Alternative Splicing of Tcfl5 Fine-tunes Notch1 Transcriptional Output and Cell Fate Decisions in T-cell Acute Lymphoblastic Leukemias

Alternative Splicing of Tcfl5 Fine-tunes Notch1 Transcriptional Output and Cell Fate Decisions in T-cell Acute Lymphoblastic Leukemias | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Alternative Splicing of Tcfl5 Fine-tunes Notch1 Transcriptional Output and Cell Fate Decisions in T-cell Acute Lymphoblastic Leukemias Angel Gutiérrez, Javier Galán-Martínez, Angela García-Rendueles, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7695228/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aberrant NOTCH1 signaling drives T-cell acute lymphoblastic leukemia (T-ALL). TCFL5 is highly expressed in T-ALL clinical samples, producing four isoforms with distinct expression patterns and risk associations. In leukemic cells, TCFL5 isoforms play opposing roles: the CHA isoform promotes proliferation, while TCFL5_E6 and TCFL5_E8 suppress tumor growth. Mechanistically, TCFL5 acts as a transcriptional effector and feedback regulator of NOTCH1, modulating its transcriptional output by cobinding promoters of key target genes like BCL2 and HES1. NOTCH1 reciprocally regulates TCFL5 isoform expression, forming a feedback loop that governs leukemic cell fate. Disrupting this loop—via pan-TCFL5 knockout or isoform-specific modulation—alters proliferation and survival in T-ALL models. These findings identify TCFL5 as a critical node in the NOTCH1 signaling network and underscore the therapeutic potential of targeting TCFL5 isoform dynamics to modulate oncogenic signaling in T-ALL. Hematology Immunology Cancer Biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by the proliferation and accumulation of immature T-cell precursors arrested in differentiation, primarily affecting the bone marrow, blood, lymphoid organs, mediastinum, and central nervous system. Although T-ALL can affect individuals of any age, most cases occur in children. T-ALL accounting for approximately 10–15% of pediatric and 25% of adult acute ALL cases ( 1 , 2 ). Despite advances in treatment, refractory and relapsed T-ALL cases have poor prognoses ( 3 , 4 ). T-ALL is genetically heterogeneous and driven by a multistep leukemogenic process involving both intrinsic genetic lesions and extrinsic microenvironmental factors ( 5 , 6 ). These alterations disrupt the normal proliferation, differentiation and survival during thymocyte development. Key molecular hallmarks include activating mutations in NOTCH1, found in over 60% of T-ALL cases, making it a major oncogenic driver ( 7 ). In this regard it has been shown that constitutively active NOTCH1 promotes leukemogenesis by activating the NF-κB pathway, both transcriptionally and via the IκB kinase (IKK) complex ( 8 ). This activation leads to the upregulation of antiapoptotic genes such as Bcl2, enhancing cell survival. Several others NOTCH1 target genes have been identified, including DTX1 (Deltex1) ( 9 ) and HES1 ( 10 ). Transcription Factor Like 5 (TCFL5) is a member of the basic Helix-Loop-Helix (bHLH) family of transcription factors. Initially identified in the testis ( 11 ), it has been predominantly studied in spermatogenesis, being expressed in primary spermatocytes during the pachytene stage ( 12 ). In humans, the TCFL5 gene comprises eight exons, with the class C bHLH domain located between exons 4 and 5 ( 13 ). A shorter isoform, CHA, was first described in the T-ALL derived Jurkat cell line and identified as an autoantigen in Chagas disease, due to its homology with Trypanosoma cruzi proteins ( 13 , 14 ). Recently, an extensive characterization of the locus has been performed leading to the identification of four different isoforms: TCFL5_E8, commonly referred to as the canonical TCFL5 isoform, TCFL5_E7, TCFL5_E6, and CHA (E2B-E8) ( 15 ). Although all isoforms retain the bHLH domain, suggesting shared a core functionality, they differ in their 5' and 3' ends, with two possible initial exons (E1 and E2b) and three alternative terminal exons (E8, E7, and E6). The presence of two initial exons suggests the existence of alternative promoters, potentially contributing to differential regulation. CHA (TCFL5 E2B-E8) interacts with another bHLH factor, Upstream Stimulatory Factor 1 (USF1), and binds to the CD2 promoter repressing its expression in T cells where it plays a role in T cell activation ( 13 ). TCFL5 is upregulated in colorectal cancer (CRC), being its expression higher in carcinomas than in adenomas, likely due to chromosome 20q amplification ( 16 ). Silencing TCFL5 in colon cancer cells reduces proliferation, multicellular tumor spheroid formation, and increases cell viability ( 15 , 17 , 18 ). TCFl5 mRNA expression has also a prognostic value in hepatocellular carcinoma ( 19 ). TCFL5 has also been suggested as a target of the NOTCH1 signaling pathway in thymocytes and its expression correlates with an increase in double-positive thymocytes ( 20 ). In B cells, activation of Toll-Like Receptors 7 and 9 induces TCFL5 expression ( 21 ). Conversely, during monocyte differentiation, TCFL5 expression is downregulated ( 22 ) Interestingly TCFL5 is notably upregulated in B-ALL (ETV6-RUNX1) ( 23 ) and in ALL cases resistant to vincristine treatment ( 24 , 25 ). In those B-ALL samples, the TCFL5 promoter was significantly hypomethylated, correlating with increased gene expression ( 26 ). In this study. we investigated the role of TCFL5 in T-ALL. This transcription factor was highly elevated patient samples and in T-ALL cell lines. All four isoforms are present, with CHA and TCFL5_E8 being the most abundant. Functional assays reveal that TCFL5_E8 and TCFL5_E6 inhibit, while CHA promotes leukemia cell growth. However, knocking out all isoforms reduces cell proliferation. Mechanistically, TCFL5 interacts with NOTCH1, binding to the same promoter regions of several genes, including BCL2 and HES-1 as well as those of CHA and TCFL5. Growth-promoting isoforms enhance NOTCH1 activity, while inhibitory ones suppress it. Inhibiting NOTCH1 increases TCFL5_E8 but decreases CHA expression, highlighting TCFL5’s significant role in T-ALL progression through its interplay with NOTCH. Materials and Methods Human samples In the present study, 38 bone marrow samples from paediatric T-ALL patients were collected at the Hospital Infantil Universitario Niño Jesús (HUNJ) under the SEHOP-PETHEMA 2013 protocol guidelines (Supplemental information). All participants signed the informed consent following the institutional Ethic Committee approved protocol (internal code R-0017/13). Bone marrow mononucleated cells (BMMCs) were isoloted using Ficoll-plaque and frozen in liquid nitrogen in a solution of 90% fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO). Healthy human PBMC and thymocytes were used as controls. Cell growth Cell growth was evañuated by cell counting with trypan blue exclusion or by MTT assay as described ( 27 ). Briefly, 100 µL of cell suspension was plated in 96-well plates (triplicates), incubated at 37°C with 5% CO₂, and treated with MTT reagent. After 5h, absorbance was measured at 550 nm wavelength. Blank wells contained all reagents except cells. Genetic manipulation of T-ALL cell lines Electroporation, nucleofection and lentiviral transduction were alternatively used for introducing foreign genetic material in T-ALL lines. Briefly, cells (5x10⁶) were electroporated with plasmid DNA encoding the various isoforms (Supplemental information) (15 µg of the plasmid of interest and 25–35 µg of carrier DNA), using a modified protocol optimized for high-efficiency DNA delivery ( 28 ). Electroporation was performed using a square-wave pulse of 960 µF and 260 mV for approximately 22 ms. Following electroporation, cells were transferred to pre-warmed RPMI-1640 medium supplemented with 5–20% FCS and incubated at 37°C. Transgene expression was analyzed at 24–72 h. For Tet-ON inducible expression in Jurkat, cells, 1,2×10⁶ cells were resuspended in 100 µL of Cell Line Nucleofector Solution V (Amaxa-Lonza) at room temperature, pre-mixed with 20 µg of indicated plasmid DNA with the reverse tetracycline-controlled transactivator (rtTA), followed by a tetracycline-responsive element (TRE) (VectorBuilder) upstream of the TCFL5 isoforms (Supplemental information) ( 29 ). The cell-DNA suspension was subjected to electroporation using program O-006 on the Nucleofector I device (Lonza). Post-nucleofection, cells were transferred to 12-well plates containing 1.5 mL of complete medium per well and collected 24 hours later for subsequent analyses ( 29 ). Stable clones were selected with hygromycin and induced with doxycycline (1 µg/mL). Lentiviral particles were generated in HEK293 cells via transient co-transfection with the packaging plasmids pCMV-Vsvg and pCspV, along with expression constructs (Supplemental information) including pLenti-CMV/TO-Hygro or pLenti-CMV/TO-Hygro-TCFL5 for overexpression studies, and pLenti-KO1-puro or pLenti-KO1-shTCFL5 variants (OriGene) for gene knockdown experiments or pHHRSin_IRES_EGFP constructs for NOTCH pathway (Supplemental information). Viral supernatants were collected at 24 and 48 h post-transfection, filtered, and used to transduce cells. Selection was performed with hygromycin (200 µg/mL) for overexpression line or puromycin (4 µg/mL) for knockdown lines. Chromatin Immunoprecipitation (ChIP). ChIP was performed as described ( 15 , 30 ). 2 × 10⁷ cells cross-linked with 1% formaldehyde in PBS for 10 minutes at room temperature, quenched, and washed with cold PBS- Nuclei were isolated and lysed, followed by chromatin shearing via sonication (10 cycles of 30 s at high power). Lysates were centrifuged at 14,000 rpm for 15 m at 4°C. A fraction corresponding to 5 × 10⁵ cells was reserved as input control. The remaining chromatin was incubated overnight at 4°C with 8 µg of antibody (anti-TCFL5, anti-ICN1 ( 31 ), or rabbit IgG control) under gentle agitation. Immunocomplexes were captured using protein A-agarose beads (Millipore) according to the manufacturer’s protocol. Chromatin was eluted at 65°C for 16 h, followed by RNase A and proteinase K treatment. DNA was purified using phenol/chloroform:/isoamyl alcohol extraction and analyzed by quantitative PCR (qPCR) using promoter-specific primers (Supplemental Table 2). RNA and Protein analysis RNA was isolated and reverse transcribed as described ( 15 ). cDNA was used for PCR using GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA) or quantitative PCR using GoTaq1 Master Mix (Promega) using specific gene primers for indicated mRNAS, entire TCFL5 isoforms mRNAs (Supplemental Table 3) or exon junctions (Supplemental Table 4) ( 15 ). qPCR was conducted using the ABI PRISM 7900HT system (Applied Biosystems Relative mRNA expression was calculated using the ΔΔCt method, normalized to GAPDH as described ( 15 ). Each reaction was performed in triplicate. Immunoblotting was performed basically as described ( 15 , 32 ). Equal amounts (15 µg) were separated by SDS-PAGE, transferred to nitrocellulose membranes (BioRad) and probed with anti-TCFL5, anti ICN1, anti-HSP90 and anti-beta actin (Santa Cruz) Supplemental Table 5) in 5% BSA-TBST overnight at 4°C. Detection was performed using HRP-conjugated secondaries and chemiluminescence (Pierce). Band intensity was quantified with ImageJ software. Luciferase reporter activity assays Jurkat cells were seeded in 24-well plates at 70% confluency and transfected by electroporation with the Notch responsive element (NRE) luciferase reporter plasmid (BPS Bioscience) and appropriate pCDNA3-TCFL5-Flag isoforms (Supplemental information) alongside a constitutively expressed Renilla luciferase plasmid as a transfection control. CHA ad TCFL5 promoter regions were cloned in front of a luciferase reporter gene (Supplemental information) and co transfected with Notch1 pathway plasmids. Transfections were performed using Metafectene Pro (Biontex) according to the manufacturer’s protocol. After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega), following the manufacturer’s instructions. Luminescence was quantified using the FLUOstar OPTIMA plate reader (BMG LabTech). Statistics Statistical analyses were performed using the GraphPad Prism v6.0 program (GraphPad Software, LLC). Student's t-test was used to compare the means of quantitative data. In the case of large amounts of data, outliers were identified and removed from the statistical analysis. All experiments were represented as the mean ± SD (standard deviation). ns, non significant *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Results TCFL5 is overexpressed in T-ALL clinical samples. By analyzing several databases, we observed higher expression levels of TCFL5 mRNA in T-ALL pediatric patients compared to healthy PBMCs or thymocytes controls (Supplemental Figure S1). We confirmed this finding in 38 T-ALL samples obtained from children. QPCR analysis detecting all TCFL5 transcripts indicated a variable expression, although on average much higher-than- control healthy patients (Fig. 1 B). However, these studies did not discriminate among the different transcript variants, so we performed a detailed isoform analysis in this cohort based on our previous identification of four TCFL5 isoforms in CRC ( 15 ) (Fig. 1 C). Amplification of E3-E4 was able to detect all TCFL5 transcripts and showed a similar expression pattern than TCFL5 mRNA by PCR. However, specific exon amplification indicates, a different behavior of the four alternative transcripts. Quantitative levels of TCFL5_E6 mRNA, detected by detection of E5-E6 exon junction were low in control samples (not shown) but were significantly higher in T-ALL patients. TCFL5_E7 mRNA, detected by E5-E7, was also low in control samples but tend to be relatively lower in T-ALL. Unfortunately, TCFL5_E8 mRNA could not be specifically detected through exons junction. However, E1-E2 and E5-E8 levels show also a higher highest expression in T-ALL, w hereas E2b-E3 (detecting only Cha) was relatively a little lower (Fig. 1 C). We then explored whether the relative expression of the different isoforms was associated with several clinical parameters commonly employed for risk stratification of T-ALL pediatric patients. Notably, high risk cases showed increased levels of E5-E6 and E5-E8 isoforms but reduced levels of the E2b-E3·isoform (Fig. 1 D). TCFL5 Isoform-Specific functions in T-ALL Cell Lines To investigate the role of TCFL5 in T-ALL, we first analyzed isoform expression in T-ALL cell lines. Higher expression of CHA (E2B-E8) followed by TCFL5 E1_E8 and much lower levels of TCFL5_E6 and TCFL5_E7 were detected by RT-QPCR in Jurkat cells (Fig. 1 E) and Molt 4 (Figure S2). Western blot analysis also revealed robust CHA (E2B-E8) expression and of TCFL5 E1_E8 in both Jurkat (Fig. 1 F) and MOLT-4 cells (Figure S2A). Isoforms TCFL5_E6 and TCFL5_E7 were also detected albeit at much lower levels. Next, we employed lentiviral shRNAs targeting different regions of the TCFL5 transcripts (Fig. 1 A). Two constructs (sh12 and sh90), designed to silence all isoforms, significantly inhibited Jurkat cell proliferation (Fig. 2 A). In contrast, shRNAs targeting the 3′UTR of E8-containing isoforms (CHA and TCFL5 E1-E8) had only modest effects. qPCR confirmed that sh12 and sh90 effectively suppressed all isoforms, while the 3′UTR-targeting shRNAs selectively reduced, but not suppressed, E8 and to a lesser extent, E6 and E7 containing isoforms (Fig. 2 B). These finding suggest a potential regulatory interplay between isoforms. To dissect isoform-specific functions, we generated T-ALL lines individually overexpressing each isoform. TCFL5_E8 and TCFL5_E6 overexpression by electroporation markedly impaired proliferation and viability. CHA modestly enhanced growth, while TCFL5_E7 had no significant effect in both Jurkat (Fig. 2 C) and MOLT-4 cells (Figure S2B). All transfected cells resulted in the expression of the transfected proteins, by FLAG detection (Figure S3). On the other hand, stable overexpression of TCFL5_E8 isoform by a different approach as lentivirus also resulted in a complete loss of proliferation, decreased cell viability and eventually cell death (Fig. 2 A). Jurkat Tet-ON lines were established with doxycycline-inducible expression of each isoform. All established lines grew similarly without doxycycline (data not shown). Upon induction, all isoforms variably increased in a time-dependent manner (Figure S3C). Consistent with above observations, induction of TCFL5_E6 or TCFL5_E8 halted cell proliferation, whereas CHA, which was highly overexpressed after doxycycline, strongly enhanced it (Fig. 2 D). Interestingly, overexpression of some isoforms also influenced endogenous levels of the rest (Fig. 2 E, S3). Thus, the stable (Figure S3B) or induced (Figure S3 C,D) overexpression of TCFL5_E8 significantly reduced CHA. TCFL5_E6 transfection resulted in a very high increase in its own levels in all transfected cell and decreased CHA. In contrast, TCFL5_E7 transfection had no significant impact on the other isoforms. These findings indicate isoform-specific cross-regulation. To explore the transcriptional impact of TCFL5 isoforms, we analyzed the expression of some genes implicated in T-ALL pathogenesis. Overexpression of CHA (E2B–E8) in TALL cell lines significantly upregulated HES1 and BCL2 and DTX1 mRNA levels, whereas overexpression of TCFL5_E8 and TCFL5_E6 repressed their expression (Fig. 2 F, S2D). Conversely, NFKBIA was induced by TCFL5_E8 and TCFL5_E6 but downregulated by CHA. DUX4 was mostly induced by TCFL5_E8 but was downregulated by TCFL5_E6. The results revealed distinct and sometimes opposing effects depending on the isoform. Crosstalk Between TCFL5 and NOTCH1. Interestingly, those TCFL5 induced transcriptional changes were accompanied by alterations in the NOTCH1 pathway. Upon ligand binding, NOTCH1 undergoes sequential proteolytic cleavages, releasing the Notch intracellular domain, which translocate to the nucleus and forms a transcriptional activation complex with CSL and MAML. DAPT, a γ-secretase inhibitor, blocks the final cleavage step thereby inhibiting downstream signaling ( 33 – 35 ). Activation of the NOTCH1 pathway via enforced stable ICN1 or MAML1 overexpression enhanced Jurkat cell proliferation, whereas dominant-negative MAML1 (dnMAML) suppressed this effect (Fig. 3 A). Inhibition of NOTCH1 signaling using the γ-secretase inhibitor DAPT reduced cell growth in a dose-dependent manner (Fig. 3 B). CHA overexpression increased ICN1 protein levels, while TCFL5_E8 and TCFL5_E6 slightly reduced them although not statistically significant (Fig. 3 C, S3E). These changes were functionally relevant: luciferase reporter assays in Jurkat cells showed that CHA overexpression enhanced NOTCH1-responsive transcription, whereas TCFL5_E8 and TCFL5_E6 suppressed it (Fig. 3 D). The addition of DAPT decreased, as expected, the transcriptional activity of NOTCH-1 in a dose-dependent manner. The repressor activity of TCFL5_E8 or TCFL5_E6 on NRE was not significantly further affected by DAPT whereas the CHA-induced increase in activity could be still reduced by DAPT (Fig. 3 E). These data suggest that TCFL5 and DAPT converge at the same point in the NOTCH-1 pathway. Conversely, manipulation of NOTCH pathway altered TCFL5 isoform expression. Thus, DAPT treatment induced TCFL5_E8 while significantly repressing CHA in a dose-dependent manner (Fig. 3 F). Dual Promoter Occupancy by TCFL5 and NOTCH1 To investigate the transcriptional regulation of TCFL5 isoforms, we analyzed their promoter regions. Previous work from our group identified a transcription start site for the CHA isoform that is distinct from the E1 containing mRNA isoforms ( 15 ) (see Fig. 4 A). In silico analysis revealed three putative regulatory regions within the TCFL5 locus, upstream of exon1, in exon 1 and upstream of CHA (Fig. 4 A). Chromatin immunoprecipitation (ChIP) assays using anti-TCFL5 antibodies specifically bound to two regions in the TCFL5 locus: one upstream of E1-containing transcripts and another upstream of the CHA mRNA transcription start sites (Fig. 4 B), supporting the hypothesis of both isoform-specific promoter regulation, and cross regulation among TCFL5 isoforms. Next, we explored whether NOTCH1 also participates in the transcriptional regulation of the TCFL5 and CHA isoforms. Bioinformatic analysis identified transcription factor binding motifs for TCFL5 (E-box) and NOTCH1 (RBPJ) within these small DNA regions bound by TCFL5 shown above. ChIP assays using ICN1 antibodies confirmed that NOTCH1 binds to the same two promoter regions in TCFL5 upstream of the E1 and E2B–E8 transcription start sites (Fig. 4 B). This co-occupancy suggests that both transcription factors may regulate these promoters either cooperatively or competitively. Similar dual TCFL5 and NOTCH motifs were also found in the promoters of canonical NOTCH1 target genes such as HES1 and BCL2 . ChIP analysis detected cobinding of ICN1 and TCFL5 to those regions in HES1 and BCL2 but only TCFL5 bound to CD2 promoter previously shown to bind TCFL5/USF1 in Jurkat cells ( 13 ), but that does not contain RBPJ sites, supporting the specificity of this regulatory interaction (Fig. 4 C). To assess the functional impact of NOTCH1 signaling on TCFL5 isoform and promoters, we performed luciferase reporter assays in Jurkat with complete pTCFL5 and pCHA promoters. Transfection of ICN1 with or MAML1, a co-activator of NOTCH1, significantly increased the activity of CHA promoter. Conversely, a dominant-negative MAML construct (dnMAML) significantly repressed its activity (Fig. 4 D). In contrast, no significant effects were observed forTCFL5 promoter. Discussion T-ALL is a biologically and clinically complex disease and despite advances in treatment, refractory and relapsed T-ALL cases have poor prognosis ( 3 , 4 ). Aberrant activation of the NOTCH1 signaling pathway plays a central role in the pathogenesis of T-ALL ( 7 ). Thus, a deeper understanding of the molecular drivers, particularly the transcriptional networks downstream of NOTCH1 is essential for developing targeted therapies and improving prognosis in relapsed or refractory cases. In this study, we identified TCFL5 as a novel effector of NOTCH1 in T-ALL. We found a high expression of TCFL5 mRNA consistently exceeding that observed in control cells. Despite the absence of significant number of alterations in this gene described in leukemia cases, our findings suggest elevated expression of this gene in T-ALL. In B-ALL samples with the t(12;21) chromosomal translocation, the TCFL5 promoter was significantly hypomethylated, correlating with increased gene expression ( 23 , 26 ). However, those studies did not distinguish between TCFL5 isoforms, which were previously shown to have opposite functional effects on colon cancer ( 15 ). We detected higher expression of all TCFL5 isoforms in clinical T-ALL samples relative to healthy controls, with the exception of TCFL5_E7. Notably, the TCFL5_E6 isoform was significantly enriched in high-risk clinical samples, whereas CHA was expressed at lower levels in these cases. Using microarrays, TCFL5 was found elevated in T and B ALL cases resistant to vincristine treatment ( 24 , 25 ) and has significant association with risk ( 36 ) and bone marrow status at day 14 ( 37 ). Interestingly, functional assays have revealed isoform-specific effects in T-ALL biology. Thus, the growth of Jurkat and MOLT4 cells was enhanced by constitutive or inducible expression of CHA and arrested by that of TCFL5_E8 and TCFL5_E6. However, growth is also inhibited by near-complete inhibition of all mRNA isoforms by lentiviral shRNAs suggesting that CHA is the primary growth-promoting isoform. These findings mirror our previous observations in colon cancer ( 15 ), highlighting the complex regulatory dynamics among TCFL5 isoforms. Cross regulation among isoforms is supported by the binding of TCFL5 to both TCFL5 and CHA promoters. Interestingly, increasing the expression of the growth-inhibitory isoform TCFL5_E8, reduced CHA levels, suggesting a competitive or negative regulatory interplay. Thus, both overexpression of TCFL5_E8 and complete TCFL5 knockdown may converge on a common growth arresting phenotype by suppressing CHA. Analysis of the potential molecular mechanisms by which TCFL5 regulates T-ALL growth has revealed an interesting crosstalk with NOTCH1. Upon ligand binding, NOTCH1—a transmembrane receptor—undergoes two sequential proteolytic cleavages: first by a metalloprotease and then by γ-secretase. This releases the Notch intracellular domain (ICN), which translocates to the nucleus and forms a transcriptional activation complex with CSL and MAML, driving the expression of target genes ( 38 , 39 ). The γ-secretase inhibitor DAPT blocks this final cleavage, thereby inhibiting downstream signaling ( 33 – 35 ). TCFL5 isoforms exert disparate effects on NOTCH1, processing, transcriptional activity and downstream target gene expression in T-ALL. We found a direct relationship between the ability of the different TCFL5 isoforms to induce T-ALL growth and modulate NOTCH1 transcription. CHA promoted growth, in line with its ability to enhance NOTCH1 transcriptional activity as evidenced by increased NRE dependent luciferase activity and expression of canonical NOTCH1 target genes, whereas TCFL5_E8 and TCFL5_E6 inhibited those activities. This may result from the observed increase by CHA and decrease by the other two isoforms in active ICN1 levels. Importantly, the CHA-mediated activation of NRE was inhibited by DAPT, indicating that this effect is dependent on active NOTCH1 signaling. Conversely, pharmacological inhibition of NOTCH1 processing with the γ-secretase inhibitor DAPT inhibited growth of T-ALL lines associated with a decrease in CHA and an increase in TCFL5_E8 respectively, suggesting that NOTCH1 activity not only regulates TCFL5 expression but also influences isoform balance. Previous studies have suggested that TCFL5 is a transcriptional target of NOTCH1 in double-negative thymocytes ( 20 ). Our findings extend this observation by demonstrating that NOTCH1 binds to the promoters of both canonical TCFL5 (E1-containing) and alternative CHA isoforms. However, activation of the NOTCH1 pathway results in different outcomes for both promoters, activating pCHA without affecting pTCFL5. The TCFL5 isoforms also differentially regulate key NOTCH-related genes involved in T-ALL biology. CHA overexpression induces HES1 expression, a critical NOTCH1 target and mediator of leukemogenesis, and promotes cell proliferation, whereas TCFL5_E8 and TCFL5_E6 isoforms repress HES1 and inhibit growth, consistent with its role in maintaining T-ALL cell survival ( 40 , 41 ). BCL2 , a critical antiapoptotic gene was also differentially regulated by TCFL5 isoforms; enhanced by CHA and suppressed by TCFL5_E8 and TCFL5_E6. Similarly, DUX4, implicated in leukemogenesis ( 42 ), is also differentially regulated by TCFL5 isoforms, further highlighting their distinct transcriptional programs. The E3 ubiquitin ligase DTX1 , which promotes NOTCH1 degradation ( 43 ) was also differentially regulated. CHA increased DTX1 expression, while TCFL5_E8 and TCFL5_E6 reduced it. These changes may underlie the observed differences in ICN1 protein levels and thus, downstream signaling. In addition, the TCFL5 isoforms differentially modulate the NF-κB pathway, another critical axis in T-ALL. NOTCH1 is known to activate NF-κB leading to the expression of genes such as NFKBIA ( 8 ). Our data show that TCFL5_E8 and TCFL5_E6 induce NF-κB inhibitor IκBα ( NFKBIA ), while CHA represses it, further supporting the notion of isoform-specific transcriptional programs. The activation of NOTCH/HES1 pathway sustains NF-κB activation in T cell leukemia ( 44 ) and modulation of this pathway may partially explain the different effect of TCFL5 isoforms. TCFL5 and NOTCH1 also co-occupy the promoters of several downstream target genes, including BCL2 and HES1, further supporting a coordinated regulatory mechanism. The cobinding of TCFL5 and ICN1 to shared promoter regions, along with their opposing effects on transcriptional activity, points to a finely tuned mechanism of isoform-specific gene regulation. Together, our results suggest that NOTCH1’s pro-leukemic effects may be partly driven by its induction of the CHA isoform, supporting a model in which NOTCH1 regulates TCFL5 isoform expression to favor growth-promoting variants. In turn, the TCFL5 isoforms differentially modulate NOTCH1’s transcriptional activity and downstream gene expression. Mechanistically, this involves the direct binding of NOTCH1 and TCFL5 to shared promoter regions—including those of TCFL5, CHA, HES1, and BCL2 mRNAs—highlighting a transcriptional interplay that influences the balance between proliferation and apoptosis in T-ALL cells. Overall, the data reveal a complex regulatory network where NOTCH1 activates CHA, and TCFL5 isoforms modulate NOTCH1 degradation and activity and T-ALL cell fate. The differential regulation of BCL2 and HES1 by TCFL5 isoforms links transcriptional control to cell survival, positioning TCFL5 as a key node in the NOTCH1 signaling pathway and a promising therapeutic target in T-ALL. Declarations Acknowledgments We are grateful to Maria Chorro, Mari Carmen Maza, Ana Flores and Carolina Maroto for their excellent technical assistance. This research was funded by “Ministerio de Ciencia e Innovación” (Spain), grant numbers PID2019-104760RB-I00) and PID2022-137487OB-I00. and ‘Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM) to MF and Institutional grants from ‘Fundación RamonAreces’ and ‘Banco de Santander’. Author Contributions M.F. conceptualization, funding acquisition and final writing; KS conceptualization and final writing. J.G., A.G., A.G.R., A.L. and S.P. designed and performed different in vitro experiments, the statistical analysis and prepared the illustrations, and helped drafting the manuscript; F.A. performed in vitro experiments; A.G., E.G.S., M.R and M.V, provided and analyzed clinical data. All the authors have revised and agreed to the published version of the manuscript. Competing interest The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Institutional Review Board Statement This study was carried out in strict accordance with the European Commission legislation. All participants signed the informed consent following the institutional Ethic Committee approved protocol (internal code R-0017/13). 213.6/21). 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Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives. Cells. 2020;9(6). Hernandez Tejada FN, Galvez Silva JR, Zweidler-McKay PA. The challenge of targeting notch in hematologic malignancies. Front Pediatr. 2014;2:54. Silveira VS, Scrideli CA, Moreno DA, Yunes JA, Brandalise SR, Toledo S, et al. Expression Profile Analysis of Genes Related to Resistance/Sensibility to Prednisolone, Daunorubicin, L-Asparaginase and Vincristine in Childhood Acute Lymphoblastic Leukemia. Blood. 2007;110(11):3463-. Silveira VS, Scrideli CA, Moreno DA, Yunes JA, Queiroz RGP, Toledo SC, et al. Gene expression pattern contributing to prognostic factors in childhood acute lymphoblastic leukemia. Leukemia & Lymphoma. 2013;54(2):310-4. Kopan R. Notch signaling. Cold Spring Harb Perspect Biol. 2012;4(10). Sachan N, Sharma V, Mutsuddi M, Mukherjee A. Notch signalling: multifaceted role in development and disease. FEBS J. 2024;291(14):3030-59. Schnell SA, Ambesi-Impiombato A, Sanchez-Martin M, Belver L, Xu L, Qin Y, et al. Therapeutic targeting of HES1 transcriptional programs in T-ALL. Blood. 2015;125(18):2806-14. Magesh V, Sekar R, AlZahrani A, Balasubramanian R, Abdelsalam SA, Rajendran P. HES1 in cancer: a key player in tumorigenesis and its prognostic significance. Molecular Genetics and Genomics. 2025;300(1):49. Davis K, Sheikh T, Aggarwal N. Emerging molecular subtypes and therapies in acute lymphoblastic leukemia. Semin Diagn Pathol. 2023;40(3):202-15. Revici R, Hosseini-Alghaderi S, Haslam F, Whiteford R, Baron M. E3 Ubiquitin Ligase Regulators of Notch Receptor Endocytosis: From Flies to Humans. Biomolecules. 2022;12(2):224. Espinosa L, Cathelin S, D'Altri T, Trimarchi T, Statnikov A, Guiu J, et al. The Notch/Hes1 pathway sustains NF-kappaB activation through CYLD repression in T cell leukemia. Cancer Cell. 2010;18(3):268-81. Additional Declarations The authors declare no competing interests. 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B) Expression of TCFL5 mRNA in T-ALL clinical samples compared with normal PBMCs. A probe detecting all isoform was used in qPCR, p\u0026lt; 0,0001 between groups. C) Expression of different TCFL5 mRNA exon junctions in T-ALL clinical samples normalized to their expression in control PBMCs; mean ± SD, *p\u0026lt; 0,001 in all except E5-E7. D) Expression of different TCFL5 mRNA exon junctions in T-ALL clinical samples in function to the risk. E1-2: Unpaired t test P value 0,3953, ns; E3-4: Unpaired t test P value 0,0172, *; E5-8: Unpaired t test: P value 0,7844, ns; E2b-E3·: Unpaired t test P value 0,0443 *; E5-7: Unpaired t test, P value 0,010, *; E5-6: Unpaired t test P value 0,0083 **. E) Expression of TCFL5 isoforms mRNA in Jurkat cell line samples normalized to endogenous HSP0 mRNA levels. data are presented as mean ± SD of the three independent experiments performed. F) Protein levels of TCFL5 isoforms in Jurkat cells. Western blot with anti-pan TCFL5 antibody. A representative experiment is shown.\u003c/p\u003e","description":"","filename":"Diapositiva1.png","url":"https://assets-eu.researchsquare.com/files/rs-7695228/v1/f50dd4c8fc7992d84c6cb8a3.png"},{"id":93019297,"identity":"e66e46c7-21b6-442a-a1ab-dab97ec11f1e","added_by":"auto","created_at":"2025-10-08 08:37:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":337361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional effects of TCFL5 genetic manipulation in Jurkat.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Effect of lentiviral transduction of shRNA and TCFL5 _E8 on growth rate. Data are presented as mean ± SD of the 3 independent experiments performed. \u0026nbsp;B) Quantification of the different exon junctions by qPCR. E5-E6, E5-E7 and E5-E8 exon junctions were quantified. pLKO is a replication-incompetent lentiviral vector chosen for expression of shRNAs. Indicated are the exons targeted by the different shRNAs. C) Effect of TCFL5 isoform overexpression, on Jurkat growth. Data are presented as mean ± SD of the 4 independent experiments performed. D) Doxycycline induction of TCFL5 isoforms on Jurkat Tet-On cells. Data are presented as mean ± SD of the 4 independent experiments performed. E) Effect of the overexpression of the individual TCFL5 isoforms on the protein expression of other isoforms in by Western blot. A representative experiment is shown. F) Gene expression analyses. qPCR of the mRNA of the indicated genes normalized to EV cells. Mean ± SD of the 4 independent experiments performed. ns, not significant *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 and ****p \u0026lt; 0.0001. EV indicates empty vector, E1-E8 (TCFL5_E8), E1-E7 (TCFL5_E7), E1-E6 (TCFL5_E6) and CHA transfected vectors or protein expression.\u003c/p\u003e","description":"","filename":"Diapositiva2.png","url":"https://assets-eu.researchsquare.com/files/rs-7695228/v1/30c682e0340071f7ff917d23.png"},{"id":93019294,"identity":"7ca7185a-fcb5-45cb-81d9-f8106ac69e99","added_by":"auto","created_at":"2025-10-08 08:37:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":263055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrosstalk of NOTCH1 and TCFL5 in Jurkat Cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Effect of overexpressing NOTCH1 pathway genes on Jurkat growth. Relative growth index of Jurkat cells transduced with lentiviral particles containing active Notch (ICN1), MAML o dominant negative dnMAML. Data are presented as mean ± SD of the 3 experiments performed., *p \u0026lt; 0.05, **p \u0026lt; 0.01. B) Effect of inhibiting NOTCH1 by DAPT pharmacological inhibitor on Jurkat growth. Relative growth index of Jurkat cells cultured in presence of the indicated doses of DAPT or solvent DMSO. Mean ± SD of the 3 experiments performed. C) Effect of TCFL5 isoform overexpression on ICN1 levels. A representative experiment is shown. D) Effect of TCFL5 isoforms overexpressed in Jurkat on NOCTH transcriptional response. Activity was measured by a NRE dependent luciferase reporter and normalized to control vector responses. Data are presented as mean ± SD of the 4 experiments performed, *p \u0026lt; 0.05. E) Effect of pharmacological inhibition by DAPT on NOCTH transcriptional response activity (NRE) modulated by TCFL5 isoforms overexpression Data are presented as mean ± SD of the 2 experiments performed. ***p \u0026lt; 0.001 differences among DAPT treatment in each group and \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 between different control groups. F) Effect on endogenous TCFL5 isoform expression levels of pharmacological inhibition by DAPT in Jurkat cells after 48 hrs of treatment. A representative experiment is shown. EV or control indicates empty vector, E1-E8 (TCFL5_E8), E1-E7 (TCFL5_E7), E1-E6 (TCFL5_E6) and CHA transfected vectors or protein expression.\u003c/p\u003e","description":"","filename":"Diapositiva3.png","url":"https://assets-eu.researchsquare.com/files/rs-7695228/v1/bbcbcfa9a09b34c1a8abb1d1.png"},{"id":93019289,"identity":"6d7b773e-d129-4731-8390-9bdfd6e702af","added_by":"auto","created_at":"2025-10-08 08:37:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":285033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrosstalk NOTCH1/TCFL5 in promoter regulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Promoter structure of TCFL5/CHA. Regions identified as possible promoters and the regions used as controls are indicated. B) Quantification of the CHIP assay (n=4) to the indicated region of TCFL5 or CHA promoters (shown in A) with anti-ICN1, anti-pan TCFL5 or normal IgG relative to no antibody added. Data are presented as mean of the 4 independent experiments performed. C) Quantification of CHIP assay (n=4) of BCL2, CD2 or HES11 promoters with anti-ICN1, anti-pan TCFL5 or normal IgG relative to no antibody added. Data are presented as mean of the 3 independent experiments performed D) Effect of active NOTCH1 and its activators or repressors, MAML or dnMAML on pCHA and pTCFL5 promoter activity measured by luciferase activity. Data are presented as mean of the 3 (pCHA) or 5 (pTCFL5) independent experiments performed. *p \u0026lt; 0.05, **p \u0026lt; 0.01 and,***p \u0026lt; 0.001 ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Diapositiva4.png","url":"https://assets-eu.researchsquare.com/files/rs-7695228/v1/5ca85a5dcbc48a6d5d2ef495.png"},{"id":93022159,"identity":"92158a6c-04a9-4ab5-bf7a-4e067a20aee5","added_by":"auto","created_at":"2025-10-08 09:01:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1969476,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7695228/v1/73edc5cc-ff3b-40e1-b5e3-2fbceeac806d.pdf"},{"id":93021405,"identity":"8720194d-d173-4724-be21-5e30c1b611d9","added_by":"auto","created_at":"2025-10-08 08:53:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":657883,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting information. Supplementary methods and Figures\u003c/p\u003e","description":"","filename":"SUPPORTINGINFORMATIONrevisedBloodcopia.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7695228/v1/9777b4319f39bcf2298b47a5.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAlternative Splicing of Tcfl5 Fine-tunes Notch1 Transcriptional Output and Cell Fate Decisions in T-cell Acute Lymphoblastic Leukemias\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by the proliferation and accumulation of immature T-cell precursors arrested in differentiation, primarily affecting the bone marrow, blood, lymphoid organs, mediastinum, and central nervous system. Although T-ALL can affect individuals of any age, most cases occur in children. T-ALL accounting for approximately 10\u0026ndash;15% of pediatric and 25% of adult acute ALL cases (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Despite advances in treatment, refractory and relapsed T-ALL cases have poor prognoses (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eT-ALL is genetically heterogeneous and driven by a multistep leukemogenic process involving both intrinsic genetic lesions and extrinsic microenvironmental factors (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These alterations disrupt the normal proliferation, differentiation and survival during thymocyte development. Key molecular hallmarks include activating mutations in NOTCH1, found in over 60% of T-ALL cases, making it a major oncogenic driver (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In this regard it has been shown that constitutively active NOTCH1 promotes leukemogenesis by activating the NF-κB pathway, both transcriptionally and via the IκB kinase (IKK) complex (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This activation leads to the upregulation of antiapoptotic genes such as Bcl2, enhancing cell survival. Several others NOTCH1 target genes have been identified, including DTX1 (Deltex1) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and HES1 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTranscription Factor Like 5 (TCFL5) is a member of the basic Helix-Loop-Helix (bHLH) family of transcription factors. Initially identified in the testis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), it has been predominantly studied in spermatogenesis, being expressed in primary spermatocytes during the pachytene stage (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In humans, the TCFL5 gene comprises eight exons, with the class C bHLH domain located between exons 4 and 5 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). A shorter isoform, CHA, was first described in the T-ALL derived Jurkat cell line and identified as an autoantigen in Chagas disease, due to its homology with \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e proteins (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Recently, an extensive characterization of the locus has been performed leading to the identification of four different isoforms: TCFL5_E8, commonly referred to as the canonical TCFL5 isoform, TCFL5_E7, TCFL5_E6, and CHA (E2B-E8) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Although all isoforms retain the bHLH domain, suggesting shared a core functionality, they differ in their 5' and 3' ends, with two possible initial exons (E1 and E2b) and three alternative terminal exons (E8, E7, and E6). The presence of two initial exons suggests the existence of alternative promoters, potentially contributing to differential regulation. CHA (TCFL5 E2B-E8) interacts with another bHLH factor, Upstream Stimulatory Factor 1 (USF1), and binds to the CD2 promoter repressing its expression in T cells where it plays a role in T cell activation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTCFL5 is upregulated in colorectal cancer (CRC), being its expression higher in carcinomas than in adenomas, likely due to chromosome 20q amplification (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Silencing TCFL5 in colon cancer cells reduces proliferation, multicellular tumor spheroid formation, and increases cell viability (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). TCFl5 mRNA expression has also a prognostic value in hepatocellular carcinoma (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTCFL5 has also been suggested as a target of the NOTCH1 signaling pathway in thymocytes and its expression correlates with an increase in double-positive thymocytes (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In B cells, activation of Toll-Like Receptors 7 and 9 induces TCFL5 expression (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Conversely, during monocyte differentiation, TCFL5 expression is downregulated (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) Interestingly TCFL5 is notably upregulated in B-ALL (ETV6-RUNX1) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) and in ALL cases resistant to vincristine treatment (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In those B-ALL samples, the TCFL5 promoter was significantly hypomethylated, correlating with increased gene expression (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study. we investigated the role of TCFL5 in T-ALL. This transcription factor was highly elevated patient samples and in T-ALL cell lines. All four isoforms are present, with CHA and TCFL5_E8 being the most abundant. Functional assays reveal that TCFL5_E8 and TCFL5_E6 inhibit, while CHA promotes leukemia cell growth. However, knocking out all isoforms reduces cell proliferation. Mechanistically, TCFL5 interacts with NOTCH1, binding to the same promoter regions of several genes, including BCL2 and HES-1 as well as those of CHA and TCFL5. Growth-promoting isoforms enhance NOTCH1 activity, while inhibitory ones suppress it. Inhibiting NOTCH1 increases TCFL5_E8 but decreases CHA expression, highlighting TCFL5\u0026rsquo;s significant role in T-ALL progression through its interplay with NOTCH.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHuman samples\u003c/h2\u003e\u003cp\u003e In the present study, 38 bone marrow samples from paediatric T-ALL patients were collected at the Hospital Infantil Universitario Ni\u0026ntilde;o Jes\u0026uacute;s (HUNJ) under the SEHOP-PETHEMA 2013 protocol guidelines (Supplemental information). All participants signed the informed consent following the institutional Ethic Committee approved protocol (internal code R-0017/13). Bone marrow mononucleated cells (BMMCs) were isoloted using Ficoll-plaque and frozen in liquid nitrogen in a solution of 90% fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO). Healthy human PBMC and thymocytes were used as controls.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell growth\u003c/h3\u003e\n\u003cp\u003eCell growth was eva\u0026ntilde;uated by cell counting with trypan blue exclusion or by MTT assay as described (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Briefly, 100 \u0026micro;L of cell suspension was plated in 96-well plates (triplicates), incubated at 37\u0026deg;C with 5% CO₂, and treated with MTT reagent. After 5h, absorbance was measured at 550 nm wavelength. Blank wells contained all reagents except cells.\u003c/p\u003e\n\u003ch3\u003eGenetic manipulation of T-ALL cell lines\u003c/h3\u003e\n\u003cp\u003eElectroporation, nucleofection and lentiviral transduction were alternatively used for introducing foreign genetic material in T-ALL lines. Briefly, cells (5x10⁶) were electroporated with plasmid DNA encoding the various isoforms (Supplemental information) (15 \u0026micro;g of the plasmid of interest and 25\u0026ndash;35 \u0026micro;g of carrier DNA), using a modified protocol optimized for high-efficiency DNA delivery (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Electroporation was performed using a square-wave pulse of 960 \u0026micro;F and 260 mV for approximately 22 ms. Following electroporation, cells were transferred to pre-warmed RPMI-1640 medium supplemented with 5\u0026ndash;20% FCS and incubated at 37\u0026deg;C. Transgene expression was analyzed at 24\u0026ndash;72 h.\u003c/p\u003e\u003cp\u003eFor Tet-ON inducible expression in Jurkat, cells, 1,2\u0026times;10⁶ cells were resuspended in 100 \u0026micro;L of Cell Line Nucleofector Solution V (Amaxa-Lonza) at room temperature, pre-mixed with 20 \u0026micro;g of indicated plasmid DNA with the reverse tetracycline-controlled transactivator (rtTA), followed by a tetracycline-responsive element (TRE) (VectorBuilder) upstream of the TCFL5 isoforms (Supplemental information) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The cell-DNA suspension was subjected to electroporation using program O-006 on the Nucleofector I device (Lonza). Post-nucleofection, cells were transferred to 12-well plates containing 1.5 mL of complete medium per well and collected 24 hours later for subsequent analyses (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Stable clones were selected with hygromycin and induced with doxycycline (1 \u0026micro;g/mL).\u003c/p\u003e\u003cp\u003eLentiviral particles were generated in HEK293 cells via transient co-transfection with the packaging plasmids pCMV-Vsvg and pCspV, along with expression constructs (Supplemental information) including pLenti-CMV/TO-Hygro or pLenti-CMV/TO-Hygro-TCFL5 for overexpression studies, and pLenti-KO1-puro or pLenti-KO1-shTCFL5 variants (OriGene) for gene knockdown experiments or pHHRSin_IRES_EGFP constructs for NOTCH pathway (Supplemental information). Viral supernatants were collected at 24 and 48 h post-transfection, filtered, and used to transduce cells. Selection was performed with hygromycin (200 \u0026micro;g/mL) for overexpression line or puromycin (4 \u0026micro;g/mL) for knockdown lines.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromatin Immunoprecipitation (ChIP).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChIP was performed as described (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). 2 \u0026times; 10⁷ cells cross-linked with 1% formaldehyde in PBS for 10 minutes at room temperature, quenched, and washed with cold PBS- Nuclei were isolated and lysed, followed by chromatin shearing via sonication (10 cycles of 30 s at high power). Lysates were centrifuged at 14,000 rpm for 15 m at 4\u0026deg;C. A fraction corresponding to 5 \u0026times; 10⁵ cells was reserved as input control. The remaining chromatin was incubated overnight at 4\u0026deg;C with 8 \u0026micro;g of antibody (anti-TCFL5, anti-ICN1 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), or rabbit IgG control) under gentle agitation. Immunocomplexes were captured using protein A-agarose beads (Millipore) according to the manufacturer\u0026rsquo;s protocol. Chromatin was eluted at 65\u0026deg;C for 16 h, followed by RNase A and proteinase K treatment. DNA was purified using phenol/chloroform:/isoamyl alcohol extraction and analyzed by quantitative PCR (qPCR) using promoter-specific primers (Supplemental Table\u0026nbsp;2).\u003c/p\u003e\n\u003ch3\u003eRNA and Protein analysis\u003c/h3\u003e\n\u003cp\u003eRNA was isolated and reverse transcribed as described (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). cDNA was used for PCR using GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA) or quantitative PCR using GoTaq1 Master Mix (Promega) using specific gene primers for indicated mRNAS, entire TCFL5 isoforms mRNAs (Supplemental Table\u0026nbsp;3) or exon junctions (Supplemental Table\u0026nbsp;4) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). qPCR was conducted using the ABI PRISM 7900HT system (Applied Biosystems Relative mRNA expression was calculated using the ΔΔCt method, normalized to GAPDH as described (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Each reaction was performed in triplicate.\u003c/p\u003e\u003cp\u003eImmunoblotting was performed basically as described (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Equal amounts (15 \u0026micro;g) were separated by SDS-PAGE, transferred to nitrocellulose membranes (BioRad) and probed with anti-TCFL5, anti ICN1, anti-HSP90 and anti-beta actin (Santa Cruz) Supplemental Table\u0026nbsp;5) in 5% BSA-TBST overnight at 4\u0026deg;C. Detection was performed using HRP-conjugated secondaries and chemiluminescence (Pierce). Band intensity was quantified with ImageJ software.\u003c/p\u003e\n\u003ch3\u003eLuciferase reporter activity assays\u003c/h3\u003e\n\u003cp\u003eJurkat cells were seeded in 24-well plates at 70% confluency and transfected by electroporation with the Notch responsive element (NRE) luciferase reporter plasmid (BPS Bioscience) and appropriate pCDNA3-TCFL5-Flag isoforms (Supplemental information) alongside a constitutively expressed Renilla luciferase plasmid as a transfection control. CHA ad TCFL5 promoter regions were cloned in front of a luciferase reporter gene (Supplemental information) and co transfected with Notch1 pathway plasmids. Transfections were performed using Metafectene Pro (Biontex) according to the manufacturer\u0026rsquo;s protocol. After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega), following the manufacturer\u0026rsquo;s instructions. Luminescence was quantified using the FLUOstar OPTIMA plate reader (BMG LabTech).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using the GraphPad Prism v6.0 program (GraphPad Software, LLC). Student's t-test was used to compare the means of quantitative data. In the case of large amounts of data, outliers were identified and removed from the statistical analysis. All experiments were represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation). ns, non significant *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eTCFL5 is overexpressed in T-ALL clinical samples.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBy analyzing several databases, we observed higher expression levels of TCFL5 mRNA in T-ALL pediatric patients compared to healthy PBMCs or thymocytes controls (Supplemental Figure S1). We confirmed this finding in 38 T-ALL samples obtained from children. QPCR analysis detecting all \u003cem\u003eTCFL5\u003c/em\u003e transcripts indicated a variable expression, although on average much higher-than- control healthy patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, these studies did not discriminate among the different transcript variants, so we performed a detailed isoform analysis in this cohort based on our previous identification of four TCFL5 isoforms in CRC (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Amplification of E3-E4 was able to detect all TCFL5 transcripts and showed a similar expression pattern than \u003cem\u003eTCFL5\u003c/em\u003e mRNA by PCR. However, specific exon amplification indicates, a different behavior of the four alternative transcripts. Quantitative levels of TCFL5_E6 mRNA, detected by detection of E5-E6 exon junction were low in control samples (not shown) but were significantly higher in T-ALL patients. TCFL5_E7 mRNA, detected by E5-E7, was also low in control samples but tend to be relatively lower in T-ALL. Unfortunately, TCFL5_E8 mRNA could not be specifically detected through exons junction. However, E1-E2 and E5-E8 levels show also a higher highest expression in T-ALL, \u003cb\u003ew\u003c/b\u003ehereas E2b-E3 (detecting only Cha) was relatively a little lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We then explored whether the relative expression of the different isoforms was associated with several clinical parameters commonly employed for risk stratification of T-ALL pediatric patients. Notably, high risk cases showed increased levels of E5-E6 and E5-E8 isoforms but reduced levels of the E2b-E3\u0026middot;isoform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTCFL5 Isoform-Specific functions in T-ALL Cell Lines\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of TCFL5 in T-ALL, we first analyzed isoform expression in T-ALL cell lines. Higher expression of CHA (E2B-E8) followed by TCFL5 E1_E8 and much lower levels of TCFL5_E6 and TCFL5_E7 were detected by RT-QPCR in Jurkat cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and Molt 4 (Figure S2). Western blot analysis also revealed robust CHA (E2B-E8) expression and of TCFL5 E1_E8 in both Jurkat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and MOLT-4 cells (Figure S2A). Isoforms TCFL5_E6 and TCFL5_E7 were also detected albeit at much lower levels.\u003c/p\u003e\u003cp\u003eNext, we employed lentiviral shRNAs targeting different regions of the TCFL5 transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Two constructs (sh12 and sh90), designed to silence all isoforms, significantly inhibited Jurkat cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, shRNAs targeting the 3\u0026prime;UTR of E8-containing isoforms (CHA and TCFL5 E1-E8) had only modest effects. qPCR confirmed that sh12 and sh90 effectively suppressed all isoforms, while the 3\u0026prime;UTR-targeting shRNAs selectively reduced, but not suppressed, E8 and to a lesser extent, E6 and E7 containing isoforms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These finding suggest a potential regulatory interplay between isoforms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo dissect isoform-specific functions, we generated T-ALL lines individually overexpressing each isoform. TCFL5_E8 and TCFL5_E6 overexpression by electroporation markedly impaired proliferation and viability. CHA modestly enhanced growth, while TCFL5_E7 had no significant effect in both Jurkat (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and MOLT-4 cells (Figure S2B). All transfected cells resulted in the expression of the transfected proteins, by FLAG detection (Figure S3). On the other hand, stable overexpression of TCFL5_E8 isoform by a different approach as lentivirus also resulted in a complete loss of proliferation, decreased cell viability and eventually cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eJurkat Tet-ON lines were established with doxycycline-inducible expression of each isoform. All established lines grew similarly without doxycycline (data not shown). Upon induction, all isoforms variably increased in a time-dependent manner (Figure S3C). Consistent with above observations, induction of TCFL5_E6 or TCFL5_E8 halted cell proliferation, whereas CHA, which was highly overexpressed after doxycycline, strongly enhanced it (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eInterestingly, overexpression of some isoforms also influenced endogenous levels of the rest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, S3). Thus, the stable (Figure S3B) or induced (Figure S3 C,D) overexpression of TCFL5_E8 significantly reduced CHA. TCFL5_E6 transfection resulted in a very high increase in its own levels in all transfected cell and decreased CHA. In contrast, TCFL5_E7 transfection had no significant impact on the other isoforms. These findings indicate isoform-specific cross-regulation.\u003c/p\u003e\u003cp\u003eTo explore the transcriptional impact of TCFL5 isoforms, we analyzed the expression of some genes implicated in T-ALL pathogenesis. Overexpression of CHA (E2B\u0026ndash;E8) in TALL cell lines significantly upregulated \u003cem\u003eHES1\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e and \u003cem\u003eDTX1\u003c/em\u003e mRNA levels, whereas overexpression of TCFL5_E8 and TCFL5_E6 repressed their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, S2D). Conversely, \u003cem\u003eNFKBIA\u003c/em\u003e was induced by TCFL5_E8 and TCFL5_E6 but downregulated by CHA. \u003cem\u003eDUX4\u003c/em\u003e was mostly induced by TCFL5_E8 but was downregulated by TCFL5_E6. The results revealed distinct and sometimes opposing effects depending on the isoform.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCrosstalk Between TCFL5 and NOTCH1.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInterestingly, those TCFL5 induced transcriptional changes were accompanied by alterations in the NOTCH1 pathway. Upon ligand binding, NOTCH1 undergoes sequential proteolytic cleavages, releasing the Notch intracellular domain, which translocate to the nucleus and forms a transcriptional activation complex with CSL and MAML. DAPT, a γ-secretase inhibitor, blocks the final cleavage step thereby inhibiting downstream signaling (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Activation of the NOTCH1 pathway via enforced stable ICN1 or MAML1 overexpression enhanced Jurkat cell proliferation, whereas dominant-negative MAML1 (dnMAML) suppressed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Inhibition of NOTCH1 signaling using the γ-secretase inhibitor DAPT reduced cell growth in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCHA overexpression increased ICN1 protein levels, while TCFL5_E8 and TCFL5_E6 slightly reduced them although not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, S3E). These changes were functionally relevant: luciferase reporter assays in Jurkat cells showed that CHA overexpression enhanced NOTCH1-responsive transcription, whereas TCFL5_E8 and TCFL5_E6 suppressed it (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The addition of DAPT decreased, as expected, the transcriptional activity of NOTCH-1 in a dose-dependent manner. The repressor activity of TCFL5_E8 or TCFL5_E6 on NRE was not significantly further affected by DAPT whereas the CHA-induced increase in activity could be still reduced by DAPT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These data suggest that TCFL5 and DAPT converge at the same point in the NOTCH-1 pathway. Conversely, manipulation of NOTCH pathway altered TCFL5 isoform expression. Thus, DAPT treatment induced TCFL5_E8 while significantly repressing CHA in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDual Promoter Occupancy by TCFL5 and NOTCH1\u003c/h2\u003e\u003cp\u003eTo investigate the transcriptional regulation of TCFL5 isoforms, we analyzed their promoter regions. Previous work from our group identified a transcription start site for the CHA isoform that is distinct from the E1 containing mRNA isoforms (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In silico analysis revealed three putative regulatory regions within the TCFL5 locus, upstream of exon1, in exon 1 and upstream of CHA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Chromatin immunoprecipitation (ChIP) assays using anti-TCFL5 antibodies specifically bound to two regions in the TCFL5 locus: one upstream of E1-containing transcripts and another upstream of the CHA mRNA transcription start sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), supporting the hypothesis of both isoform-specific promoter regulation, and cross regulation among TCFL5 isoforms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we explored whether NOTCH1 also participates in the transcriptional regulation of the TCFL5 and CHA isoforms. Bioinformatic analysis identified transcription factor binding motifs for TCFL5 (E-box) and NOTCH1 (RBPJ) within these small DNA regions bound by TCFL5 shown above. ChIP assays using ICN1 antibodies confirmed that NOTCH1 binds to the same two promoter regions in TCFL5 upstream of the E1 and E2B\u0026ndash;E8 transcription start sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This co-occupancy suggests that both transcription factors may regulate these promoters either cooperatively or competitively. Similar dual TCFL5 and NOTCH motifs were also found in the promoters of canonical NOTCH1 target genes such as \u003cem\u003eHES1\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e. ChIP analysis detected cobinding of ICN1 and TCFL5 to those regions in \u003cem\u003eHES1\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e but only TCFL5 bound to \u003cem\u003eCD2\u003c/em\u003e promoter previously shown to bind TCFL5/USF1 in Jurkat cells (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), but that does not contain RBPJ sites, supporting the specificity of this regulatory interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo assess the functional impact of NOTCH1 signaling on TCFL5 isoform and promoters, we performed luciferase reporter assays in Jurkat with complete pTCFL5 and pCHA promoters. Transfection of ICN1 with or MAML1, a co-activator of NOTCH1, significantly increased the activity of CHA promoter. Conversely, a dominant-negative MAML construct (dnMAML) significantly repressed its activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, no significant effects were observed forTCFL5 promoter.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eT-ALL is a biologically and clinically complex disease and despite advances in treatment, refractory and relapsed T-ALL cases have poor prognosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Aberrant activation of the NOTCH1 signaling pathway plays a central role in the pathogenesis of T-ALL (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Thus, a deeper understanding of the molecular drivers, particularly the transcriptional networks downstream of NOTCH1 is essential for developing targeted therapies and improving prognosis in relapsed or refractory cases. In this study, we identified TCFL5 as a novel effector of NOTCH1 in T-ALL. We found a high expression of TCFL5 mRNA consistently exceeding that observed in control cells. Despite the absence of significant number of alterations in this gene described in leukemia cases, our findings suggest elevated expression of this gene in T-ALL. In B-ALL samples with the t(12;21) chromosomal translocation, the TCFL5 promoter was significantly hypomethylated, correlating with increased gene expression (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). However, those studies did not distinguish between TCFL5 isoforms, which were previously shown to have opposite functional effects on colon cancer (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe detected higher expression of all TCFL5 isoforms in clinical T-ALL samples relative to healthy controls, with the exception of TCFL5_E7. Notably, the TCFL5_E6 isoform was significantly enriched in high-risk clinical samples, whereas CHA was expressed at lower levels in these cases. Using microarrays, TCFL5 was found elevated in T and B ALL cases resistant to vincristine treatment (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and has significant association with risk (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and bone marrow status at day 14 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, functional assays have revealed isoform-specific effects in T-ALL biology. Thus, the growth of Jurkat and MOLT4 cells was enhanced by constitutive or inducible expression of CHA and arrested by that of TCFL5_E8 and TCFL5_E6. However, growth is also inhibited by near-complete inhibition of all mRNA isoforms by lentiviral shRNAs suggesting that CHA is the primary growth-promoting isoform. These findings mirror our previous observations in colon cancer (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), highlighting the complex regulatory dynamics among TCFL5 isoforms. Cross regulation among isoforms is supported by the binding of TCFL5 to both TCFL5 and CHA promoters. Interestingly, increasing the expression of the growth-inhibitory isoform TCFL5_E8, reduced CHA levels, suggesting a competitive or negative regulatory interplay. Thus, both overexpression of TCFL5_E8 and complete TCFL5 knockdown may converge on a common growth arresting phenotype by suppressing CHA.\u003c/p\u003e\u003cp\u003eAnalysis of the potential molecular mechanisms by which TCFL5 regulates T-ALL growth has revealed an interesting crosstalk with NOTCH1. Upon ligand binding, NOTCH1\u0026mdash;a transmembrane receptor\u0026mdash;undergoes two sequential proteolytic cleavages: first by a metalloprotease and then by γ-secretase. This releases the Notch intracellular domain (ICN), which translocates to the nucleus and forms a transcriptional activation complex with CSL and MAML, driving the expression of target genes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The γ-secretase inhibitor DAPT blocks this final cleavage, thereby inhibiting downstream signaling (\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). TCFL5 isoforms exert disparate effects on NOTCH1, processing, transcriptional activity and downstream target gene expression in T-ALL. We found a direct relationship between the ability of the different TCFL5 isoforms to induce T-ALL growth and modulate NOTCH1 transcription. CHA promoted growth, in line with its ability to enhance NOTCH1 transcriptional activity as evidenced by increased NRE dependent luciferase activity and expression of canonical NOTCH1 target genes, whereas TCFL5_E8 and TCFL5_E6 inhibited those activities. This may result from the observed increase by CHA and decrease by the other two isoforms in active ICN1 levels. Importantly, the CHA-mediated activation of NRE was inhibited by DAPT, indicating that this effect is dependent on active NOTCH1 signaling. Conversely, pharmacological inhibition of NOTCH1 processing with the γ-secretase inhibitor DAPT inhibited growth of T-ALL lines associated with a decrease in CHA and an increase in TCFL5_E8 respectively, suggesting that NOTCH1 activity not only regulates TCFL5 expression but also influences isoform balance.\u003c/p\u003e\u003cp\u003ePrevious studies have suggested that TCFL5 is a transcriptional target of NOTCH1 in double-negative thymocytes (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Our findings extend this observation by demonstrating that NOTCH1 binds to the promoters of both canonical TCFL5 (E1-containing) and alternative CHA isoforms. However, activation of the NOTCH1 pathway results in different outcomes for both promoters, activating pCHA without affecting pTCFL5. The TCFL5 isoforms also differentially regulate key NOTCH-related genes involved in T-ALL biology. CHA overexpression induces \u003cem\u003eHES1\u003c/em\u003e expression, a critical NOTCH1 target and mediator of leukemogenesis, and promotes cell proliferation, whereas TCFL5_E8 and TCFL5_E6 isoforms repress \u003cem\u003eHES1\u003c/em\u003e and inhibit growth, consistent with its role in maintaining T-ALL cell survival (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). \u003cem\u003eBCL2\u003c/em\u003e, a critical antiapoptotic gene was also differentially regulated by TCFL5 isoforms; enhanced by CHA and suppressed by TCFL5_E8 and TCFL5_E6. Similarly, DUX4, implicated in leukemogenesis (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), is also differentially regulated by TCFL5 isoforms, further highlighting their distinct transcriptional programs. The E3 ubiquitin ligase \u003cem\u003eDTX1\u003c/em\u003e, which promotes NOTCH1 degradation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) was also differentially regulated. CHA increased \u003cem\u003eDTX1\u003c/em\u003e expression, while TCFL5_E8 and TCFL5_E6 reduced it. These changes may underlie the observed differences in ICN1 protein levels and thus, downstream signaling.\u003c/p\u003e\u003cp\u003eIn addition, the TCFL5 isoforms differentially modulate the NF-κB pathway, another critical axis in T-ALL. NOTCH1 is known to activate NF-κB leading to the expression of genes such as \u003cem\u003eNFKBIA\u003c/em\u003e (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Our data show that TCFL5_E8 and TCFL5_E6 induce NF-κB inhibitor IκBα (\u003cem\u003eNFKBIA\u003c/em\u003e), while CHA represses it, further supporting the notion of isoform-specific transcriptional programs. The activation of NOTCH/HES1 pathway sustains NF-κB activation in T cell leukemia (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) and modulation of this pathway may partially explain the different effect of TCFL5 isoforms. TCFL5 and NOTCH1 also co-occupy the promoters of several downstream target genes, including BCL2 and HES1, further supporting a coordinated regulatory mechanism. The cobinding of TCFL5 and ICN1 to shared promoter regions, along with their opposing effects on transcriptional activity, points to a finely tuned mechanism of isoform-specific gene regulation.\u003c/p\u003e\u003cp\u003eTogether, our results suggest that NOTCH1\u0026rsquo;s pro-leukemic effects may be partly driven by its induction of the CHA isoform, supporting a model in which NOTCH1 regulates TCFL5 isoform expression to favor growth-promoting variants. In turn, the TCFL5 isoforms differentially modulate NOTCH1\u0026rsquo;s transcriptional activity and downstream gene expression. Mechanistically, this involves the direct binding of NOTCH1 and TCFL5 to shared promoter regions\u0026mdash;including those of TCFL5, CHA, HES1, and BCL2 mRNAs\u0026mdash;highlighting a transcriptional interplay that influences the balance between proliferation and apoptosis in T-ALL cells. Overall, the data reveal a complex regulatory network where NOTCH1 activates CHA, and TCFL5 isoforms modulate NOTCH1 degradation and activity and T-ALL cell fate. The differential regulation of BCL2 and HES1 by TCFL5 isoforms links transcriptional control to cell survival, positioning TCFL5 as a key node in the NOTCH1 signaling pathway and a promising therapeutic target in T-ALL.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe are grateful to Maria Chorro, Mari Carmen Maza, Ana Flores and Carolina Maroto for their excellent technical assistance. This research was funded by \u0026ldquo;Ministerio de Ciencia e Innovaci\u0026oacute;n\u0026rdquo; (Spain), grant numbers PID2019-104760RB-I00) and PID2022-137487OB-I00. and \u0026lsquo;Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM) to MF and Institutional grants from \u0026lsquo;Fundaci\u0026oacute;n RamonAreces\u0026rsquo; and \u0026lsquo;Banco de Santander\u0026rsquo;.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eM.F. conceptualization, funding acquisition and final writing; KS conceptualization and final writing. J.G., A.G., A.G.R., A.L. and S.P. designed and performed different in vitro experiments, the statistical analysis and prepared the illustrations, and helped drafting the manuscript; F.A. performed in vitro experiments; A.G., E.G.S., M.R and M.V, provided and analyzed clinical data. All the authors have revised and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e\n\u003cp\u003eThis study was carried out in strict accordance with the European Commission legislation. All participants signed the informed consent following the institutional Ethic Committee approved protocol (internal code R-0017/13). 213.6/21).\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGianni F, Belver L, Ferrando A. The Genetics and Mechanisms of T-Cell Acute Lymphoblastic Leukemia. 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Semin Diagn Pathol. 2023;40(3):202-15.\u003c/li\u003e\n\u003cli\u003eRevici R, Hosseini-Alghaderi S, Haslam F, Whiteford R, Baron M. E3 Ubiquitin Ligase Regulators of Notch Receptor Endocytosis: From Flies to Humans. Biomolecules. 2022;12(2):224.\u003c/li\u003e\n\u003cli\u003eEspinosa L, Cathelin S, D\u0026apos;Altri T, Trimarchi T, Statnikov A, Guiu J, et al. The Notch/Hes1 pathway sustains NF-kappaB activation through CYLD repression in T cell leukemia. Cancer Cell. 2010;18(3):268-81.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Centro de Biología Molecular Severo Ochoa","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7695228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7695228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAberrant NOTCH1 signaling drives T-cell acute lymphoblastic leukemia (T-ALL). TCFL5 is highly expressed in T-ALL clinical samples, producing four isoforms with distinct expression patterns and risk associations. In leukemic cells, TCFL5 isoforms play opposing roles: the CHA isoform promotes proliferation, while TCFL5_E6 and TCFL5_E8 suppress tumor growth. Mechanistically, TCFL5 acts as a transcriptional effector and feedback regulator of NOTCH1, modulating its transcriptional output by cobinding promoters of key target genes like BCL2 and HES1. NOTCH1 reciprocally regulates TCFL5 isoform expression, forming a feedback loop that governs leukemic cell fate. Disrupting this loop\u0026mdash;via pan-TCFL5 knockout or isoform-specific modulation\u0026mdash;alters proliferation and survival in T-ALL models. These findings identify TCFL5 as a critical node in the NOTCH1 signaling network and underscore the therapeutic potential of targeting TCFL5 isoform dynamics to modulate oncogenic signaling in T-ALL.\u003c/p\u003e","manuscriptTitle":"Alternative Splicing of Tcfl5 Fine-tunes Notch1 Transcriptional Output and Cell Fate Decisions in T-cell Acute Lymphoblastic Leukemias","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 08:37:28","doi":"10.21203/rs.3.rs-7695228/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b92c24a0-5968-4d98-9523-0a8c7a1f5c1c","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55944147,"name":"Hematology"},{"id":55944148,"name":"Immunology"},{"id":55944149,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2025-10-08T08:37:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 08:37:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7695228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7695228","identity":"rs-7695228","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}