Journal: bioRxiv

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

doi: 10.64898/2026.01.13.699235

Figure Lengend Snippet: (A) Volcano plot and (B) MA plot of circular RNA expression comparing ALK(+) ALCL primary biopsies (n=39) and healthy tissues (reactive lymph nodes, RLN, n=9). (C) expression of circZBTB46 (left) and ZBTB46 mRNA (right) assessed by RNA-Seq in ALK(+) ALCL primary samples versus RLN. (D) 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. (E) 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). (F) Quantitative real-time PCR (RTLqPCR) 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: P < 0.01 ( ** ), P < 0.001 (*** ), P < 0.0001 (**** ), ns = not significant. Data are expressed as means ± SD. (G) ZBTB46 mRNA expression across hematological malignancies in the Cancer Cell Line Encyclopedia (CCLE) dataset. (H) 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.

Article Snippet: 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 .

Techniques: RNA Expression, Expressing, RNA Sequencing, Microarray, Real-time Polymerase Chain Reaction, Control, Two Tailed Test, Immunohistochemical staining

Journal: bioRxiv

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

doi: 10.64898/2026.01.13.699235

Figure Lengend Snippet: (A) Relative 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 RTLqPCR. (B) Viability (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). (C) Relative 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 RTLqPCR. (D) Viability (Annexin V-Pacific Blue/PI flow cytometry) of COST, SUPM2 and Pio cells transduced as described in (C) and treated with crizotinib (100 or 200 nM) for 7 days. ( E ) 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 RTLqPCR. ( F-G ) 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 ( F ) or after crizotinib exposure ( G ) (400 nM, 48 h). ( H ) Relative expression of circZBTB46 and ZBTB46 mRNAs in COST crizotinib-resistant cells (COSTR200) after lentiviral transduction with circZBTB46-targeting shRNAs, as measured by RTLqPCR. ( I ) 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). MLN51 was used as an internal control for RTLqPCR. Expression data are displayed as 2-ΔΔCt relative values. Statistical significance was assessed via an unpaired two-tailed Student t test with Welch correction: P < 0.05 ( * ), P < 0.01 (** ), P < 0.0001 ( **** ), ns = not significant. Data are expressed as means ± SEMs.

Article Snippet: 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 .

Techniques: Expressing, Transfection, Control, Flow Cytometry, Transduction, Derivative Assay, Staining, shRNA, Two Tailed Test

Journal: bioRxiv

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

doi: 10.64898/2026.01.13.699235

Figure Lengend Snippet: circZBTB46 modulates PIP5K1C by sponging miR-25. (A) Volcano plot showing differentially expressed genes in crizotinib-resistant cells established from MTK PDX-derived cells transfected with control siRNA (siCTL) or siRNAs targeting circZBTB46 (siCircZBTB46#1 and #2). (B) Expression of PIP5K1C at the mRNA (top panel, RTLqPCR) and protein (bottom panel, Western blot) levels in COST, MTK-PDX and COSTR200 ALK(+) ALCL cells after circZBTB46 siRNA transfection (48 h). (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 RTLqPCR 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 RTLqPCR (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 RNAs in circZBTB46 pull-down compared to control, highlighting specific microRNAs enriched in the circZBTB46 fraction. (G) Relative 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. (H) Relative expression of miR25-3p in crizotinib-sensitive (Pio) and resistant COSTR200 cells transfected with a negative control mimic (mimicCTL) or mimic_25-3p measured by RT-qPCR. Snord44 was used as an internal control. Data are shown as 2-ΔΔCt relative values . (I) Western 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. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student t test with Welch correction: P < 0.05 ( * ), P < 0.001 ( ** ), ns = not significant

Article Snippet: 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 .

Techniques: Derivative Assay, Transfection, Control, Expressing, Western Blot, shRNA, Stable Transfection, RNA Sequencing, Negative Control, Quantitative RT-PCR, Two Tailed Test