CircZBTB46, a promising therapeutic target in crizotinib resistant ALK-positive T lymphomas

CircZBTB46, a promising therapeutic target in crizotinib resistant ALK-positive T lymphomas | 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 Article CircZBTB46, a promising therapeutic target in crizotinib resistant ALK-positive T lymphomas Fabienne Meggetto, Loelia Babin, Elissa Andraos, Steffen Fuchs, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8593634/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Circular RNAs (circRNAs) are increasingly recognized as functional non-coding transcripts with oncogenic potential. Here, a comprehensive analysis of circRNA expression in primary ALK(+) anaplastic large-cell lymphoma (ALK(+) ALCL) is presented. Integrated transcriptomic profiling revealed that aberrant expression of circZBTB46 and of its linear host transcript, normally restricted to dendritic cells, is exclusive to ALK(+) lymphoma cells and driven by the oncogenic NPM1-ALK/STAT3 axis. Functional studies showed that circZBTB46, unlike its protein-coding counterpart, promotes resistance to the ALK inhibitor crizotinib. Silencing circZBTB46 restored crizotinib sensitivity in resistant ALCL cells both in vitro and in vivo . Transcriptomic analyses identified PIP5K1C as a downstream effector regulated through a competitive endogenous RNA mechanism in which circZBTB46 acts as a sponge to miR-25-3p, alleviating its repression of PIP5K1C. These findings uncover a previously unrecognized mechanism of drug resistance in ALK(+) ALCL and establish circZBTB46 as a promising therapeutic target. Biological sciences/Cancer/Haematological cancer Biological sciences/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/T-cell lymphoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The purpose of this study was to investigate circRNAs in the specific context of ALK(+) ALCL. Anaplastic large-cell lymphoma (ALCL) is a rare and aggressive peripheral T-cell non-Hodgkin lymphoma (NHL) belonging to CD30 + lymphoproliferative disorders( 1 ). ALCL is divided into two subtypes based on the expression of the anaplastic lymphoma kinase (ALK)( 2 ). The ALK(+) subtype primarily affects children and young adults. In approximately 80–85% of ALK(+) cases, the t(2;5)(p23;q35) chromosomal translocation is present, driving the fusion of the intracytoplasmic domain of ALK, on chromosome 2p23, with the N-terminal portion of nucleophosmin (NPM1), on chromosome 5q35( 3 ). This NPM1-ALK fusion protein constitutively activates key oncogenic signaling pathways, including RAS-ERK, JAK3-STAT3 and PI3K-Akt, thereby driving cancer cell proliferation, differentiation and survival( 4 ). In ALK(+) ALCL, current first-line multidrug chemotherapy (CT) regimens achieve a progression-free survival of approximately 70% at ten years after diagnosis. Crizotinib, the first ALK-targeted tyrosine kinase inhibitor (TKI), was introduced for the treatment of ALK(+) ALCL refractory or relapsing (R/R) after CT. However, the clinical efficacy of crizotinib has been hampered by the emergence of resistance. The prognosis for R/R patients remains poor, highlighting the need for a better understanding of the aggressive behavior of ALCL and the development of novel prognostic markers to identify high-risk patients at an early stage( 5 , 6 ). Previous studies have disclosed the important role of small noncoding RNAs, particularly microRNAs (miRNAs), in ALCL( 7 ). We and others have shown that specific miRNAs are associated with an increased risk of relapse in ALK(+) ALCL, after CT or crizotinib treatment( 8 – 13 ). Circular RNAs (circRNAs) are a recently recognized class of stable and conserved noncoding RNAs characterized by a covalently closed loop structure lacking 5′ and 3′ ends. Predominantly localized in the cytoplasm, circRNAs act as molecular sponges for microRNAs and RNA-binding proteins, thereby modulating post-transcriptional gene regulation. They are highly stable and often exhibit tissue- or stage-specific expression patterns. Dysregulation of circRNAs has been shown to be involved in the pathophysiology of several solid tumors and hematological malignancies, especially B-NHL and leukemias ( 14 , 15 ). A few reports have addressed the implication of circRNAs in peripheral T-cell NHL such as T-cell lymphoblastic lymphoma ( 15 , 16 ), but to date, no studies appear to have examined their role in ALK(+) ALCL. Deciphering the functions and mechanisms of circRNAs in ALK(+) ALCL could lead to the identification of innovative biomarkers development of therapeutic Results Aberrant expression of circRNA and mRNA ZBTB46 in NPM1-ALK(+) lymphoma cells. This work was initiated by investigating genome-wide circRNA expression profiles in ALK(+) ALCL via various approaches. First, a ribo-minus RNA sequencing (RNAseq) dataset, previously generated from a cohort of 39 ALK(+) ALCL biopsies, was analyzed, with 9 reactive lymph nodes (RLNs) as controls( 17 ). A circRNA expression landscape was established in ALK(+) ALCL (log2Fold Change ≥ 2 and a P value < 0.05, Fig. 1 A ) . Among upregulated circRNAs, circZBTB46 displayed the highest expression (L2FC: 3.6; Fig. 1 A and 1 B). This circRNA stood out for several reasons. First, using long-read Oxford Nanopore RNA-seq, our group has already identified circZBTB46 as one of the most abundant circRNAs expressed in four human ALK(+) ALCL cell lines( 18 ). Second, the expression of its host gene, ZBTB46 , is known to be restricted to human progenitor and conventional dendritic cells but has not been reported in T-lymphocytes( 19 , 20 ). Finally, using the RNAseq dataset, it was found that both ZBTB46 circRNA and mRNA were undetectable in RLN (Fig. 1 C). In a second step, published microarray data from peripheral T-cell lymphomas (PTCL)( 17 ) were analyzed. ZBTB46 mRNA levels were compared in samples from ALCL (n = 61), PTCL-not-otherwise specified ( PTCL-NOS, n = 71), and angioimmunoblastic T-cell lymphoma ( AITL, n = 83)( 21 , 22 ). The linear ZBTB46 transcript was found to be significantly upregulated in ALCL compared with other PTCL subtypes (Fig. 1 D ) . Next, expression levels of ZBTB46 were analyzed in four ALK(+) ALCL cell lines (COST, SU-DHL-1, SUPM2 and KARPAS-299), two ALK(-) cell lines (FEPD and Mac2a) as well as in stimulated (S) and non-stimulated (NS) normal CD3 + lymphocytes (n = 3 donors). Both RNAseq (Fig. 1 E) and RT‒qPCR (Fig. 1 F) analyses consistently revealed a strong upregulation of ZBTB46 mRNA in all ALK(+) ALCL cell lines compared with ALK(-) cell lines and normal CD3 + lymphocytes, independently of stimulation. In addition, RT-qPCR analysis showed a marked overexpression of circZBTB46 in ALK(+) ALCL cell lines compared with controls (Fig. 1 F). These findings were confirmed at the protein level via Western blotting ( Supplementary Fig. 1 ). Using the Cancer Cell Line Encyclopedia (CCLE) database and the DepMap portal website( 23 ) we found again that expression of the linear ZBTB46 transcript was far the highest in ALK(+) ALCL cell lines among lymphomas cell lines (Fig. 1 G). Finally, immunohistochemistry on primary biopsies from 19 ALK(+) and 3 ALK(-) ALCL confirmed that ZBTB46 protein expression was restricted to ALK(+) lymphoma cells (Fig. 1 H), and that no ZBTB46 expression was detected in CD68 + cells (macrophages and dendritic cells) (Fig. 1 H), Collectively, these results demonstrated that both circZBTB46 and its linear counterpart are aberrantly and quasi-exclusively expressed in ALK(+) ALCL tumoral cells. Basic information and characteristics of circZBTB46 in ALK(+) lymphoma cells. As previously reported( 18 ), circZBTB46 is a 1255-nucleotide-long circRNA produced by backsplicing exons 2 and 3 of the linear ZBTB46 transcript (ENST00000245663.9, Fig. 2 A). CircZBTB46 is listed in circBase and circBank as hsa_circ_0002805, and mapped to the genomic coordinates chr20:62407030–62422143 (hg19). Moreover, it is annotated as circZBTB46( 2 , 3 ).1 according to the standardized nomenclature for eukaryotic circular RNAs( 24 ). The circular nature of circZBTB46 in the ALK(+) ALCL cell line SU-DHL-1, was confirmed by resistance to exonuclease digestion (RNase R), unlike linear ZBTB46 mRNA (Fig. 2 B). Furthermore, upon transcription arrest by actinomycin D, circZBTB46 remained stable for 8h while its linear counterpart declined rapidly (Fig. 2 C). Finally, subcellular fractionation revealed that circZBTB46 was predominantly localized in the cytoplasm (Fig. 2 D). Collectively, these results support that circZBTB46 is a bona fide circular RNA transcript with a cytoplasmic localization. The NPM1-ALK/STAT3 axis is responsible for the aberrant accumulation of circRNA and linear ZBTB46 transcripts in ALK(+) lymphoma cells. The consistent upregulation of both circRNA and linear ZBTB46 transcripts in NPM1-ALK(+) cell lines and patient primary biopsies suggests that the NPM1-ALK fusion protein plays a central role in driving this overexpression. This was investigated by analyzing ZBTB46 linear mRNA levels in T-cells ectopically expressing NPM1-ALK, using two complementary models, i) primary human CD4 + T-lymphocytes transduced with a lentiviral vector encoding NPM1-ALK (M1 model)( 25 ) and ii) T-cells engineered to carry the canonical t(2;5)(p23;q35) translocation via CRISPR-Cas9 genome editing (ALKIma1 model)( 18 ). In both models, previously generated RNAseq datasets were used to demonstrate robust induction of ZBTB46 mRNA following immortalization and transformation of CD4 + T-cells by the NPM1-ALK oncogene (Supplementary Supplementary Fig. 2A and 2B ). To further determine whether the tyrosine kinase activity of NPM1-ALK is required for this regulation, the ALK(+) ALCL cell line COST was treated with crizotinib. TKI treatment indeed efficiently inhibited ALK kinase activity, as indicated by the loss of NPM1-ALK autophosphorylation (p-ALK). A concomitant reduction in STAT3 activation was evidenced by decreased p-STAT3 levels in Western blot (Fig. 3 A). RT‒qPCR and Western blot further demonstrated a significant downregulation of both circZBTB46 and linear ZBTB46 transcripts (Fig. 3 B), as well as reduced ZBTB46 protein levels. (Fig. 3 C). siRNAs against NPM1-ALK (siALK) or STAT3 (siSTAT3) were then used in two ALK(+) ALCL cell lines, COST and SUP-M2. Efficient knockdown of NPM1-ALK and STAT3 was confirmed at the protein level (Fig. 3 D). RT‒qPCR analysis demonstrated that knockdown of either NPM1-ALK or STAT3 strongly decreased circZBTB46 and linear ZBTB46 expression in both cell lines (Fig. 3 E), indicating that NPM1-ALK-dependent STAT3 signaling is critical for driving ZBTB46 accumulation and circZBTB46 production. Whether STAT3 binds directly to the ZBTB46 locus was investigated by analyzing publicly available STAT3 ChIP-seq datasets (GSE117164) generated from two ALK(+) ALCL cell lines (JB6 and SU-DHL-1) exposed or not to crizotinib( 26 ). As a positive control, strong STAT3 binding peaks were observed at the regulatory regions of IRF4 , a known direct STAT3 target in ALK(+) ALCL cells( 27 ). Two main STAT3 binding peaks were detected in the ZBTB46 locus in untreated cells, whereas TKI treatment significantly reduced STAT3 occupancy at these sites, consistent with the loss of STAT3 activation ( Supplementary Fig. 2C ). ChIP‒qPCR, using a STAT3-specific antibody in the ALCL cell lines SUP-M2 and COST, and two independent primer pairs within the ZBTB46 locus, allowed for precise quantifications of STAT3 binding (ZBTB46_1 and ZBTB46_2, Fig. 3 F). Enrichment of STAT3 binding at its two binding sites was confirmed in the ZBTB46 locus (Fig. 3 G). Together, these data support a direct regulatory role of the NPM1-ALK/STAT3 axis in the aberrant accumulation of linear and circRNA ZBTB46 transcripts in ALK (+) ALCL cells. CircZBTB46 promotes crizotinib resistance in ALK(+) lymphoma cells. To dissect the respective roles of circZBTB46 and its linear mRNA counterpart, CRISPR-Cas9 knockout models were generated in the COST cell line. Since both the zinc finger domain (ZNF) and nuclear localization signal (NLS) of ZBTB46 are encoded by exon 4, two guide RNAs targeting exons 4 and 5 were used, to induce loss of function of the ZBTB46 protein (Fig. 4 A). Deletion of exon 4 in three independent clones, Del4-5#C2, #C5 and #E2, was confirmed via genomic PCR (supplementary Supplementary Fig. 3A ) and RT‒qPCR ( Fig. 4 B ) . This strategy resulted truncated ZBTB46 protein expression, as confirmed in Western blot, using an antibody directed against an epitope encoded by exon 2 (supplementary Supplementary Fig. 3B ). Additional clones lacking both the functional protein (i.e. mRNA) and circZBTB46 were generated using guide RNAs targeting exons 2 and 5 (Fig. 4 C), resulting in the deletion of exons 2 to 4. DNA PCR and RT‒qPCR confirmed successful deletion in three clones, Del2-5#A2, #A3, and #E2 (Fig. 4 D and supplementary Supplementary Fig. 3A ). As expected, these clones exhibited loss of both ZBTB46 protein (supplementary Supplementary Fig. 3B ) and circRNA (Fig. 4 D). In immunofluorescence, the full-length ZBTB46 transcription factor was localized in the nucleus of parental wild-type COST cells (Fig. 4 E), while it was in the cytoplasm of Del4-5 clones (Supplementary Supplementary Fig. 3C ) and absent in Del2-5 clones (Fig. 4 E and Supplementary Supplementary Fig. 3B-C ). The proliferation and survival rates of parental Del4-5 and Del2-5 clones were comparable (Supplementary Supplementary Fig. 3D-E ). However, after 48h of treatment with crizotinib, both Del4-5 and Del2-5 clones exhibited increased survival compared with parental wild-type cells (Fig. 4 F). Importantly, Del2-5 clones, lacking circZBTB46 expression, presented significantly less TKI resistance than clones retaining this circRNA (Del4-5). To gain deeper insight into this observation, selective siRNA downregulation of circZBTB46 in the COST and KARPAS-299 cell-lines, was designed to target the back-splice junction and specifically deplete circZBTB46 without affecting the linear ZBTB46 transcript (siCircZBTB46#1 and #2; Fig. 5 A ) . After 48h of crizotinib treatment, both cell lines transfected with circZBTB46-targeting siRNAs exhibited reduced survival compared with control siRNA-treated cells (Fig. 5 B), supporting the involvement of circZBTB46 in crizotinib resistance in ALK(+) ALCL. Based on these results, a CRISPR/Cas13 lentiviral system was used to stably suppress the expression of circZBTB46 (Cas13_circZBTB46) in the COST, SUPM2 and Pio cell lines. This approach resulted in an approximately 50% reduction in circZBTB46 levels without affecting ZBTB46 mRNA or protein expression (Fig. 5 C, Supplementary Supplementary Fig. 4 ). Stable circZBTB46 knockdowns were used to assess the effect of prolonged crizotinib exposure (7 days, 100 or 200 nM). Compared with controls (Cas13_CTL), the three transduced cell lines (Cas13_circZBTB46) exhibited significantly reduced viability upon treatment (Fig. 5 D), indicating increased sensitivity to TKI. Loss of circZBTB46 restores the sensitivity of resistant ALK(+) ALCL cells to crizotinib. To explore the therapeutic potential of targeting circZBTB46 in crizotinib-resistant ALK(+) ALCL, siRNA-mediated inactivation of circZBTB46 was performed in MTK PDX-derived cells, obtained from a patient with crizotinib-refractory disease( 28 ). Two independent siRNAs targeting circZBTB46 were used to ensure specificity of the observed effects (Fig. 5 E). Silencing circZBTB46 did not affect cell viability in the absence of treatment (Fig. 5 F). However, after 48h of crizotinib exposure, transfected cells showed increased sensitivity to crizotinib compared with those treated with control siRNAs (Fig. 5 G). We next generate stable circZBTB46-knockdown cell lines via shRNA lentiviral transduction (shCircZBTB46). Owing to the low transduction efficiency of MTK PDX-derived cells, a previously established crizotinib-resistant derivative of the COST cell line (COSTR200), was used( 29 ). The effective and specific downregulation of circZBTB46 was confirmed (Fig. 5 H). As expected, circZBTB46 knockdown sensitized COSTR200 cells to crizotinib in vitro (Fig. 5 I ) . CircZBTB46 induces crizotinib resistance by modulating PIP5K1C gene expression in ALK(+) ALCL To identify molecular pathways regulated by circZBTB46, RNA-Seq was performed after the transfection of two independent siRNAs targeting circZBTB46 in crizotinib-resistant MTK PDX-derived cells. Both siRNAs induced comparable transcriptional changes, particularly downregulation of PIP5K1C and DHRS9 (|log₂Fold Change| ≥ 0.5, P < 0.05) (Fig. 6 A). The downregulation of PIP5K1C was validated by RT‒qPCR and Western blotting, confirming reduced expression at both mRNA and protein levels in all cellular models previously described (Fig. 6 B-D). By contrast, DHRS9 downregulation was not consistently observed (data not shown). Contribution of PIP5K1C to crizotinib resistance in ALK(+) ALCL was explored by CRISPR interference (CRISPRi) technology. COSTR200 cell line were transduced with dCas9-KRAB-MeCP2 fusion proteins using guide RNAs targeting the PIP5K1C promoter to achieve transcriptional silencing. As shown in Supplementary Fig. 5A , gRNA-mediated silencing of PIP5K1C resulted in a significant reduction in PIP5K1C protein expression. Compared with control cells, PIP5K1C-silenced cells exhibited a slight decreased viability in vitro ( Supplementary Fig. 5B ). These results indicate that regulation of PIP5K1C by circZBTB46 participates in promoting resistance to ALK inhibition in ALK(+) ALCL. CircZBTB46 regulates PIP5K1C by sponging miR25-3 in ALK(+) ALCL cells. After RNA pull-down was performed using a back-splice junction-specific probe in ALK(+) ALCL cells, RT‒qPCR (Fig. 6 E) confirmed a successful enrichment of circZBTB46. Mass spectrometry analysis of associated proteins did not reveal any specific interactors (data not shown), whereas small RNA sequencing identified four miRNAs (cutoff of |log2Fold Change|≥ 2 and a P value < 0.05), namely, hsa-miR-22-3p, hsa-miR-222-5p, hsa-miR-10a-5p, and hsa-miR-25-3p, that were significantly enriched in the circZBTB46 pull-down compared with control conditions (Fig. 6 F). With the exception of hsa_miR_222-5p, the other three miRNAs were expressed in ALK(+) ALCL patients (Fig. 6 G), hsa-miR-25-3p being notably overexpressed compared with RLN. Using miRNA–mRNA interaction prediction algorithms (miRDB, TargetScanHuman and miRTargetLink 2.0), PIP5K1C was identified as a putative target of miR-25-3p ( Supplementary Fig. 5C-D ). Consistently, transfection of a miR-25-3p mimic in crizotinib-sensitive (Pio) and crizotinib-resistant (COSTR200) cells, as validated by RT‒qPCR analysis (Fig. 6 H), led to a reduction in PIP5K1C protein level (Fig. 6 I). Conversely, transfection of a miR-25-3p inhibitor significantly increased PIP5K1C mRNA and protein levels as observed in in COSTR200 cells, further validating this regulatory pathway ( Supplementary Fig. 5E-F) . Together, these data indicate that circZBTB46 modulates PIP5K1C levels via miR-25-3p sequestration, uncovering a novel competing endogenous RNA axis in ALK(+) ALCL. Targeting circZBTB46 in ALK(+) ALCL cells reduces tumor growth in vivo. In vitro experiments revealed that circZBTB46 is involved in crizotinib resistance in ALK(+) ALCL cells. To evaluate its potential as therapeutic target, the effect of circZBTB46 targeting on tumor growth was analyzed after subcutaneous injection in NSG mice. First, CRIPSR/Cas9 crizotinib-sensitive models were engrafted. Del2-5 and Del4-5 clones formed tumors at comparable rates (Fig. 7 A). Daily administration of crizotinib, started five days after transplantation, initially reduced the tumor volume in all groups. However, by day 15, all tumors derived from ZBTB46 RNA-depleted clones resumed growth, whereas those derived from parental (WT) cells continued to regress (Fig. 7 B). Notably, tumors derived from Del2-5 clones lacking circZBTB46 exhibited slower growth than those derived from circZBTB46(+) clones (Del4-5), corroborating in vitro findings. Next, Cas13-modified crizotinib-sensitive Pio cells were xenografted into NSG mice and treated daily with crizotinib since nine days after injection. Tumor growth was delayed in mice bearing circZBTB46-deficient tumors (Fig. 7 C). As PIP5K1C was identified as a downstream target of circZBTB46, PIP5K1C CRISPRi models were also injected in NSG mice. This resulted in a slight reduction of tumor growth upon crizotinib administration ( Supplementary Fig. 6 ), highlighting that PIP5K1C as a mediator in TKI-resistance phenotype. Finally, the crizotinib resistant shCircZBTB46 models were injected to determine whether targeting circZBTB46 in crizotinib-resistant cells could restore drug sensitivity. Mice were daily treated with crizotinib or vehicle. In the absence of TKI treatment, tumor growth did not differ between the groups. However, upon crizotinib treatment, tumors derived from shCircZBTB46 cells exhibited significantly reduced growth (Fig. 7 D ) . All these results support the contribution of circZBTB46 to crizotinib resistance in ALK(+) ALCL and identify circZBTB46 as an interesting therapeutic target of TKI-resistance to in ALK(+) ALCL disease. Discussion This study identifies the circular RNA, hsa_circ_0002805 (circZBTB46), as a key driver of crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK(+) ALCL). This circRNA, derived from exons 2 and 3 of the ZBTB46 gene, was found to be significantly and specifically upregulated in this lymphoma type. The upregulation is driven transcriptionally by the STAT3 pathway, which is activated by the oncogenic NPM1-ALK fusion protein. Using a combination of CRISPR/Cas and RNAi technologies, we functionally dissected the role of circZBTB46, finding that it, unlike its linear protein-coding counterpart, promotes resistance to crizotinib, an ALK inhibitor. This was demonstrated in both in vitro and in vivo models. Depleting circZBTB46 in resistant cells restored their sensitivity to crizotinib, highlighting its potential as a therapeutic target. Transcriptomic analysis revealed that PIP5K1C is a downstream effector of the circZBTB46-mediated resistance. Mechanistically, circZBTB46 acts as a molecular sponge for miR-25-3p, a microRNA that normally suppresses PIP5K1C expression. By sequestering miR-25-3p, circZBTB46 de-represses PIP5K1C, promoting survival signals and contributing to drug resistance. This proposed circZBTB46–miR-25-3p–PIP5K1C axis is a novel resistance mechanism in hematologic malignancies. ZBTB46 is a transcription factor normally restricted to dendritic cell precursors, where it maintains their immature state( 20 , 30 , 31 ) ,34,35 . It is also found in non-proliferative endothelial cells, where it inhibits cell proliferation( 32 ). Emerging evidence, however, points to a context-dependent role for ZBTB46 in cancer( 33 – 35 ). In acute myeloid leukemia (AML), elevated ZBTB46 is associated with poor outcomes( 36 ), and despite not being essential for normal hematopoiesis, it is crucial for the survival and proliferation of AML cells( 37 ). Similarly, circZBTB46 exhibits context-dependent functions across different diseases. In atherosclerosis, circZBTB46 promotes disease progression by activating the AKT/mTOR pathway( 38 ). In this context, circZBTB46 depletion induces apoptosis and impairs cell migration, highlighting its importance in vascular pathology. In contrast, this study found no effect on AKT/mTOR signaling in ALK(+) ALCL cells upon circZBTB46 silencing (data not shown). In AML, circZBTB46 has been identified as an oncogenic effector that promotes cell survival. It acts as a competing endogenous RNA, sponging both hsa-miR-326 and hsa-miR-671-5p. This activity increases the expression of stearoyl-CoA desaturase 1 (SCD1), an enzyme that protects cells from lipid peroxidation and ferroptotic cell death.These findings establish circZBTB46 as a novel oncogenic effector in AML and reveal an unrecognized ZBTB46 -derived noncoding RNA axis that governs cell fate through ferroptosis-related mechanisms( 36 ). Notably, both ZBTB46 linear RNA and circZBTB46 were found to be expressed at markedly higher levels in ALK(+) ALCL than in AML. This was demonstrated by comparative transcriptomic analyses using a k-mer-based approach( 39 , 40 ) applied to in-house RNA-seq data from primary biopsy samples, including ALK(+) ALCL and AML samples (IUCT-AML cohort) ( Supplementary Fig. 7 )( 41 , 42 ). RLN and established cell lines (AML and ALK(+) ALCL), including NPM1-ALK –transformed immortalized T-cell models( 18 , 25 ), and healthy T lymphocytes confirmed these data. This unexpected expression pattern underscores the context-dependent transcriptional regulation and functional plasticity of the ZBTB46 locus across distinct hematological malignancies. In fact, this study expands knowledge on the pathophysiological oncogenic potential of ALK(+) ALCL by demonstrating the role of circZBTB46 in promoting resistance to crizotinib. It was shown here that circZBTB46 is transcriptionally upregulated via the NPM1-ALK/STAT3 signaling axis, thereby linking oncogenic kinase activity to circular RNA-mediated drug resistance. These findings suggest that circZBTB46 acts as a context-specific regulator of therapy resistance by engaging survival pathways, making it a potential therapeutic target in ALK-driven hematological malignancies. In this context, PIP5K1C appears as a downstream target of circZBTB46. miR-25-3p was identified as a candidate intermediary. miR-25-3p has previously been shown to target PIP5K1C in prostate cancer( 43 ). These findings support a model in which circZBTB46 may act as a molecular sponge for miR-25-3p, thereby derepressing PIP5K1C expression and promoting survival signaling. Thus, circZBTB46 might contribute to crizotinib resistance in ALK(+) ALCL through a miR-25-3p–PIP5K1C axis, a mechanism not previously appreciated in hematologic malignancies. Interestingly, PIP5K1C was found to play a role in conferring resistance to ALK inhibition in ALCL. This lipid kinase catalyzes the phosphorylation of phosphatidylinositol 4-phosphate (PI4P) to produce phosphatidylinositol 4,5-bisphosphate (PI( 4 , 5 )P₂), which plays a key role in remodeling the actin cytoskeleton, intracellular trafficking and cell migration( 44 ). PIP5K1C phosphorylation at serine 448 has been suggested to be a potential biomarker for breast cancer progression, particularly in invasive breast cancer( 45 ). Compared with their sensitive counterparts, melanoma cells that are resistant to the PIKFYVE inhibitor WX8 exhibit elevated expression of PIP5K1C, and this resistance can be reversed by either siRNA-mediated knockdown of PIP5K1C or pharmacological inhibition of its kinase activity( 46 ). These findings suggest that PIP5K1C could mediate therapeutic resistance in multiple cancer types including hematological neoplasms. Therefore, focusing on PIP5K1C in crizotinib-resistant ALCL cells could be an effective way to overcome resistance. Methods Cell line culture The ALCL cell lines KARPAS-299, SU-DHL-1 SUPM2, Pio and COST, which carry the t(2;5)(p23;q35) translocation, were obtained from the DSMZ (German Collection of Microorganisms and Cell Culture, Leibnitz, Germany) or established locally ( 29 ). The MTK PDX-derived cell line was provided by J. Mathews and S. Turner ( 28 ), and the crizotinib-resistant KARPAS-299-CR06 cell line was provided by C. Gambacorti-Passerini ( 47 ). COST cells were exposed to increasing concentrations of crizotinib, ranging from 25 nM to 200 nM. At each step, the dose was increased only when treated cells achieved a proliferation rate comparable to that of untreated wild-type (WT) cells. All ALK(+) ALCL cell lines and PDXs were cultured in RPMI-1640 medium (Invitrogen, Waltham, MA #61870044) supplemented with 20% FBS (Pan Biotech, Aidenbach, Germany #500105M1M). For crizotinib-resistant cell lines, crizotinib was added to the medium twice a week at a concentration of 200 nM for COSTR200 and 600 nM for KARPAS-299-CR06. Crizotinib, siRNA and mimic treatments Cells were seeded at 2x10 5 cells/mL and treated at 24h with different doses of crizotinib (Selleckchem, Houston, TX, PF-02341066 #S1068). Aliquotes of 3.10 6 cells were transfected with 70 to 100 nmol siRNA/mimic (listed in Supplementary Table S1 ) using Amaxa4D nucleofector (Primary Cell kit P3, CA-137 program, Lonza Bioscience, Antwerp, Belgium). RNA pulldown iDRIP was performed as previously described ( 48 ). Probe sequences are listed in Supplementary Data Table S1 . Xenograft tumor assay Mice were housed under pathogen-free conditions in an animal room at constant temperature (20°C-22°C) with a 12-h light/dark cycle and free access to food and water. A total of 3×10 6 cells were injected subcutaneously into one flank of 5-week-old female NSG mice (Janvier Labs, Le Genest Saint Isle, France). Mouse body weight and tumor volumes were measured 3 times a week with calipers via the following formula: length × width 2 × π/6. At the end of the experiment, the mice (8 per group) were humanely sacrificed. For crizotinib-resistant cells, mice were treated with 100 mg/kg/day crizotinib starting on the day following injection. For crizotinib-sensitive cells, mice were treated with 50 mg/kg/day crizotinib, starting when the tumor size reached 300 mm 3 . Statistical analyses Data are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. Graphs and statistical analyses were performed via GraphPad Prism 10 (GraphPad Software Inc., La Jolla, CA). P values were calculated by using an unpaired two-tailed Student T test with Welch correction. CircRNA validation by PCR and Sanger sequencing The validation of circRNA back-splice junctions, circularity, and stability were performed as previously. In brief, PCR amplification was followed by Sanger sequencing to confirm the back splicing junction and the full circRNA sequence. Circularity and exonuclease resistance were assessed via RNase R treatment, whereas transcript stability was evaluated following exposure to actinomycin D. Relative expression levels were determined via RT‒qPCR via the 2-∆∆Ct method. Declarations Acknowledgments The study was supported by INSERM, Labex Toucan, Association Eva pour la vie, Féderation Grandir sans Cancer, RACCE Lions Club de Lourdes, the French Ministry of Health and the French National Cancer Institute (CircOma, PRT-K 2022-184), Fondation ARC et Fondation pour la Recherche Médicale supported by its sponsor Nagui and his production company, Banijay Production (EQU202403018019). All grants were obtained by F.M. E.A. and were supported by Labex Toucan/Laboratoire d’Excellence Toulouse Cancer, the association Cassandra, and Labex Toucan/Laboratoire d’Excellence Toulouse Cancer, EUR CARe N°ANR-18-EURE-0003 in the framework of the Programme des Investissements d'Avenir, L.B. and C.B. were supported by the Fondation de France. L.B. was supported by Fondation l’Oréal for Women in Science, Prolific Graine de Chercheur and Association Ellye. L.C. and S.F. were supported by a grant from the French Ministry of Health and the French National Cancer Institute (CircOma, PRT-K 2022--184). S.F. was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, grant no. 439441203). S.F. is a participant in the BIH-Charité Clinician Scientist Programme funded by the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health. C.P. and I.D. were supported by FONROGA. The authors thank the staff of the animal facility of UMS US006/CREFRE (Toulouse, France) for their technical assistance. This work benefited from the equipment and services of the iGenSeq core facility (genotyping and sequencing) at the ICM. Part of this work was carried out by the Paris Brain Institute (ICM) Data Analysis Core (DAC) plateform (RRID:SCR_026138, https://dac.institutducerveau.org/). We gratefully acknowledge Thomas Gareau for assistance with RNAseq data analysis. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Toulouse Hospital Biobank (DC-2020-4074; AC-2020-4031, 2020). Medical writing for this manuscript was assisted by MPIYP (MC Béné), Paris, France. Data availability The ribo zero full RNA sequencing, the long-read Oxford Nanopore RNA sequencing and STAT3 ChIP-seq datasets are available in the GEO repository (GSE160123, GSE197872 and GSE117164 respectively). The RNA-sequencing datasets generated are available from the corresponding author on reasonable request. Author contributions E.A., L.B. and F.M. designed the research study. E.A., S.F., C.P., I.D., C.Q., R.F., A.Z. and L.B. performed the experiments. E.A., L.B., S.F., C.Q. and F.M. analyzed the data. L.L. and C.Q. were involved in the diagnosis of ALCL. C.B., L.B. and C.G. performed the bioinformatic analysis. S.P. and M.B. helped with discussions and critical reading. L.B. and F.M. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Conflicts of interest Authors declare no conflict of interest. References Stein H, Foss HD, Durkop H, Marafioti T, Delsol G, Pulford K, et al. CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood. 2000;96(12):3681–95. Falini B, Bigerna B, Fizzotti M, Pulford K, Pileri SA, Delsol G, et al. ALK expression defines a distinct group of T/null lymphomas ("ALK lymphomas") with a wide morphological spectrum. Am J Pathol. 1998;153(3):875–86. Alaggio R, Amador C, Anagnostopoulos I, Attygalle AD, Araujo IBO, Berti E, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia. 2022;36(7):1720–48. Bonzheim I, Steinhilber J, Fend F, Lamant L, Quintanilla-Martinez L. ALK-positive anaplastic large cell lymphoma: an evolving story. Front Biosci (Schol Ed). 2015;7(2):248–59. Prokoph N, Larose H, Lim MS, Burke GAA, Turner SD. Treatment Options for Paediatric Anaplastic Large Cell Lymphoma (ALCL): Current Standard and beyond. Cancers (Basel). 2018;10(4). Zhang XR, Chien PN, Nam SY, Heo CY. Anaplastic Large Cell Lymphoma: Molecular Pathogenesis and Treatment. Cancers (Basel). 2022;14(7). Fuchs S, Naderi J, Meggetto F. Non-Coding RNA Networks in ALK-Positive Anaplastic-Large Cell Lymphoma. Int J Mol Sci. 2019;20(9). Dejean E, Renalier MH, Foisseau M, Agirre X, Joseph N, de Paiva GR, et al. Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas. Leukemia. 2011;25(12):1882–90. Desjobert C, Renalier MH, Bergalet J, Dejean E, Joseph N, Kruczynski A, et al. MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression. Blood. 2011;117(24):6627–37. Hoareau-Aveilla C, Merkel O, Meggetto F. MicroRNA and ALK-positive anaplastic large cell lymphoma. Front Biosci (Schol Ed). 2015;7(2):217–25. Merkel O, Hamacher F, Laimer D, Sifft E, Trajanoski Z, Scheideler M, et al. Identification of differential and functionally active miRNAs in both anaplastic lymphoma kinase (ALK) + and ALK- anaplastic large-cell lymphoma. Proc Natl Acad Sci U S A. 2010;107(37):16228–33. Montes-Mojarro IA, Steinhilber J, Griessinger CM, Rau A, Gersmann AK, Kohlhofer U, et al. CD147 a direct target of miR-146a supports energy metabolism and promotes tumor growth in ALK + ALCL. Leukemia. 2022;36(8):2050–63. Sorrentino D, Frentzel J, Mitou G, Blasco RB, Torossian A, Hoareau-Aveilla C, et al. High Levels of miR-7-5p Potentiate Crizotinib-Induced Cytokilling and Autophagic Flux by Targeting RAF1 in NPM-ALK Positive Lymphoma Cells. Cancers (Basel). 2020;12(10). Li Q, Ren X, Wang Y, Xin X. CircRNA: a rising star in leukemia. PeerJ. 2023;11:e15577. Perez de Acha O, Rossi M, Gorospe M. Circular RNAs in Blood Malignancies. Front Mol Biosci. 2020;7:109. Bonizzato A, Gaffo E, Te Kronnie G, Bortoluzzi S. CircRNAs in hematopoiesis and hematological malignancies. Blood Cancer J. 2016;6(10):e483. Daugrois C, Bessiere C, Dejean S, Anton-Leberre V, Commes T, Pyronnet S, et al. Gene Expression Signature Associated with Clinical Outcome in ALK-Positive Anaplastic Large Cell Lymphoma. Cancers (Basel). 2021;13(21). Fuchs S, Babin L, Andraos E, Bessiere C, Willier S, Schulte JH, et al. Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing. PLoS One. 2022;17(9):e0273253. Robbins SH, Walzer T, Dembele D, Thibault C, Defays A, Bessou G, et al. Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling. Genome Biol. 2008;9(1):R17. Satpathy AT, Kc W, Albring JC, Edelson BT, Kretzer NM, Bhattacharya D, et al. Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J Exp Med. 2012;209(6):1135–52. de Leval L, Rickman DS, Thielen C, Reynies A, Huang YL, Delsol G, et al. The gene expression profile of nodal peripheral T-cell lymphoma demonstrates a molecular link between angioimmunoblastic T-cell lymphoma (AITL) and follicular helper T (TFH) cells. Blood. 2007;109(11):4952–63. de Leval L, Parrens M, Le Bras F, Jais JP, Fataccioli V, Martin A, et al. Angioimmunoblastic T-cell lymphoma is the most common T-cell lymphoma in two distinct French information data sets. Haematologica. 2015;100(9):e361-4. Arafeh R, Shibue T, Dempster JM, Hahn WC, Vazquez F. The present and future of the Cancer Dependency Map. Nat Rev Cancer. 2025;25(1):59–73. Chen LL, Bindereif A, Bozzoni I, Chang HY, Matera AG, Gorospe M, et al. A guide to naming eukaryotic circular RNAs. Nat Cell Biol. 2023;25(1):1–5. Congras A, Hoareau-Aveilla C, Caillet N, Tosolini M, Villarese P, Cieslak A, et al. ALK-transformed mature T lymphocytes restore early thymus progenitor features. J Clin Invest. 2020;130(12):6395–408. Menotti M, Ambrogio C, Cheong TC, Pighi C, Mota I, Cassel SH, et al. Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma. Nat Med. 2019;25(1):130–40. Bandini C, Pupuleku A, Spaccarotella E, Pellegrino E, Wang R, Vitale N, et al. IRF4 Mediates the Oncogenic Effects of STAT3 in Anaplastic Large Cell Lymphomas. Cancers (Basel). 2018;10(1). Prokoph N, Matthews JD, Trigg RM, Montes-Mojarro IA, Burke GAA, Fend F, et al. Patient-derived xenograft models of ALK + ALCL reveal preclinical promise for therapy with brigatinib. Br J Haematol. 2023;202(5):985–94. Lamant L, Espinos E, Duplantier M, Dastugue N, Robert A, Allouche M, et al. Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL. Leukemia. 2004;18(10):1693–8. Boto P, Gerzsenyi TB, Lengyel A, Szunyog B, Szatmari I. Zbtb46-dependent altered developmental program in embryonic stem cell-derived blood cell progenitors. Stem Cells. 2021;39(10):1322–34. Shao T, Ji JF, Zheng JY, Li C, Zhu LY, Fan DD, et al. Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model. J Immunol. 2022;208(12):2686–701. Wang Y, Sun HY, Kumar S, Puerta MDM, Jo H, Rezvan A. ZBTB46 is a shear-sensitive transcription factor inhibiting endothelial cell proliferation via gene expression regulation of cell cycle proteins. Lab Invest. 2019;99(3):305–18. Smebye ML, Agostini A, Johannessen B, Thorsen J, Davidson B, Trope CG, et al. Involvement of DPP9 in gene fusions in serous ovarian carcinoma. BMC Cancer. 2017;17(1):642. Song X, Zhou K, Zhao Y, Huai C, Zhao Y, Yu H, et al. Fine mapping analysis of a region of 20q13.33 identified five independent susceptibility loci for glioma in a Chinese Han population. Carcinogenesis. 2012;33(5):1065–71. Xu N, Wang F, Lv M, Cheng L. Microarray expression profile analysis of long non-coding RNAs in human breast cancer: a study of Chinese women. Biomed Pharmacother. 2015;69:221–7. Long F, Lin Z, Long Q, Lu Z, Zhu K, Zhao M, et al. CircZBTB46 Protects Acute Myeloid Leukemia Cells from Ferroptotic Cell Death by Upregulating SCD. Cancers (Basel). 2023;15(2). Liu YY, Xiao FF, Yang BJ, Li X, Xu SN, Chen ZW, et al. Zinc finger and BTB domain-containing protein 46 is essential for survival and proliferation of acute myeloid leukemia cell line but dispensable for normal hematopoiesis. Chin Med J (Engl). 2020;133(14):1688–95. Fu Y, Jia Q, Ren M, Bie H, Zhang X, Zhang Q, et al. Circular RNA ZBTB46 depletion alleviates the progression of Atherosclerosis by regulating the ubiquitination and degradation of hnRNPA2B1 via the AKT/mTOR pathway. Immun Ageing. 2023;20(1):66. Marchet C, Iqbal Z, Gautheret D, Salson M, Chikhi R. REINDEER: efficient indexing of k-mer presence and abundance in sequencing datasets. Bioinformatics. 2020;36(Suppl_1):i177-i85. Bessiere C, Xue H, Guibert B, Boureux A, Ruffle F, Viot J, et al. Transipedia.org: k-mer-based exploration of large RNA sequencing datasets and application to cancer data. Genome Biol. 2024;25(1):266. Corre E, Soum C, Pfeifer R, Bessiere C, Dailhau S, Marboeuf C, et al. Differential prognostic values of the three AKT isoforms in acute myeloid leukemia. Sci Rep. 2024;14(1):7070. De Clara E, Gourvest M, Ma H, Vergez F, Tosolini M, Dejean S, et al. Long non-coding RNA expression profile in cytogenetically normal acute myeloid leukemia identifies a distinct signature and a new biomarker in NPM1-mutated patients. Haematologica. 2017;102(10):1718–26. Zoni E, van der Horst G, van de Merbel AF, Chen L, Rane JK, Pelger RC, et al. miR-25 Modulates Invasiveness and Dissemination of Human Prostate Cancer Cells via Regulation of alphav- and alpha6-Integrin Expression. Cancer Res. 2015;75(11):2326–36. Lin S, Tao C, Yan Q, Gao H, Qin L, Zhong Y, et al. Pip5k1c expression in osteocytes regulates bone remodeling in mice. J Orthop Translat. 2024;45:36–47. Durand N, Borges S, Hall T, Bastea L, Doppler H, Edenfield BH, et al. The phosphorylation status of PIP5K1C at serine 448 can be predictive for invasive ductal carcinoma of the breast. Oncotarget. 2018;9(91):36358–70. Roy A, Chakraborty AR, Nomanbhoy T, DePamphilis ML. PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells. Autophagy. 2023;19(9):2464–84. Ceccon M, Mologni L, Bisson W, Scapozza L, Gambacorti-Passerini C. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors. Mol Cancer Res. 2013;11(2):122–32. Chu HP, Minajigi A, Chen Y, Morris R, Guh CY, Hsieh YH, et al. iDRiP for the systematic discovery of proteins bound directly to noncoding RNA. Nat Protoc. 2021;16(7):3672–94. Additional Declarations There is NO conflict of interest to disclose. 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18:43:59","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155167,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/351694fc8b41e1299c9bf51f.html"},{"id":100622789,"identity":"c8d9ee93-552c-41cb-bf92-2eeadc4c937d","added_by":"auto","created_at":"2026-01-19 18:43:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":732328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of circZBTB46 and ZBTB46 mRNAsin ALK(+) ALCL primary tumors and normal tissues.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Volcano plot and \u003cstrong\u003e(B)\u003c/strong\u003e MA plot of circular RNA expression comparing ALK(+) ALCL primary biopsies (n=39) and healthy tissues (reactive lymph nodes, RLN, n=9). \u003cstrong\u003e(C) \u003c/strong\u003eexpression of circZBTB46 (left) and ZBTB46 mRNA (right) assessed by RNA-Seq in ALK(+) ALCL primary samples versus RLN. \u003cstrong\u003e(D)\u003c/strong\u003e ZBTB46 mRNA expression (RMA) from microarray datasets in ALK(+) ALCL (n=61), angioimmunoblastic T-cell lymphoma (AITL, n=83) and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS, n=71) primary biopsies. \u003cstrong\u003e(E)\u003c/strong\u003e ZBTB46 mRNA expression (log2 of the transcript count per million(lenghScaled TPM) from RNA-Seq data in five ALK(+) ALCL cell lines (KARPAS-299, SU-DHL-1, SUP-M2, Pio, COST), two ALK(-) ALCL cell lines (FEPD, MAC-2A) and CD3(+) lymphocytes stimulated (S, n=3) or not (NS, n=3). \u003cstrong\u003e(F)\u003c/strong\u003e Quantitative real-time PCR (RT‒qPCR) analysis of circZBTB46 and ZBTB46 mRNAs in the same cell types. MLN51 was used as an internal control. Values are expressed as 2^(–Δ)Ct relative ratios. Experiments were performed at least in triplicate. Statistical significance was assessed via an unpaired two-tailed Student’s t test with Welch’s correction: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 (\u003cstrong\u003e**\u003c/strong\u003e), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 \u003cstrong\u003e(***\u003c/strong\u003e), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 \u003cstrong\u003e(****\u003c/strong\u003e), ns = not significant. Dataare expressed as means ± SD.\u003cstrong\u003e(G)\u003c/strong\u003eZBTB46 mRNA expression across hematologicalmalignancies in the Cancer Cell Line Encyclopedia (CCLE) dataset. \u003cstrong\u003e(H)\u003c/strong\u003e Representative immunohistochemical image of an ALK(+) ALCL primary tumor showing ZBTB46 (brown, arrowhead) and CD68 (red, arrow) expression. Cell nuclei were counterstained with hematoxylin(blue). Original magnification, ×24.6.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/f5f057ebf747c2d6cab43380.png"},{"id":100622903,"identity":"87bfb899-e45b-47e5-a60e-f50e0660e437","added_by":"auto","created_at":"2026-01-19 18:44:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of circZBTB46 in the SU-DHL-1 ALK(+) ALCL cell line.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eSchematic representation of the circZBTB46 structure with Sanger sequencing of the back-splice junction. \u003cstrong\u003e(B) \u003c/strong\u003eQuantitative real-time PCR (RT‒qPCR) analysis of circZBTB46 and ZBTB46 mRNA expression in cells treated or not with RNase R, a 3′-5′ exonuclease that degrades linear RNAs but spares circular RNAs.\u003cstrong\u003e (C) \u003c/strong\u003eRT‒qPCR analysis after treatment with actinomycin D, an inhibitor of RNA synthesis, to assess transcript stability. 18S rRNA was used as an internal control.\u003cstrong\u003e \u003c/strong\u003eExpression values are displayed as 2-∆∆Ct relative ratios.\u003cstrong\u003e (D) \u003c/strong\u003eSubcellular localization of circZBTB46 and ZBTB46 mRNAsdetermined by cellular fractionation. MALAT1 and GAPDH mRNAs were used as nuclear and cytoplasmic controls, respectively. All experiments were performed in triplicate. Statistical significance was assessed via an unpaired two-tailed Student t test with Welch correction: \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 (\u003cstrong\u003e*\u003c/strong\u003e). Data are expressed as means ± SD.\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/cc3ca1fde1aeaf00e7dd0786.png"},{"id":100622826,"identity":"75e6ccc7-0adb-46a5-bf14-830c15ed7d44","added_by":"auto","created_at":"2026-01-19 18:43:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":317829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecircZBTB46\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZBTB46\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression by ALK and STAT3 signaling in ALK(+) ALCL.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Western blot analysis of total and phosphorylated ALK (ALK, p-ALK) and STAT3 (STAT3, p-STAT3) protein levels in COST cells treated for 48 h with crizotinib (400 μM). (\u003cstrong\u003eB\u003c/strong\u003e) RT‒qPCR analysis of \u003cem\u003ecircZBTB46\u003c/em\u003e and \u003cem\u003eZBTB46\u003c/em\u003e mRNA levels following crizotinib treatment (Crizo). (\u003cstrong\u003eC\u003c/strong\u003e) Western blot analysis of ZBTB46 and p-ALK protein expression under the same conditions. (\u003cstrong\u003eD\u003c/strong\u003e) Western blot and (\u003cstrong\u003eE\u003c/strong\u003e) RT‒qPCR analysis of ZBTB46, ALK, and STAT3 expression in COST and SUP-M2 cells transfected for 48 h with control siRNA (siCTL), ALK-targeting siRNA (siALK), or STAT3-targeting siRNA (siSTAT3). GAPDH or actin were used as a loading controls. MLN51 served as an internal control for RT‒qPCR. mRNA expression values are shown as 2-∆∆Ct relative ratios. Experiments were performed at least in triplicate. (\u003cstrong\u003eF\u003c/strong\u003e) STAT3 ChIP-seq data from the JB6 cell line showing enrichment of STAT3 at the \u003cem\u003eZBTB46\u003c/em\u003elocus (data from GSE117164; (26)). (\u003cstrong\u003eG\u003c/strong\u003e) ChIP‒qPCR analysis using two primer pairs (ZBTB46_1 and ZBTB46_2) showing STAT3 enrichment at the \u003cem\u003eZBTB46\u003c/em\u003e promoter in SUP-M2 and COST cells, represented as % input. STAT3 was a positive control and ALK and GAPDH were used as negative controls. IgG was used as a nonspecific binding control. Experiments were performed at least in triplicate. Representative Western blots from three independent experiments are shown. Statistical significance was assessed via an unpaired two-tailed Student t test with Welch correction: P \u0026lt; 0.05 (*), P \u0026lt; 0.0001 (****). Data are expressed as means ± SD.\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/89cc58c43a0b2bb96efe0e16.png"},{"id":100622900,"identity":"a2f3527c-3f11-4074-938a-c9fb96299f1d","added_by":"auto","created_at":"2026-01-19 18:44:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":471856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and characterization of ZBTB46 CRISPR/Cas9 knockout models.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A) \u003c/strong\u003eSchematic representation of the genomic deletion between exons 4 and 5 (Del4–5). \u003cstrong\u003e(B) \u003c/strong\u003eRelative expression levels of \u003cem\u003ecircZBTB46\u003c/em\u003e and \u003cem\u003eZBTB46\u003c/em\u003e mRNAs determined by RT‒qPCR in COST Del4–5 clones.\u003cstrong\u003e (C) \u003c/strong\u003eSchematic representation of the genomic deletion between exons 2 and 5 (Del2–5). \u003cstrong\u003e(D)\u003c/strong\u003e Relative expression levels of \u003cem\u003ecircZBTB46\u003c/em\u003e and \u003cem\u003eZBTB46\u003c/em\u003e mRNAs determined by RT‒qPCR in COST Del2–5 clones. \u003cem\u003eMLN51\u003c/em\u003ewas used as an internal control for RT‒qPCR. Data are expressed as \u0026nbsp;2-∆∆Ct relative values. All experiments were performed withat least three biological replicates. (\u003cstrong\u003eE\u003c/strong\u003e) Immunofluorescence analysis of ZBTB46 protein expression in parental (wild-type) COST cells and Del4–5#A2 and Del2–5#E2 clones. Nuclei were counterstained with DAPI (blue). Original magnification, 63×. \u003cstrong\u003e(F) \u003c/strong\u003eCell viability was assessed by Annexin V-Pacific blue/PI staining (flow cytometry) in parental COST cells and Del4-5 and Del2-5 clones treated with increasing concentrations of crizotinib (1, 2, 3, 5 and 10 µM) for 48 h.\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/5a61bea4ba511ebe14ed44b2.png"},{"id":100622788,"identity":"8e334798-4f0d-49de-accf-a027846506cd","added_by":"auto","created_at":"2026-01-19 18:43:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional effects of circZBTB46 depletion and sensitivity to crizotinib.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eRelative expression of circZBTB46 and ZBTB46 mRNAs in COST and KARPAS-299 cells transfected for 48 h with control siRNA (siCTL) or two different circZBTB46-targeting siRNAs (siCircZBTB46#1 and #2), as measured by RT‒qPCR. \u003cstrong\u003e(B) \u003c/strong\u003eViability (Annexin V-Pacific Blue/PI flow cytometry) of COST and KARPAS-299 cells transfected with siCircZBTB46#1 or #2 and subsequently treated with crizotinib (3 µM, 48 h).\u003cstrong\u003e (C) \u003c/strong\u003eRelative expression of circZBTB46 and ZBTB46 mRNAs in COST, SUPM2 and Pio cells transduced with Cas13 and either control gRNA (CTL) or gRNA targeting circZBTB46, as assessed by RT‒qPCR. \u003cstrong\u003e(D) \u003c/strong\u003eViability (Annexin V-Pacific Blue/PI flow cytometry) of COST, SUPM2 and Pio cells transduced as described in \u003cstrong\u003e(C) \u003c/strong\u003eand treated with crizotinib (100 or 200 nM) for 7 days. (\u003cstrong\u003eE\u003c/strong\u003e) Relative expression of circZBTB46 and ZBTB46 mRNAs in crizotinib-resistant cells established from MTK PDX-derived cells transfected for 48 h with control siRNA (siCTL) or two different siRNAs targeting circZBTB46 (siCircZBTB46#1 and #2), as measured by RT‒qPCR. (\u003cstrong\u003eF-G\u003c/strong\u003e) Cell viability assessed by Annexin V-Pacific Blue/PI staining (flow cytometry) in crizotinib-resistant cells established from MTK PDX-derived cells transfected with siCircZBTB46#1 or #2, without treatment (\u003cstrong\u003eF\u003c/strong\u003e) or after crizotinib exposure (\u003cstrong\u003eG\u003c/strong\u003e) (400 nM, 48 h). (\u003cstrong\u003eH\u003c/strong\u003e) Relative expression of circZBTB46 and ZBTB46 mRNAs in COST crizotinib-resistant cells (COSTR200) after lentiviral transduction with circZBTB46-targeting shRNAs, as measured by RT‒qPCR. (\u003cstrong\u003eI\u003c/strong\u003e) Viability of COSTR200 cells determined by Annexin V/PI staining (flow cytometry) after transduction with circZBTB46 shRNA and subsequent treatment with crizotinib (1,000 nM, 7 days).\u003c/p\u003e\n\u003cp\u003eMLN51 was used as an internal control for RT‒qPCR. Expression data are displayed as 2-∆∆Ct relative values. Statistical significance was assessed via an unpaired two-tailed Student t test with Welch correction: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (\u003cstrong\u003e*\u003c/strong\u003e), \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cstrong\u003e(**\u003c/strong\u003e), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (\u003cstrong\u003e****\u003c/strong\u003e), ns = not significant. Data are expressed as means ± SEMs.\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/5ddba69647089dce056729ca.png"},{"id":100623135,"identity":"8a62cf14-0959-4c86-adec-2b83e290c098","added_by":"auto","created_at":"2026-01-19 18:46:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":300242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircZBTB46 modulates PIP5K1C by sponging miR-25.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Volcano plot showing differentially expressed genes in \u003c/strong\u003e\u0026nbsp;crizotinib-resistant cells established from MTK PDX-derived\u003cstrong\u003e cells transfected with control siRNA (siCTL) or siRNAs targeting circZBTB46 (siCircZBTB46#1 and #2). (B) Expression of PIP5K1C at the mRNA (top panel, RT‒qPCR) and protein (bottom panel, Western blot) levels in COST, MTK-PDX and COSTR200 ALK(+) ALCL cells after circZBTB46 siRNA transfection (48 h).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) Expression of PIP5K1C at the mRNA (left panel) and protein (right panel) levels in stable COSTR200 cells expressing either control shRNA (CTL) or shRNA targeting circZBTB46, as assessed by RT‒qPCR and Western blot, respectively. (D) Expression of PIP5K1C in Pio ALK(+) ALCL cells stably expressing Cas13 with control gRNA (CTL) or gRNA targeting circZBTB46, as determined by RT‒qPCR (left panel) and immunoblotting (right panel), respectively. (E) RNA enrichment after RNA pull-down using biotinylated probes targeting circZBTB46 or control probes. (F) Volcano plot showing in the COST cell line differentially enriched \u003c/strong\u003eRNAs in circZBTB46 pull-down compared to control, highlighting specific microRNAs enriched in the circZBTB46 fraction\u003cstrong\u003e. (G) \u003c/strong\u003eRelative expression levels of microRNAs (miR_497-5p, miR_222-5p, miR_22-3p, miR_10a-5p, and miR_25-3p) in ALK(+) ALCL primary samples versus reactive lymph nodes (RLNs), as assessed by small RNA sequencing.\u003cstrong\u003e (H) R\u003c/strong\u003eelative expression of miR25-3p in crizotinib-sensitive (Pio) and resistant COSTR200 cells transfected with a negative control mimic (mimicCTL) or mimic_25-3p \u003cstrong\u003emeasured by RT-qPCR.\u003c/strong\u003e \u003cstrong\u003eSnord44 was used as an internal control. Data are shown as 2-∆∆Ct relative values.\u003c/strong\u003e \u003cstrong\u003e(I) \u003c/strong\u003eWestern blot analysis of PIP5K1C protein levels in PIO and COSTR200 cells transfected with a miR_25-3p mimic or control mimic. GAPDH served as a loading control. Experiments were performed in triplicate. \u003cstrong\u003eData are presented as mean ± SEM. \u003c/strong\u003eStatistical significance wasdetermined using an \u003cstrong\u003eunpaired two-tailed Student t test with Welch correction\u003c/strong\u003e: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (\u003cstrong\u003e*\u003c/strong\u003e), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (\u003cstrong\u003e**\u003c/strong\u003e), ns = not significant\u003c/p\u003e","description":"","filename":"Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/49fe04e786ef2e78f6b86ddc.png"},{"id":100623125,"identity":"0b61a164-fc49-459f-9d31-09630d017a64","added_by":"auto","created_at":"2026-01-19 18:46:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":75309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecircZBTB46 modulates crizotinib resistance \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTumor growth in NSG mice (n=8) injected subcutaneously \u003cstrong\u003e(A-B) \u003c/strong\u003ewith COST cells \u003cstrong\u003e(A) \u003c/strong\u003euntreated or\u003cstrong\u003e (B) \u003c/strong\u003etreated with crizotinib (5 mg/kg/day); \u003cstrong\u003eC\u003c/strong\u003e) with Pio cells expressing Cas13 with either a nontargetingcontrol gRNA (Cas13_CTL) or a gRNA targeting circZBTB46 (Cas13_circZBTB46) and \u0026nbsp;(\u003cstrong\u003eD\u003c/strong\u003e) with COSTR200 cells transduced with or without circZBTB46 shRNA with or without crizotinib treatment (100 mg/kg/day). Parental cells were included as a control. Tumor volume was monitored over time via calipers and is expressed as the mean ± SD. Statistical significance was assessed via an unpaired two-tailed Student’s t test with Welch correction: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (\u003cstrong\u003e*\u003c/strong\u003e), \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cstrong\u003e(**\u003c/strong\u003e), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (\u003cstrong\u003e****\u003c/strong\u003e), ns = not significant. Dataare expressed as means ± SD.\u003c/p\u003e","description":"","filename":"Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/aade28336eaa144893f52719.png"},{"id":100796233,"identity":"7dfff7f8-c62c-4d18-8041-78977bcd80fd","added_by":"auto","created_at":"2026-01-21 13:42:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4184104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/723c2e18-d217-459c-a9a7-b5d5a4fdc0b5.pdf"},{"id":100622961,"identity":"b8196b83-f1f4-43db-a941-3d05dd2a6040","added_by":"auto","created_at":"2026-01-19 18:44:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":246003,"visible":true,"origin":"","legend":"supplementary tables","description":"","filename":"Supplementarytables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/ce259bab06757823e9eb2df8.pdf"},{"id":100622921,"identity":"02235c5e-4c2c-4850-9e2a-ed86a3f0ccd7","added_by":"auto","created_at":"2026-01-19 18:44:38","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":658457,"visible":true,"origin":"","legend":"supplementary figure legends","description":"","filename":"Supplementaryfigurestextfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/93fd26edaf9631706649c627.pdf"},{"id":100622966,"identity":"180c39ae-1362-42cd-8508-7403ea585c52","added_by":"auto","created_at":"2026-01-19 18:45:01","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":46704,"visible":true,"origin":"","legend":"supplemental mat\u0026#x00E9;rial and methods","description":"","filename":"SupplementaryDatatextFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/80d598e130883776ac602579.docx"},{"id":100622775,"identity":"a47cfa65-547f-4947-b503-d83abb03750f","added_by":"auto","created_at":"2026-01-19 18:43:30","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1221550,"visible":true,"origin":"","legend":"supplementary figures","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8593634/v1/675c69016a4ed58c5c1a8802.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCircZBTB46, a promising therapeutic target in crizotinib resistant ALK-positive T lymphomas\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cb\u003eThe purpose of this study was to investigate circRNAs in the specific context of ALK(+) ALCL.\u003c/b\u003e Anaplastic large-cell lymphoma (ALCL) is a rare and aggressive peripheral T-cell non-Hodgkin lymphoma (NHL) belonging to CD30\u003csup\u003e+\u003c/sup\u003e lymphoproliferative disorders(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). ALCL is divided into two subtypes based on the expression of the anaplastic lymphoma kinase (ALK)(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The ALK(+) subtype primarily affects children and young adults. In approximately 80\u0026ndash;85% of ALK(+) cases, the t(2;5)(p23;q35) chromosomal translocation is present, driving the fusion of the intracytoplasmic domain of ALK, on chromosome 2p23, with the N-terminal portion of nucleophosmin (NPM1), on chromosome 5q35(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). This NPM1-ALK fusion protein constitutively activates key oncogenic signaling pathways, including RAS-ERK, JAK3-STAT3 and PI3K-Akt, thereby driving cancer cell proliferation, differentiation and survival(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn ALK(+) ALCL, current first-line multidrug chemotherapy (CT) regimens achieve a progression-free survival of approximately 70% at ten years after diagnosis. Crizotinib, the first ALK-targeted tyrosine kinase inhibitor (TKI), was introduced for the treatment of ALK(+) ALCL refractory or relapsing (R/R) after CT. However, the clinical efficacy of crizotinib has been hampered by the emergence of resistance. The prognosis for R/R patients remains poor, highlighting the need for a better understanding of the aggressive behavior of ALCL and the development of novel prognostic markers to identify high-risk patients at an early stage(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Previous studies have disclosed the important role of small noncoding RNAs, particularly microRNAs (miRNAs), in ALCL(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). We and others have shown that specific miRNAs are associated with an increased risk of relapse in ALK(+) ALCL, after CT or crizotinib treatment(\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). \u003cb\u003eCircular RNAs (circRNAs) are a recently recognized class of stable and conserved noncoding RNAs characterized by a covalently closed loop structure lacking 5\u0026prime; and 3\u0026prime; ends. Predominantly localized in the cytoplasm, circRNAs act as molecular sponges for microRNAs and RNA-binding proteins, thereby modulating post-transcriptional gene regulation. They are highly stable and often exhibit tissue- or stage-specific expression patterns. Dysregulation of circRNAs has been shown to be involved in the pathophysiology of several solid tumors and hematological malignancies, especially B-NHL and leukemias\u003c/b\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). \u003cb\u003eA few reports have addressed the implication of circRNAs in peripheral T-cell NHL such as T-cell lymphoblastic lymphoma\u003c/b\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), \u003cb\u003ebut to date, no studies appear to have examined their role in ALK(+) ALCL. Deciphering the functions and mechanisms of circRNAs in ALK(+) ALCL could lead to the identification of innovative biomarkers development of therapeutic\u003c/b\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAberrant expression of circRNA and mRNA\u003c/b\u003e \u003cb\u003eZBTB46\u003c/b\u003e \u003cb\u003ein NPM1-ALK(+) lymphoma cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis work was initiated by investigating genome-wide circRNA expression profiles in ALK(+) ALCL via various approaches.\u003c/p\u003e \u003cp\u003eFirst, a ribo-minus RNA sequencing (RNAseq) dataset, previously generated from a cohort of 39 ALK(+) ALCL biopsies, was analyzed, with 9 reactive lymph nodes (RLNs) as controls(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). A circRNA expression landscape was established in ALK(+) ALCL (log2Fold Change\u0026thinsp;\u0026ge;\u0026thinsp;2 and a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Among upregulated circRNAs, circZBTB46 displayed the highest expression (L2FC: 3.6; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This circRNA stood out for several reasons. First, using long-read Oxford Nanopore RNA-seq, our group has already identified circZBTB46 as one of the most abundant circRNAs expressed in four human ALK(+) ALCL cell lines(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Second, the expression of its host gene, \u003cem\u003eZBTB46\u003c/em\u003e, is known to be restricted to human progenitor and conventional dendritic cells but has not been reported in T-lymphocytes(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Finally, using the RNAseq dataset, it was found that both \u003cem\u003eZBTB46\u003c/em\u003e circRNA and mRNA were undetectable in RLN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a second step, published microarray data from peripheral T-cell lymphomas (PTCL)(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) were analyzed. \u003cem\u003eZBTB46\u003c/em\u003e mRNA levels were compared in samples from ALCL (n\u0026thinsp;=\u0026thinsp;61), PTCL-not-otherwise specified \u003cb\u003e(\u003c/b\u003ePTCL-NOS, n\u0026thinsp;=\u0026thinsp;71), and angioimmunoblastic T-cell lymphoma \u003cb\u003e(\u003c/b\u003eAITL, n\u0026thinsp;=\u0026thinsp;83)(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The linear \u003cem\u003eZBTB46\u003c/em\u003e transcript was found to be significantly upregulated in ALCL compared with other PTCL subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eNext, expression levels of \u003cem\u003eZBTB46\u003c/em\u003e were analyzed in four ALK(+) ALCL cell lines (COST, SU-DHL-1, SUPM2 and KARPAS-299), two ALK(-) cell lines (FEPD and Mac2a) as well as in stimulated (S) and non-stimulated (NS) normal CD3\u003csup\u003e+\u003c/sup\u003e lymphocytes (n\u0026thinsp;=\u0026thinsp;3 donors). Both RNAseq (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and RT‒qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) analyses consistently revealed a strong upregulation of \u003cem\u003eZBTB46\u003c/em\u003e mRNA in all ALK(+) ALCL cell lines compared with ALK(-) cell lines and normal CD3\u0026thinsp;+\u0026thinsp;lymphocytes, independently of stimulation. In addition, RT-qPCR analysis showed a marked overexpression of circZBTB46 in ALK(+) ALCL cell lines compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These findings were confirmed at the protein level via Western blotting (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eUsing the Cancer Cell Line Encyclopedia (CCLE) database and the DepMap portal website(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) we found again that expression of the linear \u003cem\u003eZBTB46\u003c/em\u003e transcript was far the highest in ALK(+) ALCL cell lines among lymphomas cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eFinally, immunohistochemistry on primary biopsies from 19 ALK(+) and 3 ALK(-) ALCL confirmed that ZBTB46 protein expression was restricted to ALK(+) lymphoma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), and that no ZBTB46 expression was detected in CD68\u003csup\u003e+\u003c/sup\u003e cells (macrophages and dendritic cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH),\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrated that both circZBTB46 and its linear counterpart are aberrantly and quasi-exclusively expressed in ALK(+) ALCL tumoral cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBasic information and characteristics of circZBTB46 in ALK(+) lymphoma cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs previously reported(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), circZBTB46 is a 1255-nucleotide-long circRNA produced by backsplicing exons 2 and 3 of the linear \u003cem\u003eZBTB46\u003c/em\u003e transcript (ENST00000245663.9, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). CircZBTB46 is listed in circBase and circBank as hsa_circ_0002805, and mapped to the genomic coordinates chr20:62407030\u0026ndash;62422143 (hg19). Moreover, it is annotated as circZBTB46(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).1 according to the standardized nomenclature for eukaryotic circular RNAs(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The circular nature of circZBTB46 in the ALK(+) ALCL cell line SU-DHL-1, was confirmed by resistance to exonuclease digestion (RNase R), unlike linear \u003cem\u003eZBTB46\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, upon transcription arrest by actinomycin D, circZBTB46 remained stable for 8h while its linear counterpart declined rapidly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Finally, subcellular fractionation revealed that circZBTB46 was predominantly localized in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results support that circZBTB46 is a bona fide circular RNA transcript with a cytoplasmic localization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe NPM1-ALK/STAT3 axis is responsible for the aberrant accumulation of circRNA and linear\u003c/b\u003e \u003cb\u003eZBTB46\u003c/b\u003e \u003cb\u003etranscripts in ALK(+) lymphoma cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe consistent upregulation of both circRNA and linear \u003cem\u003eZBTB46\u003c/em\u003e transcripts in NPM1-ALK(+) cell lines and patient primary biopsies suggests that the NPM1-ALK fusion protein plays a central role in driving this overexpression. This was investigated by analyzing \u003cem\u003eZBTB46\u003c/em\u003e \u003cb\u003elinear mRNA\u003c/b\u003e levels in T-cells ectopically expressing NPM1-ALK, using two complementary models, i) primary human CD4\u003csup\u003e+\u003c/sup\u003e T-lymphocytes transduced with a lentiviral vector encoding NPM1-ALK (M1 model)(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and ii) T-cells engineered to carry the canonical t(2;5)(p23;q35) translocation via CRISPR-Cas9 genome editing (ALKIma1 model)(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In both models, previously generated RNAseq datasets were used to demonstrate robust induction of \u003cem\u003eZBTB46\u003c/em\u003e mRNA following immortalization and transformation of CD4\u003csup\u003e+\u003c/sup\u003e T-cells by the NPM1-ALK oncogene (Supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;2A and 2B\u003c/b\u003e). To further determine whether the tyrosine kinase activity of NPM1-ALK is required for this regulation, the ALK(+) ALCL cell line COST was treated with crizotinib. TKI treatment indeed efficiently inhibited ALK kinase activity, as indicated by the loss of NPM1-ALK autophosphorylation (p-ALK). A concomitant reduction in STAT3 activation was evidenced by decreased p-STAT3 levels in Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). RT‒qPCR and Western blot further demonstrated a significant downregulation of both circZBTB46 and linear \u003cem\u003eZBTB46\u003c/em\u003e transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), as well as reduced ZBTB46 protein levels. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). siRNAs against \u003cem\u003eNPM1-ALK\u003c/em\u003e (siALK) or \u003cem\u003eSTAT3\u003c/em\u003e (siSTAT3) were then used in two ALK(+) ALCL cell lines, COST and SUP-M2. Efficient knockdown of NPM1-ALK and STAT3 was confirmed at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). RT‒qPCR analysis demonstrated that knockdown of either \u003cem\u003eNPM1-ALK\u003c/em\u003e or \u003cem\u003eSTAT3\u003c/em\u003e strongly decreased circZBTB46 and linear \u003cem\u003eZBTB46\u003c/em\u003e expression in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating that NPM1-ALK-dependent STAT3 signaling is critical for driving \u003cem\u003eZBTB46\u003c/em\u003e accumulation and circZBTB46 production. Whether STAT3 binds directly to the \u003cem\u003eZBTB46\u003c/em\u003e locus was investigated by analyzing publicly available STAT3 ChIP-seq datasets (GSE117164) generated from two ALK(+) ALCL cell lines (JB6 and SU-DHL-1) exposed or not to crizotinib(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). As a positive control, strong STAT3 binding peaks were observed at the regulatory regions of \u003cem\u003eIRF4\u003c/em\u003e, a known direct STAT3 target in ALK(+) ALCL cells(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Two main STAT3 binding peaks were detected in the \u003cem\u003eZBTB46\u003c/em\u003e locus in untreated cells, whereas TKI treatment significantly reduced STAT3 occupancy at these sites, consistent with the loss of STAT3 activation (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C\u003c/b\u003e). ChIP‒qPCR, using a STAT3-specific antibody in the ALCL cell lines SUP-M2 and COST, and two independent primer pairs within the \u003cem\u003eZBTB46\u003c/em\u003e locus, allowed for precise quantifications of STAT3 binding (ZBTB46_1 and ZBTB46_2, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Enrichment of STAT3 binding at its two binding sites was confirmed in the \u003cem\u003eZBTB46\u003c/em\u003e locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTogether, these data support a direct regulatory role of the NPM1-ALK/STAT3 axis in the aberrant accumulation of linear and circRNA \u003cem\u003eZBTB46\u003c/em\u003e transcripts in ALK (+) ALCL cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCircZBTB46 promotes crizotinib resistance in ALK(+) lymphoma cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo dissect the respective roles of circZBTB46 and its linear mRNA counterpart, CRISPR-Cas9 knockout models were generated in the COST cell line. Since both the zinc finger domain (ZNF) and nuclear localization signal (NLS) of ZBTB46 are encoded by exon 4, two guide RNAs targeting exons 4 and 5 were used, to induce loss of function of the ZBTB46 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Deletion of exon 4 in three independent clones, Del4-5#C2, #C5 and #E2, was confirmed via genomic PCR (supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e) and RT‒qPCR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This strategy resulted truncated ZBTB46 protein expression, as confirmed in Western blot, using an antibody directed against an epitope encoded by exon 2 (supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3B\u003c/b\u003e). Additional clones lacking both the functional protein (i.e. mRNA) and circZBTB46 were generated using guide RNAs targeting exons 2 and 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), resulting in the deletion of exons 2 to 4. DNA PCR and RT‒qPCR confirmed successful deletion in three clones, Del2-5#A2, #A3, and #E2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e). As expected, these clones exhibited loss of both ZBTB46 protein (supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3B\u003c/b\u003e) and circRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In immunofluorescence, the full-length ZBTB46 transcription factor was localized in the nucleus of parental wild-type COST cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), while it was in the cytoplasm of Del4-5 clones (Supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3C\u003c/b\u003e) and absent in Del2-5 clones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3B-C\u003c/b\u003e). The proliferation and survival rates of parental Del4-5 and Del2-5 clones were comparable (Supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;3D-E\u003c/b\u003e). However, after 48h of treatment with crizotinib, both Del4-5 and Del2-5 clones exhibited increased survival compared with parental wild-type cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Importantly, Del2-5 clones, lacking circZBTB46 expression, presented significantly less TKI resistance than clones retaining this circRNA (Del4-5). To gain deeper insight into this observation, selective siRNA downregulation of circZBTB46 in the COST and KARPAS-299 cell-lines, was designed to target the back-splice junction and specifically deplete circZBTB46 without affecting the linear \u003cem\u003eZBTB46\u003c/em\u003e transcript (siCircZBTB46#1 and #2; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. After 48h of crizotinib treatment, both cell lines transfected with circZBTB46-targeting siRNAs exhibited reduced survival compared with control siRNA-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), supporting the involvement of circZBTB46 in crizotinib resistance in ALK(+) ALCL.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these results, a CRISPR/Cas13 lentiviral system was used to stably suppress the expression of circZBTB46 (Cas13_circZBTB46) in the COST, SUPM2 and Pio cell lines. This approach resulted in an approximately 50% reduction in circZBTB46 levels without affecting \u003cem\u003eZBTB46\u003c/em\u003e mRNA or protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, Supplementary \u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). Stable circZBTB46 knockdowns were used to assess the effect of prolonged crizotinib exposure (7 days, 100 or 200 nM). Compared with controls (Cas13_CTL), the three transduced cell lines (Cas13_circZBTB46) exhibited significantly reduced viability upon treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), indicating increased sensitivity to TKI.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLoss of circZBTB46 restores the sensitivity of resistant ALK(+) ALCL cells to crizotinib.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the therapeutic potential of targeting circZBTB46 in crizotinib-resistant ALK(+) ALCL, siRNA-mediated inactivation of circZBTB46 was performed in MTK PDX-derived cells, obtained from a patient with crizotinib-refractory disease(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Two independent siRNAs targeting circZBTB46 were used to ensure specificity of the observed effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Silencing circZBTB46 did not affect cell viability in the absence of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). However, after 48h of crizotinib exposure, transfected cells showed increased sensitivity to crizotinib compared with those treated with control siRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). We next generate stable circZBTB46-knockdown cell lines via shRNA lentiviral transduction (shCircZBTB46). Owing to the low transduction efficiency of MTK PDX-derived cells, a previously established crizotinib-resistant derivative of the COST cell line (COSTR200), was used(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The effective and specific downregulation of circZBTB46 was confirmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). As expected, circZBTB46 knockdown sensitized COSTR200 cells to crizotinib \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCircZBTB46 induces crizotinib resistance by modulating PIP5K1C gene expression in ALK(+) ALCL\u003c/h2\u003e \u003cp\u003eTo identify molecular pathways regulated by circZBTB46, RNA-Seq was performed after the transfection of two independent siRNAs targeting circZBTB46 in crizotinib-resistant MTK PDX-derived cells. Both siRNAs induced comparable transcriptional changes, particularly downregulation of PIP5K1C and DHRS9 (|log₂Fold Change| \u0026ge; 0.5, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The downregulation of PIP5K1C was validated by RT‒qPCR and Western blotting, confirming reduced expression at both mRNA and protein levels in all cellular models previously described (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). By contrast, DHRS9 downregulation was not consistently observed (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eContribution of PIP5K1C to crizotinib resistance in ALK(+) ALCL was explored by CRISPR interference (CRISPRi) technology. COSTR200 cell line were transduced with dCas9-KRAB-MeCP2 fusion proteins using guide RNAs targeting the PIP5K1C promoter to achieve transcriptional silencing. As shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;5A\u003c/b\u003e, gRNA-mediated silencing of PIP5K1C resulted in a significant reduction in PIP5K1C protein expression. Compared with control cells, PIP5K1C-silenced cells exhibited a slight decreased viability \u003cem\u003ein vitro\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;5B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThese results indicate that regulation of PIP5K1C by circZBTB46 participates in promoting resistance to ALK inhibition in ALK(+) ALCL.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCircZBTB46 regulates PIP5K1C by sponging miR25-3 in ALK(+) ALCL cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter RNA pull-down was performed using a back-splice junction-specific probe in ALK(+) ALCL cells, RT‒qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) confirmed a successful enrichment of circZBTB46. Mass spectrometry analysis of associated proteins did not reveal any specific interactors (data not shown), whereas small RNA sequencing identified four miRNAs (cutoff of |log2Fold Change|\u0026ge; 2 and a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05), namely, hsa-miR-22-3p, hsa-miR-222-5p, hsa-miR-10a-5p, and hsa-miR-25-3p, that were significantly enriched in the circZBTB46 pull-down compared with control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). With the exception of hsa_miR_222-5p, the other three miRNAs were expressed in ALK(+) ALCL patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG), hsa-miR-25-3p being notably overexpressed compared with RLN. Using miRNA\u0026ndash;mRNA interaction prediction algorithms (miRDB, TargetScanHuman and miRTargetLink 2.0), PIP5K1C was identified as a putative target of miR-25-3p (\u003cb\u003eSupplementary Fig.\u0026nbsp;5C-D\u003c/b\u003e). Consistently, transfection of a miR-25-3p mimic in crizotinib-sensitive (Pio) and crizotinib-resistant (COSTR200) cells, as validated by RT‒qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH), led to a reduction in PIP5K1C protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Conversely, transfection of a miR-25-3p inhibitor significantly increased \u003cem\u003ePIP5K1C\u003c/em\u003e mRNA and protein levels as observed in in COSTR200 cells, further validating this regulatory pathway (\u003cb\u003eSupplementary Fig.\u0026nbsp;5E-F)\u003c/b\u003e. Together, these data indicate that circZBTB46 modulates PIP5K1C levels via miR-25-3p sequestration, uncovering a novel competing endogenous RNA axis in ALK(+) ALCL.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTargeting circZBTB46 in ALK(+) ALCL cells reduces tumor growth\u003c/b\u003e \u003cb\u003ein vivo.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e experiments revealed that circZBTB46 is involved in crizotinib resistance in ALK(+) ALCL cells. To evaluate its potential as therapeutic target, the effect of circZBTB46 targeting on tumor growth was analyzed after subcutaneous injection in NSG mice.\u003c/p\u003e \u003cp\u003eFirst, CRIPSR/Cas9 crizotinib-sensitive models were engrafted. Del2-5 and Del4-5 clones formed tumors at comparable rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Daily administration of crizotinib, started five days after transplantation, initially reduced the tumor volume in all groups. However, by day 15, all tumors derived from ZBTB46 RNA-depleted clones resumed growth, whereas those derived from parental (WT) cells continued to regress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Notably, tumors derived from Del2-5 clones lacking circZBTB46 exhibited slower growth than those derived from circZBTB46(+) clones (Del4-5), corroborating \u003cem\u003ein vitro\u003c/em\u003e findings. Next, Cas13-modified crizotinib-sensitive Pio cells were xenografted into NSG mice and treated daily with crizotinib since nine days after injection. Tumor growth was delayed in mice bearing circZBTB46-deficient tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). As PIP5K1C was identified as a downstream target of circZBTB46, PIP5K1C CRISPRi models were also injected in NSG mice. This resulted in a slight reduction of tumor growth upon crizotinib administration (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e), highlighting that PIP5K1C as a mediator in TKI-resistance phenotype. Finally, the crizotinib resistant shCircZBTB46 models were injected to determine whether targeting circZBTB46 in crizotinib-resistant cells could restore drug sensitivity. Mice were daily treated with crizotinib or vehicle. In the absence of TKI treatment, tumor growth did not differ between the groups. However, upon crizotinib treatment, tumors derived from shCircZBTB46 cells exhibited significantly reduced growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. All these results support the contribution of circZBTB46 to crizotinib resistance in ALK(+) ALCL and identify circZBTB46 as an interesting therapeutic target of TKI-resistance to in ALK(+) ALCL disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study identifies the circular RNA, hsa_circ_0002805 (circZBTB46), as a key driver of crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK(+) ALCL). This circRNA, derived from exons 2 and 3 of the ZBTB46 gene, was found to be significantly and specifically upregulated in this lymphoma type. The upregulation is driven transcriptionally by the STAT3 pathway, which is activated by the oncogenic NPM1-ALK fusion protein. Using a combination of CRISPR/Cas and RNAi technologies, we functionally dissected the role of circZBTB46, finding that it, unlike its linear protein-coding counterpart, promotes resistance to crizotinib, an ALK inhibitor. This was demonstrated in both in vitro and in vivo models. Depleting circZBTB46 in resistant cells restored their sensitivity to crizotinib, highlighting its potential as a therapeutic target. Transcriptomic analysis revealed that PIP5K1C is a downstream effector of the circZBTB46-mediated resistance. Mechanistically, circZBTB46 acts as a molecular sponge for miR-25-3p, a microRNA that normally suppresses PIP5K1C expression. By sequestering miR-25-3p, circZBTB46 de-represses PIP5K1C, promoting survival signals and contributing to drug resistance. This proposed circZBTB46\u0026ndash;miR-25-3p\u0026ndash;PIP5K1C axis is a novel resistance mechanism in hematologic malignancies.\u003c/p\u003e \u003cp\u003eZBTB46 is a transcription factor normally restricted to dendritic cell precursors, where it maintains their immature state(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003csup\u003e,34,35\u003c/sup\u003e. It is also found in non-proliferative endothelial cells, where it inhibits cell proliferation(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Emerging evidence, however, points to a context-dependent role for ZBTB46 in cancer(\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In acute myeloid leukemia (AML), elevated ZBTB46 is associated with poor outcomes(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and despite not being essential for normal hematopoiesis, it is crucial for the survival and proliferation of AML cells(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, circZBTB46 exhibits context-dependent functions across different diseases. In atherosclerosis, circZBTB46 promotes disease progression by activating the AKT/mTOR pathway(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In this context, circZBTB46 depletion induces apoptosis and impairs cell migration, highlighting its importance in vascular pathology. In contrast, this study found no effect on AKT/mTOR signaling in ALK(+) ALCL cells upon circZBTB46 silencing (data not shown).\u003c/p\u003e \u003cp\u003eIn AML, circZBTB46 has been identified as an oncogenic effector that promotes cell survival. It acts as a competing endogenous RNA, sponging both hsa-miR-326 and hsa-miR-671-5p. This activity increases the expression of stearoyl-CoA desaturase 1 (SCD1), an enzyme that protects cells from lipid peroxidation and ferroptotic cell death.These findings establish circZBTB46 as a novel oncogenic effector in AML and reveal an unrecognized \u003cem\u003eZBTB46\u003c/em\u003e-derived noncoding RNA axis that governs cell fate through ferroptosis-related mechanisms(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Notably, both \u003cem\u003eZBTB46\u003c/em\u003e linear RNA and circZBTB46 were found to be expressed at markedly higher levels in ALK(+) ALCL than in AML. This was demonstrated by comparative transcriptomic analyses using a k-mer-based approach(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) applied to in-house RNA-seq data from primary biopsy samples, including ALK(+) ALCL and AML samples (IUCT-AML cohort) (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e)(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). RLN and established cell lines (AML and ALK(+) ALCL), including \u003cem\u003eNPM1-ALK\u003c/em\u003e\u0026ndash;transformed immortalized T-cell models(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and healthy T lymphocytes confirmed these data. This unexpected expression pattern underscores the context-dependent transcriptional regulation and functional plasticity of the \u003cem\u003eZBTB46\u003c/em\u003e locus across distinct hematological malignancies. In fact, this study expands knowledge on the pathophysiological oncogenic potential of ALK(+) ALCL by demonstrating the role of circZBTB46 in promoting resistance to crizotinib. It was shown here that circZBTB46 is transcriptionally upregulated via the NPM1-ALK/STAT3 signaling axis, thereby linking oncogenic kinase activity to circular RNA-mediated drug resistance. These findings suggest that circZBTB46 acts as a context-specific regulator of therapy resistance by engaging survival pathways, making it a potential therapeutic target in ALK-driven hematological malignancies. In this context, PIP5K1C appears as a downstream target of circZBTB46. miR-25-3p was identified as a candidate intermediary. miR-25-3p has previously been shown to target PIP5K1C in prostate cancer(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). These findings support a model in which circZBTB46 may act as a molecular sponge for miR-25-3p, thereby derepressing PIP5K1C expression and promoting survival signaling. Thus, circZBTB46 might contribute to crizotinib resistance in ALK(+) ALCL through a miR-25-3p\u0026ndash;PIP5K1C axis, a mechanism not previously appreciated in hematologic malignancies.\u003c/p\u003e \u003cp\u003eInterestingly, PIP5K1C was found to play a role in conferring resistance to ALK inhibition in ALCL. This lipid kinase catalyzes the phosphorylation of phosphatidylinositol 4-phosphate (PI4P) to produce phosphatidylinositol 4,5-bisphosphate (PI(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)P₂), which plays a key role in remodeling the actin cytoskeleton, intracellular trafficking and cell migration(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). PIP5K1C phosphorylation at serine 448 has been suggested to be a potential biomarker for breast cancer progression, particularly in invasive breast cancer(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Compared with their sensitive counterparts, melanoma cells that are resistant to the PIKFYVE inhibitor WX8 exhibit elevated expression of PIP5K1C, and this resistance can be reversed by either siRNA-mediated knockdown of PIP5K1C or pharmacological inhibition of its kinase activity(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). These findings suggest that PIP5K1C could mediate therapeutic resistance in multiple cancer types including hematological neoplasms. Therefore, focusing on PIP5K1C in crizotinib-resistant ALCL cells could be an effective way to overcome resistance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell line culture\u003c/h2\u003e \u003cp\u003eThe ALCL cell lines KARPAS-299, SU-DHL-1 SUPM2, Pio and COST, which carry the t(2;5)(p23;q35) translocation, were obtained from the DSMZ (German Collection of Microorganisms and Cell Culture, Leibnitz, Germany) or established locally (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The MTK PDX-derived cell line was provided by J. Mathews and S. Turner (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), and the crizotinib-resistant KARPAS-299-CR06 cell line was provided by C. Gambacorti-Passerini (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). COST cells were exposed to increasing concentrations of crizotinib, ranging from 25 nM to 200 nM. At each step, the dose was increased only when treated cells achieved a proliferation rate comparable to that of untreated wild-type (WT) cells. All ALK(+) ALCL cell lines and PDXs were cultured in RPMI-1640 medium (Invitrogen, Waltham, MA #61870044) supplemented with 20% FBS (Pan Biotech, Aidenbach, Germany #500105M1M). For crizotinib-resistant cell lines, crizotinib was added to the medium twice a week at a concentration of 200 nM for COSTR200 and 600 nM for KARPAS-299-CR06.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCrizotinib, siRNA and mimic treatments\u003c/h3\u003e\n\u003cp\u003eCells were seeded at 2x10\u003csup\u003e5\u003c/sup\u003e cells/mL and treated at 24h with different doses of crizotinib (Selleckchem, Houston, TX, PF-02341066 #S1068). Aliquotes of 3.10\u003csup\u003e6\u003c/sup\u003e cells were transfected with 70 to 100 nmol siRNA/mimic (listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) using Amaxa4D nucleofector (Primary Cell kit P3, CA-137 program, Lonza Bioscience, Antwerp, Belgium).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA pulldown\u003c/h2\u003e \u003cp\u003eiDRIP was performed as previously described (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Probe sequences are listed in \u003cb\u003eSupplementary Data Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eXenograft tumor assay\u003c/h3\u003e\n\u003cp\u003eMice were housed under pathogen-free conditions in an animal room at constant temperature (20\u0026deg;C-22\u0026deg;C) with a 12-h light/dark cycle and free access to food and water. A total of 3\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were injected subcutaneously into one flank of 5-week-old female NSG mice (Janvier Labs, Le Genest Saint Isle, France). Mouse body weight and tumor volumes were measured 3 times a week with calipers via the following formula: length \u0026times; width\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026times;\u0026thinsp;π/6. At the end of the experiment, the mice (8 per group) were humanely sacrificed. For crizotinib-resistant cells, mice were treated with 100 mg/kg/day crizotinib starting on the day following injection. For crizotinib-sensitive cells, mice were treated with 50 mg/kg/day crizotinib, starting when the tumor size reached 300 mm\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analyses\u003c/strong\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of at least three independent experiments. Graphs and statistical analyses were performed via GraphPad Prism 10 (GraphPad Software Inc., La Jolla, CA). P values were calculated by using an unpaired two-tailed Student \u003cem\u003eT\u003c/em\u003e test with Welch correction.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eCircRNA validation by PCR and Sanger sequencing\u003c/h3\u003e\n\u003cp\u003eThe validation of circRNA back-splice junctions, circularity, and stability were performed as previously. In brief, PCR amplification was followed by Sanger sequencing to confirm the back splicing junction and the full circRNA sequence. Circularity and exonuclease resistance were assessed via RNase R treatment, whereas transcript stability was evaluated following exposure to actinomycin D. Relative expression levels were determined via RT‒qPCR via the 2-∆∆Ct method.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by INSERM, Labex Toucan, Association Eva pour la vie, Féderation Grandir sans Cancer, RACCE Lions Club de Lourdes, the French Ministry of Health and the French National Cancer Institute (CircOma, PRT-K 2022-184), Fondation ARC et Fondation pour la Recherche Médicale supported by its sponsor Nagui and his production company, Banijay Production (EQU202403018019).\u0026nbsp;All grants were obtained by F.M.\u0026nbsp;E.A. and were supported by\u0026nbsp;Labex Toucan/Laboratoire d’Excellence Toulouse Cancer, the association Cassandra, and\u0026nbsp;Labex Toucan/Laboratoire d’Excellence Toulouse Cancer,\u0026nbsp;EUR CARe N°ANR-18-EURE-0003 in the framework of the Programme des Investissements d'Avenir,\u0026nbsp;\u0026nbsp;L.B. and C.B. were supported by the Fondation de France. L.B. was supported by\u0026nbsp;Fondation l’Oréal for Women in Science, Prolific Graine de Chercheur and Association Ellye.\u0026nbsp;L.C. and S.F. were supported by a grant from the French Ministry of Health and the French National Cancer Institute (CircOma, PRT-K 2022--184).\u0026nbsp;S.F. was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, grant no. 439441203). S.F. is a participant in the BIH-Charité Clinician Scientist Programme funded by the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health. C.P. and I.D. were supported by FONROGA.\u0026nbsp;The authors thank the staff of the animal facility of UMS US006/CREFRE (Toulouse, France) for their technical assistance.\u0026nbsp;This work benefited from the equipment and services of the iGenSeq core facility (genotyping and sequencing) at the ICM.\u0026nbsp;Part of this work was carried out by the Paris Brain Institute (ICM) Data Analysis Core (DAC) plateform (RRID:SCR_026138, https://dac.institutducerveau.org/). We gratefully acknowledge Thomas Gareau for assistance with RNAseq data analysis. \u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Toulouse Hospital Biobank (DC-2020-4074; AC-2020-4031, 2020).\u003c/p\u003e\n\u003cp\u003eMedical writing for this manuscript was assisted by MPIYP (MC Béné), Paris, France.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ribo zero full RNA sequencing, the long-read Oxford Nanopore RNA sequencing and \u0026nbsp;STAT3 ChIP-seq datasets are available in the GEO repository (GSE160123, GSE197872 and GSE117164 respectively). The RNA-sequencing datasets generated are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.A., L.B. and F.M. designed the research study. E.A., S.F., C.P., I.D., C.Q., R.F., A.Z. and L.B. performed the experiments. E.A., L.B., S.F., C.Q. and F.M. analyzed the data. L.L. and C.Q. were involved in the diagnosis of ALCL. C.B., L.B. and C.G. performed the bioinformatic analysis. S.P. and M.B. helped with discussions and critical reading. L.B. and F.M. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStein H, Foss HD, Durkop H, Marafioti T, Delsol G, Pulford K, et al. CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood. 2000;96(12):3681\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalini B, Bigerna B, Fizzotti M, Pulford K, Pileri SA, Delsol G, et al. ALK expression defines a distinct group of T/null lymphomas (\"ALK lymphomas\") with a wide morphological spectrum. Am J Pathol. 1998;153(3):875\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlaggio R, Amador C, Anagnostopoulos I, Attygalle AD, Araujo IBO, Berti E, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia. 2022;36(7):1720\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonzheim I, Steinhilber J, Fend F, Lamant L, Quintanilla-Martinez L. ALK-positive anaplastic large cell lymphoma: an evolving story. Front Biosci (Schol Ed). 2015;7(2):248\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProkoph N, Larose H, Lim MS, Burke GAA, Turner SD. Treatment Options for Paediatric Anaplastic Large Cell Lymphoma (ALCL): Current Standard and beyond. Cancers (Basel). 2018;10(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XR, Chien PN, Nam SY, Heo CY. Anaplastic Large Cell Lymphoma: Molecular Pathogenesis and Treatment. Cancers (Basel). 2022;14(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuchs S, Naderi J, Meggetto F. Non-Coding RNA Networks in ALK-Positive Anaplastic-Large Cell Lymphoma. Int J Mol Sci. 2019;20(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDejean E, Renalier MH, Foisseau M, Agirre X, Joseph N, de Paiva GR, et al. Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas. Leukemia. 2011;25(12):1882\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesjobert C, Renalier MH, Bergalet J, Dejean E, Joseph N, Kruczynski A, et al. MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression. Blood. 2011;117(24):6627\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoareau-Aveilla C, Merkel O, Meggetto F. MicroRNA and ALK-positive anaplastic large cell lymphoma. Front Biosci (Schol Ed). 2015;7(2):217\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerkel O, Hamacher F, Laimer D, Sifft E, Trajanoski Z, Scheideler M, et al. Identification of differential and functionally active miRNAs in both anaplastic lymphoma kinase (ALK)\u0026thinsp;+\u0026thinsp;and ALK- anaplastic large-cell lymphoma. Proc Natl Acad Sci U S A. 2010;107(37):16228\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontes-Mojarro IA, Steinhilber J, Griessinger CM, Rau A, Gersmann AK, Kohlhofer U, et al. CD147 a direct target of miR-146a supports energy metabolism and promotes tumor growth in ALK\u0026thinsp;+\u0026thinsp;ALCL. Leukemia. 2022;36(8):2050\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorrentino D, Frentzel J, Mitou G, Blasco RB, Torossian A, Hoareau-Aveilla C, et al. High Levels of miR-7-5p Potentiate Crizotinib-Induced Cytokilling and Autophagic Flux by Targeting RAF1 in NPM-ALK Positive Lymphoma Cells. Cancers (Basel). 2020;12(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Ren X, Wang Y, Xin X. CircRNA: a rising star in leukemia. PeerJ. 2023;11:e15577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez de Acha O, Rossi M, Gorospe M. Circular RNAs in Blood Malignancies. Front Mol Biosci. 2020;7:109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonizzato A, Gaffo E, Te Kronnie G, Bortoluzzi S. CircRNAs in hematopoiesis and hematological malignancies. Blood Cancer J. 2016;6(10):e483.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaugrois C, Bessiere C, Dejean S, Anton-Leberre V, Commes T, Pyronnet S, et al. Gene Expression Signature Associated with Clinical Outcome in ALK-Positive Anaplastic Large Cell Lymphoma. Cancers (Basel). 2021;13(21).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuchs S, Babin L, Andraos E, Bessiere C, Willier S, Schulte JH, et al. Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing. PLoS One. 2022;17(9):e0273253.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobbins SH, Walzer T, Dembele D, Thibault C, Defays A, Bessou G, et al. Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling. Genome Biol. 2008;9(1):R17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSatpathy AT, Kc W, Albring JC, Edelson BT, Kretzer NM, Bhattacharya D, et al. Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J Exp Med. 2012;209(6):1135\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Leval L, Rickman DS, Thielen C, Reynies A, Huang YL, Delsol G, et al. The gene expression profile of nodal peripheral T-cell lymphoma demonstrates a molecular link between angioimmunoblastic T-cell lymphoma (AITL) and follicular helper T (TFH) cells. Blood. 2007;109(11):4952\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Leval L, Parrens M, Le Bras F, Jais JP, Fataccioli V, Martin A, et al. Angioimmunoblastic T-cell lymphoma is the most common T-cell lymphoma in two distinct French information data sets. Haematologica. 2015;100(9):e361-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArafeh R, Shibue T, Dempster JM, Hahn WC, Vazquez F. The present and future of the Cancer Dependency Map. Nat Rev Cancer. 2025;25(1):59\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen LL, Bindereif A, Bozzoni I, Chang HY, Matera AG, Gorospe M, et al. A guide to naming eukaryotic circular RNAs. Nat Cell Biol. 2023;25(1):1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCongras A, Hoareau-Aveilla C, Caillet N, Tosolini M, Villarese P, Cieslak A, et al. ALK-transformed mature T lymphocytes restore early thymus progenitor features. J Clin Invest. 2020;130(12):6395\u0026ndash;408.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenotti M, Ambrogio C, Cheong TC, Pighi C, Mota I, Cassel SH, et al. Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma. Nat Med. 2019;25(1):130\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBandini C, Pupuleku A, Spaccarotella E, Pellegrino E, Wang R, Vitale N, et al. IRF4 Mediates the Oncogenic Effects of STAT3 in Anaplastic Large Cell Lymphomas. Cancers (Basel). 2018;10(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProkoph N, Matthews JD, Trigg RM, Montes-Mojarro IA, Burke GAA, Fend F, et al. Patient-derived xenograft models of ALK\u0026thinsp;+\u0026thinsp;ALCL reveal preclinical promise for therapy with brigatinib. Br J Haematol. 2023;202(5):985\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamant L, Espinos E, Duplantier M, Dastugue N, Robert A, Allouche M, et al. Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL. Leukemia. 2004;18(10):1693\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoto P, Gerzsenyi TB, Lengyel A, Szunyog B, Szatmari I. Zbtb46-dependent altered developmental program in embryonic stem cell-derived blood cell progenitors. Stem Cells. 2021;39(10):1322\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao T, Ji JF, Zheng JY, Li C, Zhu LY, Fan DD, et al. Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model. J Immunol. 2022;208(12):2686\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Sun HY, Kumar S, Puerta MDM, Jo H, Rezvan A. ZBTB46 is a shear-sensitive transcription factor inhibiting endothelial cell proliferation via gene expression regulation of cell cycle proteins. Lab Invest. 2019;99(3):305\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmebye ML, Agostini A, Johannessen B, Thorsen J, Davidson B, Trope CG, et al. Involvement of DPP9 in gene fusions in serous ovarian carcinoma. BMC Cancer. 2017;17(1):642.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong X, Zhou K, Zhao Y, Huai C, Zhao Y, Yu H, et al. Fine mapping analysis of a region of 20q13.33 identified five independent susceptibility loci for glioma in a Chinese Han population. Carcinogenesis. 2012;33(5):1065\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu N, Wang F, Lv M, Cheng L. Microarray expression profile analysis of long non-coding RNAs in human breast cancer: a study of Chinese women. Biomed Pharmacother. 2015;69:221\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong F, Lin Z, Long Q, Lu Z, Zhu K, Zhao M, et al. CircZBTB46 Protects Acute Myeloid Leukemia Cells from Ferroptotic Cell Death by Upregulating SCD. Cancers (Basel). 2023;15(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu YY, Xiao FF, Yang BJ, Li X, Xu SN, Chen ZW, et al. Zinc finger and BTB domain-containing protein 46 is essential for survival and proliferation of acute myeloid leukemia cell line but dispensable for normal hematopoiesis. Chin Med J (Engl). 2020;133(14):1688\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu Y, Jia Q, Ren M, Bie H, Zhang X, Zhang Q, et al. Circular RNA ZBTB46 depletion alleviates the progression of Atherosclerosis by regulating the ubiquitination and degradation of hnRNPA2B1 via the AKT/mTOR pathway. Immun Ageing. 2023;20(1):66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchet C, Iqbal Z, Gautheret D, Salson M, Chikhi R. REINDEER: efficient indexing of k-mer presence and abundance in sequencing datasets. Bioinformatics. 2020;36(Suppl_1):i177-i85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBessiere C, Xue H, Guibert B, Boureux A, Ruffle F, Viot J, et al. Transipedia.org: k-mer-based exploration of large RNA sequencing datasets and application to cancer data. Genome Biol. 2024;25(1):266.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorre E, Soum C, Pfeifer R, Bessiere C, Dailhau S, Marboeuf C, et al. Differential prognostic values of the three AKT isoforms in acute myeloid leukemia. Sci Rep. 2024;14(1):7070.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Clara E, Gourvest M, Ma H, Vergez F, Tosolini M, Dejean S, et al. Long non-coding RNA expression profile in cytogenetically normal acute myeloid leukemia identifies a distinct signature and a new biomarker in NPM1-mutated patients. Haematologica. 2017;102(10):1718\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZoni E, van der Horst G, van de Merbel AF, Chen L, Rane JK, Pelger RC, et al. miR-25 Modulates Invasiveness and Dissemination of Human Prostate Cancer Cells via Regulation of alphav- and alpha6-Integrin Expression. Cancer Res. 2015;75(11):2326\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin S, Tao C, Yan Q, Gao H, Qin L, Zhong Y, et al. Pip5k1c expression in osteocytes regulates bone remodeling in mice. J Orthop Translat. 2024;45:36\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurand N, Borges S, Hall T, Bastea L, Doppler H, Edenfield BH, et al. The phosphorylation status of PIP5K1C at serine 448 can be predictive for invasive ductal carcinoma of the breast. Oncotarget. 2018;9(91):36358\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoy A, Chakraborty AR, Nomanbhoy T, DePamphilis ML. PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells. Autophagy. 2023;19(9):2464\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCeccon M, Mologni L, Bisson W, Scapozza L, Gambacorti-Passerini C. Crizotinib-resistant NPM-ALK mutants confer differential sensitivity to unrelated Alk inhibitors. Mol Cancer Res. 2013;11(2):122\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu HP, Minajigi A, Chen Y, Morris R, Guh CY, Hsieh YH, et al. iDRiP for the systematic discovery of proteins bound directly to noncoding RNA. Nat Protoc. 2021;16(7):3672\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8593634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8593634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCircular RNAs (circRNAs) are increasingly recognized as functional non-coding transcripts with oncogenic potential. Here, a comprehensive analysis of circRNA expression in primary ALK(+) anaplastic large-cell lymphoma (ALK(+) ALCL) is presented. Integrated transcriptomic profiling revealed that aberrant expression of circZBTB46 and of its linear host transcript, normally restricted to dendritic cells, is exclusive to ALK(+) lymphoma cells and driven by the oncogenic NPM1-ALK/STAT3 axis. Functional studies showed that circZBTB46, unlike its protein-coding counterpart, promotes resistance to the ALK inhibitor crizotinib. Silencing circZBTB46 restored crizotinib sensitivity in resistant ALCL cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Transcriptomic analyses identified PIP5K1C as a downstream effector regulated through a competitive endogenous RNA mechanism in which circZBTB46 acts as a sponge to miR-25-3p, alleviating its repression of PIP5K1C. These findings uncover a previously unrecognized mechanism of drug resistance in ALK(+) ALCL and establish circZBTB46 as a promising therapeutic target.\u003c/p\u003e","manuscriptTitle":"CircZBTB46, a promising therapeutic target in crizotinib resistant ALK-positive T lymphomas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 18:22:25","doi":"10.21203/rs.3.rs-8593634/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-05T14:52:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-04T12:12:36+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-28T18:28:45+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-20T13:13:38+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-15T09:02:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-14T20:18:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-14T16:38:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T16:30:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2026-01-13T15:17:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f35199de-3a75-48fc-b9d0-94e163e6f492","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61391890,"name":"Biological sciences/Cancer/Haematological cancer"},{"id":61391891,"name":"Biological sciences/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/T-cell lymphoma"}],"tags":[],"updatedAt":"2026-05-06T15:41:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 18:22:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8593634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8593634","identity":"rs-8593634","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}