genes in a qRT-PCR-based cohort. Relative expression of miRNA identified on the nCounter panel as being high in AITL (top panel), PTCL-TBX21 (middle panel), or PTCL-GATA3 (bottom panel). Data are expressed by the median centered Ct value and lower values correspond to higher expression.

Table S8. Unique miRNA signatures in PTCL entities

T-prolymphocytic leukemia (T-PLL) is a rare and mature T-cell malignancy with characteristic chemotherapy-refractory behavior and a poor prognosis. Molecular concepts of disease development have been restricted to protein-coding genes. Recent global microRNA (miR) expression profiles revealed miR-141-3p and miR-200c-3p (miR-141/200c) as two of the highest differentially expressed miRs in T-PLL cells versus healthy donor-derived T cells. Furthermore, miR-141/200c expression separates T-PLL cases into two subgroups with high and low expression, respectively. Evaluating the potential pro-oncogenic function of miR-141/200c deregulation, we discovered accelerated proliferation and reduced stress-induced cell death induction upon stable miR-141/200c overexpression in mature T-cell leukemia/lymphoma lines. We further characterized a miR-141/200c-specific transcriptome involving the altered expression of genes associated with enhanced cell cycle transition, impaired DNA damage responses, and augmented survival signaling pathways. Among those genes, we identified STAT4 as a potential miR-141/200c target. Low STAT4 expression (in the absence of miR-141/200c upregulation) was associated with an immature phenotype of primary T-PLL cells as well as with a shortened overall survival of T-PLL patients. Overall, we demonstrate an aberrant miR-141/200c-STAT4 axis, showing for the first time the potential pathogenetic implications of a miR cluster, as well as of STAT4, in the leukemogenesis of this orphan disease.

Peripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL–not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall survival (OS), with DNMT3A-mutated residues skewed toward the methyltransferase domain and dimerization motif (S881–R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genomewide methylation analysis of DNMT3A-mutant vs wild-type (WT) PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating interferon-γ (IFN-γ), T-cell receptor signaling, and EOMES (eomesodermin), a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in vivo) and further validated in vitro by ectopic expression of DNMT3A mutants (DNMT3A-R882, -Q886, and -V716, vs WT) in CD8+ T-cell line, resulting in T-cell activation and EOMES upregulation. Furthermore, stable, ectopic expression of the DNMT3A mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T cells with DNMT3AR882H mutation. Single-cell RNA sequencing(RNA-seq) analysis of CD3+ T cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance and thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.