Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual Proteasome and Histone Deacetylase Inhibition Overcomes Tyrosine Kinase Inhibitor Resistance in Breakpoint Cluster Region: Abelson 1‐Driven Leukaemia Cell Lines

doi: 10.1111/jcmm.71053

Figure Lengend Snippet: Responses to asciminib and ponatinib in parental and resistant chronic myelogenous leukaemia (CML) models. (A) CML cell lines (K562, K562 PR, K562 IR, Ba/F3 asc‐R, Ba/F3 PR, Ba/F3 BCR::ABL, and Ba/F3 T315I) were exposed to asciminib (0–1 μM) for 72 h, and cell viability was measured using the Cell Counting Kit‐8 (CCK‐8) assay. (B) The same CML cell lines were treated with asciminib (0–1 μM) for 48 h, and cytotoxicity was assessed using a Cytotoxicity LDH Assay Kit. Data were normalised to untreated controls and expressed as means ± SD. * p < 0.05, **** p < 0.0001 vs. control; ns, not significant. (C) Ponatinib (0–1 μM) was administered to the CML cells for 72 h, and cell viability was measured using the CCK‐8 assay. (D) Cytotoxicity in CML cells treated with ponatinib (0–1 μM) for 48 h was determined using the LDH assay. Results were normalised to controls and presented as means ± SD. * p < 0.05, **** p < 0.0001 vs. control; ns, not significant.

Article Snippet: The CML cell line K562 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Cell Counting, CCK-8 Assay, Lactate Dehydrogenase Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual Proteasome and Histone Deacetylase Inhibition Overcomes Tyrosine Kinase Inhibitor Resistance in Breakpoint Cluster Region: Abelson 1‐Driven Leukaemia Cell Lines

doi: 10.1111/jcmm.71053

Figure Lengend Snippet: Proteasome inhibitor bortezomib suppresses growth and induces apoptosis in parental and TKI‐resistant CML models. (A) CML cell lines (K562, K562 PR, K562 IR, Ba/F3 asc‐R, Ba/F3 PR, Ba/F3 BCR::ABL, and Ba/F3 T315I) were exposed to bortezomib (0–100 nM) for 72 h, and cell viability was determined using the CCK‐8 assay. (B) Cytotoxicity was measured in the same cell lines following 48‐h exposure to bortezomib (0–100 nM) using the LDH assay. Data were normalised to untreated controls and expressed as means ± SD. * p < 0.05, **** p < 0.0001 vs. control; ns, not significant. (C) Caspase‐3/7 activity was measured in CML cells treated with bortezomib (0–100 nM) for 48 h. * p < 0.05, **** p < 0.0001 vs. control; ns, not significant.

Article Snippet: The CML cell line K562 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: CCK-8 Assay, Lactate Dehydrogenase Assay, Control, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual Proteasome and Histone Deacetylase Inhibition Overcomes Tyrosine Kinase Inhibitor Resistance in Breakpoint Cluster Region: Abelson 1‐Driven Leukaemia Cell Lines

doi: 10.1111/jcmm.71053

Figure Lengend Snippet: Panobinostat exhibits broad, dose‐dependent antileukemic activity in parental and drug‐resistant CML models. (A) CML cell lines (K562, K562 PR, K562 IR, Ba/F3 asc‐R, Ba/F3 PR, Ba/F3 BCR::ABL, and Ba/F3 T315I) were treated with panobinostat (0–100 nM) for 72 h, and cell viability was assessed using the CCK‐8 assay. (B) Cytotoxicity following 48‐h exposure to panobinostat (0–100 nM) was measured using the LDH assay. Data are expressed as means ± SD. **** p < 0.0001 vs. control. (C) Caspase‐3/7 activity was evaluated in CML cells treated with panobinostat (0–100 nM) for 48 h. **** p < 0.0001 vs. control; ns, not significant.

Article Snippet: The CML cell line K562 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, CCK-8 Assay, Lactate Dehydrogenase Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual Proteasome and Histone Deacetylase Inhibition Overcomes Tyrosine Kinase Inhibitor Resistance in Breakpoint Cluster Region: Abelson 1‐Driven Leukaemia Cell Lines

doi: 10.1111/jcmm.71053

Figure Lengend Snippet: Combined bortezomib and panobinostat treatment enhances antileukemic activity in CML models. (A–C) CML cell lines (K562, K562 PR, K562 IR, Ba/F3 asc‐R, Ba/F3 PR, Ba/F3 BCR::ABL, and Ba/F3 T315I) were treated with 10 nM bortezomib and/or 10 nM panobinostat for 48 or 72 h. (A) Cell viability, (B) cytotoxicity, and (C) caspase‐3/7 activity were assessed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control; ns, not significant.

Article Snippet: The CML cell line K562 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual Proteasome and Histone Deacetylase Inhibition Overcomes Tyrosine Kinase Inhibitor Resistance in Breakpoint Cluster Region: Abelson 1‐Driven Leukaemia Cell Lines

doi: 10.1111/jcmm.71053

Figure Lengend Snippet: Bortezomib plus panobinostat suppresses clonogenicity, disrupts mitochondrial membrane potential, and induces apoptosis. (A) K562 cells were exposed to 10 nM bortezomib, with or without 10 nM panobinostat, for 7–9 days. Colonies were photographed using a digital microscope and counted. Representative images and quantification from three independent experiments are shown (scale bar: 1000 μm). Statistical significance: *** p < 0.001, **** p < 0.0001 vs. control. (B) Ba/F3 asc‐R and Ba/F3 PR cells were treated with bortezomib ± panobinostat for 48 h, and mitochondrial membrane potential was assessed using a mitochondrial staining kit. **** p < 0.0001 vs. control. (C) Ba/F3 asc‐R and Ba/F3 PR cells were treated with bortezomib ± panobinostat for 24 h. Apoptosis assays showed minimal induction with single agents, whereas the combination markedly increased apoptotic cell levels. Statistical significance: *** p < 0.001, **** p < 0.0001 vs. control.

Article Snippet: The CML cell line K562 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Membrane, Microscopy, Control, Staining

Journal: bioRxiv

Article Title: Targeting SOS1 synergistically enhances efficacy of BCR/ABL tyrosine kinase inhibitors and overcomes resistance in chronic myeloid leukemia

doi: 10.1101/2025.09.29.679122

Figure Lengend Snippet: (a–b) Time-course analysis of cell proliferation (a) and Apoptosis analysis (% Annexin V⁺ cells) (b) in K562 and KCL22 cells treated with increasing concentrations of imatinib (10–10000 nM, blue) or BI-3406 (10–10,000 nM, orange) for up to 48 h. Cell viability was measured by live object count normalized to time 0, and apoptosis was assessed by Annexin V staining. (c–d) Dose-response viability curves (c) and Annexin V apoptosis assays (d) after 48 h treatment as indicated with imatinib alone or in combination with fixed doses of BI-3406 (1, 10, or 100 nM). (e) Synergy analysis using ZIP scoring algorithm. 3D surface plots estimating synergy between BI-3406 and imatinib in both CML cell lines, with ZIP scores >20 indicating strong combinatorial effects. (f) Summary table of IC 50 values (mean ± SD) for imatinib calculated after 48 h co-treatment of both K562 and KCL22 cell lines. Data are mean ± SEM from at least three technical and biological replicates; ICDD values were calculated using non-linear regression (four-parameter logistic model) in GraphPad Prism v8.0. Statistical significance was assessed by one-way ANOVA with Tukey’s post-test; **p < 0.01; ***p<0.001.

Article Snippet: The human CML cell lines K562 (ATCC® CCL-243TM) and KCL22 (ATCC® CRL-3349TM), as well as murine Ba/F3-p210 BCR/ABL cells (control strain and derivatives carrying the T315I or the E255K BCR-ABL1 mutations), were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere with 5% COD.

Techniques: Staining

Journal: bioRxiv

Article Title: Targeting SOS1 synergistically enhances efficacy of BCR/ABL tyrosine kinase inhibitors and overcomes resistance in chronic myeloid leukemia

doi: 10.1101/2025.09.29.679122

Figure Lengend Snippet: (a) Dose–response viability curves of K562 and KCL22 cells treated with dasatinib (left panels), nilotinib (middle panels), or ponatinib (right panels), alone or in combination with fixed doses of BI-3406 (1 nM, 10 nM, or 100 nM). BI-3406 monotherapy had minimal effect on viability but markedly potentiated the cytotoxic activity of all three TKIs in both cell lines, resulting in a pronounced reduction in ICDD values (see table in panel c). (b) Synergy analysis of BI-3406 in combination with dasatinib, nilotinib, or ponatinib using the ZIP model. 3D surface plots show areas of synergy (red) across concentration matrices. ZIP synergy scores >10 denote strong combinatorial interaction. Synergistic effects were observed in all conditions, with particularly high scores for Nilotinib combinations. Data presented as mean ± SEM from three independent replicates. ICDD values were calculated after 48 h treatment.

Article Snippet: The human CML cell lines K562 (ATCC® CCL-243TM) and KCL22 (ATCC® CRL-3349TM), as well as murine Ba/F3-p210 BCR/ABL cells (control strain and derivatives carrying the T315I or the E255K BCR-ABL1 mutations), were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere with 5% COD.

Techniques: Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting SOS1 synergistically enhances efficacy of BCR/ABL tyrosine kinase inhibitors and overcomes resistance in chronic myeloid leukemia

doi: 10.1101/2025.09.29.679122

Figure Lengend Snippet: (a) Western blot analysis of serum-starved K562 and KCL22 cells stimulated with EGF (100 ng/mL) for 2, 5, or 10 minutes, either alone or following pre-treatment with BI-3406 (1 μM). Active GTP-bound RAS, RAC and MRAS were assessed by pull-down assays as described in Methods. Tubulin (TUB) was used as a loading control for all blots. Quantification of RAS-GTP, RAC-GTP, pERK, and pAKT levels normalized to total protein is shown on the right. Graphs represent mean ± SEM from at least three independent experiments. (b) Cells were treated for 24 hours with BI-3406 (1μM), imatinib (IMA, 500nM), or their combination, and phospho-ERK and phospho-AKT levels were assessed by Western blot. Tubulin was used as loading control. Representative of at least three independent experiments. Blue asterisks indicate significance vs. imatinib alone; orange asterisks indicate significance vs. BI-3406 alone and black asterisks indicate significance vs vehicle treated alone. Statistical significance was assessed by one-way ANOVA with Tukey’s post-test; * p<0.05; **p < 0.01; ***p<0.001.

Article Snippet: The human CML cell lines K562 (ATCC® CCL-243TM) and KCL22 (ATCC® CRL-3349TM), as well as murine Ba/F3-p210 BCR/ABL cells (control strain and derivatives carrying the T315I or the E255K BCR-ABL1 mutations), were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere with 5% COD.

Techniques: Western Blot, Control