ribociclib  (MedChemExpress)

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Western blots of cyclin E, CDK2, pCDK2 (Thr160), Rb, and pRb (Ser807/811) in HCC1806 parental cells, scramble control, and CCNE1 or CDK2 knockdown cells. B Representative dose-response curves and IC 50 values for BLU-222-treated HCC1806 scramble control and CCNE1 and CDK2 knockdown cell lines. Error bars represent standard error of the mean (SEM) from n = 3 experimental replicates. C Annexin V (+) quantification in HCC1806 control and CCNE1 and CDK2 knockdown cells treated with BLU-222 at 0.5 µM in for 0, 24, and 48 h. The experiments were done as two biological replicates. D Cell cycle distribution of HCC1806 control and CCNE1 and CDK2 knockdown cells treated with BLU-222 at 0.5 µM, measured by flow cytometry using propidium iodide (PI) staining. The experiments were done as two biological replicates. E Western blots of cyclin E1, CDK2, pCDK2 (Thr160), CDK4, CDK6, cyclin D1, and pRb (Ser807/811) in HCC1806 parental and CCNE1 or CDK2 knockdown cells, treated with BLU-222 at 0.3 µM, 0.6 µM, and 1.2 µM for 3 days. F Representative dose-response curves and IC 50 values for palbociclib-treated HCC1806 parental and CCNE1 and CDK2 knockdown cells. Error bars represent SEM from n = 3 experimental replicates. G Western blots of cyclin E, CDK2, and pCDK2 (Thr160) in T47D parental cells, empty vector (EV), full-length cyclin E (EL), or low-molecular-weight truncated cyclin E (LMW-E) cells. H Representative dose-response curves and IC 50 values for BLU-222-treated T47D parental cells, EL, and LMW-E. Error bars represent SEM from n = 3 experimental replicates. I Annexin V (+) quantification in T47D parental, EL, and LMW-E cells treated with palbociclib and/or BLU-222 (0.5 µM each) for 3 days. The experiments were done as 3 biological replicates. J - M IC 50 values distribution for 15 breast cancer cell lines treated with single agents of J palbociclib, K abemaciclib, L ribociclib, or M BLU-222. HR+/HER2− cell lines include MCF7, T47D, and CAMA1. The HER2-enriched cell lines include BT474, SKBR3, MDA-MB-361, UACC812, HCC1569, and HCC1954. The triple-negative (TNBC) cell lines include MDA-MB-231, BT20, HCC38, MDA-MB-157, MDA-MB-468, and HCC1806. Each box plot displays the median (center line), the 25 and 75th percentiles (bounds of the box), and the minimum and maximum values (whiskers). Individual data points represent independent cell lines. N - P Correlation between BLU-222 IC 50 values and those of N palbociclib, O abemaciclib, and P ribociclib. Differences between two groups were analyzed using a two-tailed unpaired Student’s t-test, and for multiple-group comparisons, a one-way ANOVA with Tukey’s multiple comparisons test was applied. The Pearson correlation coefficient (r) and associated p value were calculated for correlation analyses (ns, not significant).

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Western Blot, Control, Knockdown, Flow Cytometry, Staining, Plasmid Preparation, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Generation of palbociclib-resistant (PR) cell lines by stepwise dose escalation from 1.2 to 4.8 µM. B , C IC 50 values for MCF7 and T47D panels treated with single-agent palbociclib or BLU-222. Error bars represent SEM from n = 3 experimental replicates. D Representative 3D synergy plots for MCF7 parental and PR1.2 cells treated with different doses of palbociclib and BLU-222. E Representative 3D synergy plots for T47D parental and PR1.2 cells treated with different doses of palbociclib and BLU-222. F Annexin V (+) quantification in MCF7 parental and PR1.2 cells treated with single agents or combination. Error bars represent SEM from n = 3 experimental replicates. G Cell cycle distribution of MCF7 parental and PR1.2 cells treated with palbociclib and/or BLU-222 (0.3 µM, 3 days), measured by flow cytometry using PI staining. H Annexin V (+) quantification in T47D parental and PR1.2 cells treated with single agents or combination. The experiments were done as two biological replicates. I Cell cycle distribution of T47D parental and PR1.2 cells treated with palbociclib and/or BLU-222 (0.3 µM, 3 days). J Representative 3D synergy plots for HCC1806 and MDA-MB-157 cells treated with different doses of palbociclib and BLU-222. K Annexin V (+) quantification in HCC1806 and MDA-MB-157 cells treated with single agents or combination. The experiments were done as two biological replicates. L Cell cycle distribution of HCC1806 and MDA-MB-157 cells treated with palbociclib and/or BLU-222 (0.3 µM, 3 days), measured by flow cytometry using PI staining. The experiments were done as two biological replicates. M Representative 3D synergy plots for MDA-MB-231 parental and PR cells treated with different doses of palbociclib and BLU-222. N Annexin V (+) quantification in MDA-MB-231 parental and PR cells treated with single agents or combination. Error bars represent SEM from n = 3 experimental replicates. O Cell cycle distribution of MDA-MB-231 parental and PR cells under combination and single-agent treatment (0.3 µM, 3 days, n = 3). P Heatmap of highest single agent (HSA) synergic scores across HR+ , TNBC and acquired palbo-resistant breast cancer cell lines. Error bars represent SEM in at least three independent experiments. Comparisons between two treatment groups were analyzed using a two-tailed unpaired Student’s t-test; for comparisons among multiple groups, a one-way ANOVA with Tukey’s multiple comparisons test was applied.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Flow Cytometry, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A , B Representative Western blots showing expression levels of cyclin E1, E2, A, B1, D1, CDK2, CDK4, phosphorylated and total Rb, p21, p27 and actin in ( A ) MCF7 parental, PR1.2, and shRB1 knockdown cell lines and B T47D parental and PR1.2 cell lines treated with 0.3 µM of single agents or a combination of palbociclib and BLU-222 for 3 days. Blots are representative of 3 biologically independent experiments performed on separate cell preparations with similar results. The samples were derived from the same experiment, and all gels/blots were processed in parallel under identical conditions. C Western blots showing successfully CRISPR p21 knockout (KO) in MCF7 parental and PR1.2 cells. Bar graph showing the HSA synergistic scores in MCF7 parental ( n = 5) and PR1.2 cells ( n = 4) treated with the combination of BLU-222 and palbociclib, as well as pooled ( n = 4) and single clones of cells with p21 KO (A4, n = 4; C12, n = 4; CL1, n = 4; CL3, n = 3). Error bars represent SEM from n = 3 experimental replicates. D Representative 3D synergy plots illustrating the response of ( D ) MCF7 parental cells and p21 KO single clone (A4) and E MCF7 PR1.2 and p21 KO single clone (CL1) to combination treatment with different doses of palbociclib and BLU-222. F Western blots showing successfully CRISPR p27 KO in MCF7 parental ( n = 5) and PR1.2 cells ( n = 4). Bar graph showing the HSA synergistic scores in MCF7 and PR1.2 cells treated with the combination of BLU-222 and palbociclib, as well as pooled ( n = 3) and single clones of cells with p27 KO (A4, n = 2; E9, n = 4; CL1, n = 4; CL3, n = 4). Error bars represent SEM from n = 3 experimental replicates. G Representative 3D synergy plots illustrating the response of MCF7 p27 KO single clone (A4) and PR1.2 p27 KO single clone (CL1) to combination treatment with different doses of palbociclib and BLU-222. H Heatmap showing HSA scores across MCF7 parental (par) and PR1.2 CRISPR KO pools and single clones for p21 or p27. I Bar graph showing the IC 50 of MCF7 PR1.2 parental and CRISPR KO p21 pools or single clones treated with palbociclib (PR1.2, n = 4; pools, n = 4; CL1, n = 3; CL3, n = 5) or BLU-222 as single agents (PR1.2, n = 4; pools, n = 3; CL1, n = 4; CL3, n = 4). Box plots show the median (center line), 25th–75th percentiles (bounds of the box), and minimum–maximum values (whiskers). Error bars represent SEM from n = 3 experimental replicates. Comparisons between two treatment groups were analyzed using a two-tailed unpaired Student’s t-test; for comparisons among multiple groups, a one-way ANOVA with Tukey’s multiple comparisons test was applied.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Western Blot, Expressing, Knockdown, Derivative Assay, CRISPR, Knock-Out, Clone Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Representative confocal images of proximity ligation assay (PLA) on MCF7 parental cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM each, 3 days) detecting proximity and binding between p21 and CDK2. Nuclei stained with DAPI (blue); PLA performed for p21 and CDK2 (red); and actin filaments stained with phalloidin (green). Scale bars: 15 μm. B Quantification of PLA signals per cell nucleus showing p21/CDK2 binding in MCF7 parental cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM each, 3 days). C Representative confocal images of PLA on MCF7 PR1.2 cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM, 3 days) detecting binding between p21 and CDK2. D Quantification of PLA signals per cell nucleus showing p21/CDK2 binding in MCF7 PR1.2 cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM each, 3 days). E Immunoprecipitation (IP)–Western blotting analysis of p21 and p27 interactions with CDK2 in MCF7 parental (left) and PR1.2 (right) cells treated with single agents or the combination of palbociclib and BLU-222 (0.3 µM, 3 days). D, DMSO; P, palbociclib; B, BLU-222. F IP–Western blotting analysis of p21 and p27 interactions with CDK4 in MCF7 parental (left) and PR1.2 (right) cells treated with single agents or the combination of palbociclib and BLU-222 (0.3 µM, 3 days). Blots are representative of 3 biologically independent experiments performed on separate cell preparations with similar results. The samples were derived from the same experiment, and all gels/blots were processed in parallel under identical conditions. Error bars represent SEM from n = 3 experimental replicates. Differences among groups were analyzed using a one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Proximity Ligation Assay, Binding Assay, Staining, Immunoprecipitation, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – I Tumor growth curves, waterfall plots showing tumor volume changes, and Kaplan-Meier survival curves of PDX palbociclib-resistant HR+/HER2− breast cancer models. A – C PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5). D – F PR3 (vehicle, n = 8; BLU-222, n = 3; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 4), and G – I PR4 (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4), treated with vehicle, palbociclib, BLU-222, BLU-222 plus palbociclib, or triple combination with fulvestrant. Tumor pieces from each model were xenograft implanted into the fat pad of NCR-NU-F sp/sp 10-week-old female mice. Drug treatment was initiated when tumors reached approximately 100 mm³. Palbociclib: 50 mg/kg, orally, once daily; BLU-222: 60 mg/kg, orally, twice daily; fulvestrant: 2.5 mg/kg, subcutaneously, once weekly. Error bars represent the SEM from n = biological replicates. J – W Immunohistochemistry (IHC) quantification of tumors collected from treated PDX mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. J – M PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5), N – Q PR3, (vehicle, n = 8; BLU-222, n = 3; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 4), and R – U PR4 (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4). V Representative IHC images of p21 and p27 expression in the PR1 model treated with vehicle, single agents, or a combination of palbociclib and BLU-222 +/- fulvestrant. Scale bar = 50 μm. Tumor growth curves were analyzed using a two-way ANOVA to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – I Tumor growth curves and waterfall plots showing tumor volume changes and Kaplan-Meier survival curves of PDX PR HR+/HER2− breast cancer models. A – C PR1, treated with vehicle ( n = 5), ribociclib ( n = 6), fulvestrant ( n = 4), ribociclib + fulvestrant ( n = 5), palbociclib + fulvestrant ( n = 6), BLU-222 + ribociclib ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 5). D – F PR3, treated with vehicle ( n = 5), ribociclib ( n = 5), fulvestrant ( n = 5), palbociclib + fulvestrant ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 4), and G – I PR4, treated with vehicle ( n = 4), ribociclib ( n = 5), fulvestrant ( n = 4), palbociclib + fulvestrant ( n = 5), BLU-222 + ribociclib ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 5). Ribociclib: 50 mg/kg, orally, once daily; Palbociclib: 50 mg/kg, orally, once daily; BLU-222: 60 mg/kg, orally, twice daily; Fulvestrant: 2.5 mg/kg, subcutaneously, once weekly. Error bars represent the SEM from n = biological replicates. J – U IHC quantification of tumors collected from treated PDX mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. J - M PR1 (vehicle, n = 5; ribociclib, n = 6; fulvestrant, n = 4; ribociclib + fulvestrant, n = 5; palbociclib + fulvestrant, n = 6); BLU-222 + ribociclib, n = 5; BLU-222 + ribociclib + fulvestrant, n = 5). N - Q PR3 (vehicle, n = 5; ribociclib, n = 5; fulvestrant, n = 5; palbociclib + fulvestrant, n = 5; BLU-222 + ribociclib + fulvestrant, n = 4), and R - U PR4 (vehicle, n = 4; ribociclib, n = 5; fulvestrant, n = 4; palbociclib + fulvestrant, n = 5; BLU-222 + ribociclib, n = 5; BLU-222 + ribociclib + fulvestrant, n = 5). V Representative IHC images of p21 and p27 expression in the PR1 model treated with vehicle, single agents, or a combination of ribociclib and BLU-222 +/- fulvestrant. Scale bar = 50 μm. Tumor growth curves were analyzed using a two-way ANOVA test to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Staining, Expressing

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – D Tumor growth curves and waterfall plots showing tumor volume changes and Kaplan-Meier survival curves of PDX PR TNBC breast cancer models. A XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), B BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), C BCX017 (vehicle, n = 6; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 5), and D MMTV-T1p, a transgenic model overexpressing LMW-E (vehicle, n = 5; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4). Palbociclib: 50 mg/kg, orally, once daily; BLU-222: low-dose 50 mg/kg and high-dose 100 mg/kg, orally, twice daily. Error bars represent the SEM from n = biological replicates. E - T IHC quantification of tumors collected from treated mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. E, I, M, Q XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), ( F, J, N, R ) BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), ( G, K, O, S ) BCX017 (vehicle, n = 6; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 5), and ( H, L, P, T ) transgenic T1p (vehicle, n = 5; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4). Each tumor piece was quantified by the average percentage of positivity of cells in 2-3 different areas. Tumor growth curves were analyzed using a two-way ANOVA with Tukey’s multiple comparisons test to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Transgenic Assay, Staining

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Dot plot showing normalized enrichment scores (NES) of Hallmark gene sets from gene set enrichment analysis (GSEA) for the combination BLU-222 and palbociclib treatment arm across HR+/HER2− (PR1, n = 3 and PR4, n = 4) and TNBC (BCX070, n = 5 and XC5172013, n = 4) PDX models. B Venn diagram showing common and unique upregulated genes associated with pathways upon combination treatment with BLU-222 and palbociclib across 4 PDX models. C – F Heatmaps showing the standardized single-sample GSEA (ssGSEA) scores for each sample of interferon-α and -γ pathways, TNF-α, and inflammation response pathways across 4 PDX models. Bar plots on top of each heatmap indicate the average value of the standardized ssGSEA scores for each sample. G , H Enrichment plots of the interferon-γ gene set from GSEA performed on XC5172013 and PR1 tumors for the comparison of the treatment combination of BLU-222 and palbociclib versus vehicle treatment. I , J Heatmaps of fold-change values of genes associated with interferon-α and -γ pathways in treated tumors compared to vehicle-treated tumors across all PDX models. K – N Heatmaps showing the standardized ssGSEA scores for each sample of gene sets related to senescence across 4 PDX models. O , P Representative images of Sudan Black B (SBB)-stained lipofuscin (far-red, Cy5), indicative of therapy-induced senescence, in O BCX070 and P PR4 tumors from vehicle-treated and combination-treated (BLU-222+palbociclib) mice. Scale bars, 100 μm. Q Quantification of SBB-positive lipofuscin area (%) in BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5), and PR4 tumors (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4) across indicated treatment groups. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Comparison, Staining

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – I Tumor growth curves, waterfall plots showing tumor volume changes, and Kaplan-Meier survival curves of PDX palbociclib-resistant HR+/HER2− breast cancer models. A – C PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5). D – F PR3 (vehicle, n = 8; BLU-222, n = 3; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 4), and G – I PR4 (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4), treated with vehicle, palbociclib, BLU-222, BLU-222 plus palbociclib, or triple combination with fulvestrant. Tumor pieces from each model were xenograft implanted into the fat pad of NCR-NU-F sp/sp 10-week-old female mice. Drug treatment was initiated when tumors reached approximately 100 mm³. Palbociclib: 50 mg/kg, orally, once daily; BLU-222: 60 mg/kg, orally, twice daily; fulvestrant: 2.5 mg/kg, subcutaneously, once weekly. Error bars represent the SEM from n = biological replicates. J – W Immunohistochemistry (IHC) quantification of tumors collected from treated PDX mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. J – M PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5), N – Q PR3, (vehicle, n = 8; BLU-222, n = 3; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 4), and R – U PR4 (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4). V Representative IHC images of p21 and p27 expression in the PR1 model treated with vehicle, single agents, or a combination of palbociclib and BLU-222 +/- fulvestrant. Scale bar = 50 μm. Tumor growth curves were analyzed using a two-way ANOVA to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – I Tumor growth curves and waterfall plots showing tumor volume changes and Kaplan-Meier survival curves of PDX PR HR+/HER2− breast cancer models. A – C PR1, treated with vehicle ( n = 5), ribociclib ( n = 6), fulvestrant ( n = 4), ribociclib + fulvestrant ( n = 5), palbociclib + fulvestrant ( n = 6), BLU-222 + ribociclib ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 5). D – F PR3, treated with vehicle ( n = 5), ribociclib ( n = 5), fulvestrant ( n = 5), palbociclib + fulvestrant ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 4), and G – I PR4, treated with vehicle ( n = 4), ribociclib ( n = 5), fulvestrant ( n = 4), palbociclib + fulvestrant ( n = 5), BLU-222 + ribociclib ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 5). Ribociclib: 50 mg/kg, orally, once daily; Palbociclib: 50 mg/kg, orally, once daily; BLU-222: 60 mg/kg, orally, twice daily; Fulvestrant: 2.5 mg/kg, subcutaneously, once weekly. Error bars represent the SEM from n = biological replicates. J – U IHC quantification of tumors collected from treated PDX mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. J - M PR1 (vehicle, n = 5; ribociclib, n = 6; fulvestrant, n = 4; ribociclib + fulvestrant, n = 5; palbociclib + fulvestrant, n = 6); BLU-222 + ribociclib, n = 5; BLU-222 + ribociclib + fulvestrant, n = 5). N - Q PR3 (vehicle, n = 5; ribociclib, n = 5; fulvestrant, n = 5; palbociclib + fulvestrant, n = 5; BLU-222 + ribociclib + fulvestrant, n = 4), and R - U PR4 (vehicle, n = 4; ribociclib, n = 5; fulvestrant, n = 4; palbociclib + fulvestrant, n = 5; BLU-222 + ribociclib, n = 5; BLU-222 + ribociclib + fulvestrant, n = 5). V Representative IHC images of p21 and p27 expression in the PR1 model treated with vehicle, single agents, or a combination of ribociclib and BLU-222 +/- fulvestrant. Scale bar = 50 μm. Tumor growth curves were analyzed using a two-way ANOVA test to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Staining, Expressing

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Dot plot showing normalized enrichment scores (NES) of Hallmark gene sets from gene set enrichment analysis (GSEA) for the combination BLU-222 and palbociclib treatment arm across HR+/HER2− (PR1, n = 3 and PR4, n = 4) and TNBC (BCX070, n = 5 and XC5172013, n = 4) PDX models. B Venn diagram showing common and unique upregulated genes associated with pathways upon combination treatment with BLU-222 and palbociclib across 4 PDX models. C – F Heatmaps showing the standardized single-sample GSEA (ssGSEA) scores for each sample of interferon-α and -γ pathways, TNF-α, and inflammation response pathways across 4 PDX models. Bar plots on top of each heatmap indicate the average value of the standardized ssGSEA scores for each sample. G , H Enrichment plots of the interferon-γ gene set from GSEA performed on XC5172013 and PR1 tumors for the comparison of the treatment combination of BLU-222 and palbociclib versus vehicle treatment. I , J Heatmaps of fold-change values of genes associated with interferon-α and -γ pathways in treated tumors compared to vehicle-treated tumors across all PDX models. K – N Heatmaps showing the standardized ssGSEA scores for each sample of gene sets related to senescence across 4 PDX models. O , P Representative images of Sudan Black B (SBB)-stained lipofuscin (far-red, Cy5), indicative of therapy-induced senescence, in O BCX070 and P PR4 tumors from vehicle-treated and combination-treated (BLU-222+palbociclib) mice. Scale bars, 100 μm. Q Quantification of SBB-positive lipofuscin area (%) in BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5), and PR4 tumors (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4) across indicated treatment groups. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Palbociclib isethionate and fulvestrant were purchased from MedChemExpress (palbociclib #HY-A0065; fulvestrant #HY-13636).

Techniques: Comparison, Staining

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Western blots of cyclin E, CDK2, pCDK2 (Thr160), Rb, and pRb (Ser807/811) in HCC1806 parental cells, scramble control, and CCNE1 or CDK2 knockdown cells. B Representative dose-response curves and IC 50 values for BLU-222-treated HCC1806 scramble control and CCNE1 and CDK2 knockdown cell lines. Error bars represent standard error of the mean (SEM) from n = 3 experimental replicates. C Annexin V (+) quantification in HCC1806 control and CCNE1 and CDK2 knockdown cells treated with BLU-222 at 0.5 µM in for 0, 24, and 48 h. The experiments were done as two biological replicates. D Cell cycle distribution of HCC1806 control and CCNE1 and CDK2 knockdown cells treated with BLU-222 at 0.5 µM, measured by flow cytometry using propidium iodide (PI) staining. The experiments were done as two biological replicates. E Western blots of cyclin E1, CDK2, pCDK2 (Thr160), CDK4, CDK6, cyclin D1, and pRb (Ser807/811) in HCC1806 parental and CCNE1 or CDK2 knockdown cells, treated with BLU-222 at 0.3 µM, 0.6 µM, and 1.2 µM for 3 days. F Representative dose-response curves and IC 50 values for palbociclib-treated HCC1806 parental and CCNE1 and CDK2 knockdown cells. Error bars represent SEM from n = 3 experimental replicates. G Western blots of cyclin E, CDK2, and pCDK2 (Thr160) in T47D parental cells, empty vector (EV), full-length cyclin E (EL), or low-molecular-weight truncated cyclin E (LMW-E) cells. H Representative dose-response curves and IC 50 values for BLU-222-treated T47D parental cells, EL, and LMW-E. Error bars represent SEM from n = 3 experimental replicates. I Annexin V (+) quantification in T47D parental, EL, and LMW-E cells treated with palbociclib and/or BLU-222 (0.5 µM each) for 3 days. The experiments were done as 3 biological replicates. J - M IC 50 values distribution for 15 breast cancer cell lines treated with single agents of J palbociclib, K abemaciclib, L ribociclib, or M BLU-222. HR+/HER2− cell lines include MCF7, T47D, and CAMA1. The HER2-enriched cell lines include BT474, SKBR3, MDA-MB-361, UACC812, HCC1569, and HCC1954. The triple-negative (TNBC) cell lines include MDA-MB-231, BT20, HCC38, MDA-MB-157, MDA-MB-468, and HCC1806. Each box plot displays the median (center line), the 25 and 75th percentiles (bounds of the box), and the minimum and maximum values (whiskers). Individual data points represent independent cell lines. N - P Correlation between BLU-222 IC 50 values and those of N palbociclib, O abemaciclib, and P ribociclib. Differences between two groups were analyzed using a two-tailed unpaired Student’s t-test, and for multiple-group comparisons, a one-way ANOVA with Tukey’s multiple comparisons test was applied. The Pearson correlation coefficient (r) and associated p value were calculated for correlation analyses (ns, not significant).

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Western Blot, Control, Knockdown, Flow Cytometry, Staining, Plasmid Preparation, Molecular Weight, Two Tailed Test

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Generation of palbociclib-resistant (PR) cell lines by stepwise dose escalation from 1.2 to 4.8 µM. B , C IC 50 values for MCF7 and T47D panels treated with single-agent palbociclib or BLU-222. Error bars represent SEM from n = 3 experimental replicates. D Representative 3D synergy plots for MCF7 parental and PR1.2 cells treated with different doses of palbociclib and BLU-222. E Representative 3D synergy plots for T47D parental and PR1.2 cells treated with different doses of palbociclib and BLU-222. F Annexin V (+) quantification in MCF7 parental and PR1.2 cells treated with single agents or combination. Error bars represent SEM from n = 3 experimental replicates. G Cell cycle distribution of MCF7 parental and PR1.2 cells treated with palbociclib and/or BLU-222 (0.3 µM, 3 days), measured by flow cytometry using PI staining. H Annexin V (+) quantification in T47D parental and PR1.2 cells treated with single agents or combination. The experiments were done as two biological replicates. I Cell cycle distribution of T47D parental and PR1.2 cells treated with palbociclib and/or BLU-222 (0.3 µM, 3 days). J Representative 3D synergy plots for HCC1806 and MDA-MB-157 cells treated with different doses of palbociclib and BLU-222. K Annexin V (+) quantification in HCC1806 and MDA-MB-157 cells treated with single agents or combination. The experiments were done as two biological replicates. L Cell cycle distribution of HCC1806 and MDA-MB-157 cells treated with palbociclib and/or BLU-222 (0.3 µM, 3 days), measured by flow cytometry using PI staining. The experiments were done as two biological replicates. M Representative 3D synergy plots for MDA-MB-231 parental and PR cells treated with different doses of palbociclib and BLU-222. N Annexin V (+) quantification in MDA-MB-231 parental and PR cells treated with single agents or combination. Error bars represent SEM from n = 3 experimental replicates. O Cell cycle distribution of MDA-MB-231 parental and PR cells under combination and single-agent treatment (0.3 µM, 3 days, n = 3). P Heatmap of highest single agent (HSA) synergic scores across HR+ , TNBC and acquired palbo-resistant breast cancer cell lines. Error bars represent SEM in at least three independent experiments. Comparisons between two treatment groups were analyzed using a two-tailed unpaired Student’s t-test; for comparisons among multiple groups, a one-way ANOVA with Tukey’s multiple comparisons test was applied.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Flow Cytometry, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A , B Representative Western blots showing expression levels of cyclin E1, E2, A, B1, D1, CDK2, CDK4, phosphorylated and total Rb, p21, p27 and actin in ( A ) MCF7 parental, PR1.2, and shRB1 knockdown cell lines and B T47D parental and PR1.2 cell lines treated with 0.3 µM of single agents or a combination of palbociclib and BLU-222 for 3 days. Blots are representative of 3 biologically independent experiments performed on separate cell preparations with similar results. The samples were derived from the same experiment, and all gels/blots were processed in parallel under identical conditions. C Western blots showing successfully CRISPR p21 knockout (KO) in MCF7 parental and PR1.2 cells. Bar graph showing the HSA synergistic scores in MCF7 parental ( n = 5) and PR1.2 cells ( n = 4) treated with the combination of BLU-222 and palbociclib, as well as pooled ( n = 4) and single clones of cells with p21 KO (A4, n = 4; C12, n = 4; CL1, n = 4; CL3, n = 3). Error bars represent SEM from n = 3 experimental replicates. D Representative 3D synergy plots illustrating the response of ( D ) MCF7 parental cells and p21 KO single clone (A4) and E MCF7 PR1.2 and p21 KO single clone (CL1) to combination treatment with different doses of palbociclib and BLU-222. F Western blots showing successfully CRISPR p27 KO in MCF7 parental ( n = 5) and PR1.2 cells ( n = 4). Bar graph showing the HSA synergistic scores in MCF7 and PR1.2 cells treated with the combination of BLU-222 and palbociclib, as well as pooled ( n = 3) and single clones of cells with p27 KO (A4, n = 2; E9, n = 4; CL1, n = 4; CL3, n = 4). Error bars represent SEM from n = 3 experimental replicates. G Representative 3D synergy plots illustrating the response of MCF7 p27 KO single clone (A4) and PR1.2 p27 KO single clone (CL1) to combination treatment with different doses of palbociclib and BLU-222. H Heatmap showing HSA scores across MCF7 parental (par) and PR1.2 CRISPR KO pools and single clones for p21 or p27. I Bar graph showing the IC 50 of MCF7 PR1.2 parental and CRISPR KO p21 pools or single clones treated with palbociclib (PR1.2, n = 4; pools, n = 4; CL1, n = 3; CL3, n = 5) or BLU-222 as single agents (PR1.2, n = 4; pools, n = 3; CL1, n = 4; CL3, n = 4). Box plots show the median (center line), 25th–75th percentiles (bounds of the box), and minimum–maximum values (whiskers). Error bars represent SEM from n = 3 experimental replicates. Comparisons between two treatment groups were analyzed using a two-tailed unpaired Student’s t-test; for comparisons among multiple groups, a one-way ANOVA with Tukey’s multiple comparisons test was applied.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Western Blot, Expressing, Knockdown, Derivative Assay, CRISPR, Knock-Out, Clone Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Representative confocal images of proximity ligation assay (PLA) on MCF7 parental cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM each, 3 days) detecting proximity and binding between p21 and CDK2. Nuclei stained with DAPI (blue); PLA performed for p21 and CDK2 (red); and actin filaments stained with phalloidin (green). Scale bars: 15 μm. B Quantification of PLA signals per cell nucleus showing p21/CDK2 binding in MCF7 parental cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM each, 3 days). C Representative confocal images of PLA on MCF7 PR1.2 cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM, 3 days) detecting binding between p21 and CDK2. D Quantification of PLA signals per cell nucleus showing p21/CDK2 binding in MCF7 PR1.2 cells treated with DMSO, palbociclib, BLU-222, or the combination (0.3 µM each, 3 days). E Immunoprecipitation (IP)–Western blotting analysis of p21 and p27 interactions with CDK2 in MCF7 parental (left) and PR1.2 (right) cells treated with single agents or the combination of palbociclib and BLU-222 (0.3 µM, 3 days). D, DMSO; P, palbociclib; B, BLU-222. F IP–Western blotting analysis of p21 and p27 interactions with CDK4 in MCF7 parental (left) and PR1.2 (right) cells treated with single agents or the combination of palbociclib and BLU-222 (0.3 µM, 3 days). Blots are representative of 3 biologically independent experiments performed on separate cell preparations with similar results. The samples were derived from the same experiment, and all gels/blots were processed in parallel under identical conditions. Error bars represent SEM from n = 3 experimental replicates. Differences among groups were analyzed using a one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Proximity Ligation Assay, Binding Assay, Staining, Immunoprecipitation, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – I Tumor growth curves, waterfall plots showing tumor volume changes, and Kaplan-Meier survival curves of PDX palbociclib-resistant HR+/HER2− breast cancer models. A – C PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5). D – F PR3 (vehicle, n = 8; BLU-222, n = 3; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 4), and G – I PR4 (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4), treated with vehicle, palbociclib, BLU-222, BLU-222 plus palbociclib, or triple combination with fulvestrant. Tumor pieces from each model were xenograft implanted into the fat pad of NCR-NU-F sp/sp 10-week-old female mice. Drug treatment was initiated when tumors reached approximately 100 mm³. Palbociclib: 50 mg/kg, orally, once daily; BLU-222: 60 mg/kg, orally, twice daily; fulvestrant: 2.5 mg/kg, subcutaneously, once weekly. Error bars represent the SEM from n = biological replicates. J – W Immunohistochemistry (IHC) quantification of tumors collected from treated PDX mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. J – M PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5), N – Q PR3, (vehicle, n = 8; BLU-222, n = 3; palbociclib, n = 4; BLU-222 + palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 4), and R – U PR4 (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4). V Representative IHC images of p21 and p27 expression in the PR1 model treated with vehicle, single agents, or a combination of palbociclib and BLU-222 +/- fulvestrant. Scale bar = 50 μm. Tumor growth curves were analyzed using a two-way ANOVA to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – I Tumor growth curves and waterfall plots showing tumor volume changes and Kaplan-Meier survival curves of PDX PR HR+/HER2− breast cancer models. A – C PR1, treated with vehicle ( n = 5), ribociclib ( n = 6), fulvestrant ( n = 4), ribociclib + fulvestrant ( n = 5), palbociclib + fulvestrant ( n = 6), BLU-222 + ribociclib ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 5). D – F PR3, treated with vehicle ( n = 5), ribociclib ( n = 5), fulvestrant ( n = 5), palbociclib + fulvestrant ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 4), and G – I PR4, treated with vehicle ( n = 4), ribociclib ( n = 5), fulvestrant ( n = 4), palbociclib + fulvestrant ( n = 5), BLU-222 + ribociclib ( n = 5), BLU-222 + ribociclib + fulvestrant ( n = 5). Ribociclib: 50 mg/kg, orally, once daily; Palbociclib: 50 mg/kg, orally, once daily; BLU-222: 60 mg/kg, orally, twice daily; Fulvestrant: 2.5 mg/kg, subcutaneously, once weekly. Error bars represent the SEM from n = biological replicates. J – U IHC quantification of tumors collected from treated PDX mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. J - M PR1 (vehicle, n = 5; ribociclib, n = 6; fulvestrant, n = 4; ribociclib + fulvestrant, n = 5; palbociclib + fulvestrant, n = 6); BLU-222 + ribociclib, n = 5; BLU-222 + ribociclib + fulvestrant, n = 5). N - Q PR3 (vehicle, n = 5; ribociclib, n = 5; fulvestrant, n = 5; palbociclib + fulvestrant, n = 5; BLU-222 + ribociclib + fulvestrant, n = 4), and R - U PR4 (vehicle, n = 4; ribociclib, n = 5; fulvestrant, n = 4; palbociclib + fulvestrant, n = 5; BLU-222 + ribociclib, n = 5; BLU-222 + ribociclib + fulvestrant, n = 5). V Representative IHC images of p21 and p27 expression in the PR1 model treated with vehicle, single agents, or a combination of ribociclib and BLU-222 +/- fulvestrant. Scale bar = 50 μm. Tumor growth curves were analyzed using a two-way ANOVA test to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Staining, Expressing

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A – D Tumor growth curves and waterfall plots showing tumor volume changes and Kaplan-Meier survival curves of PDX PR TNBC breast cancer models. A XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), B BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), C BCX017 (vehicle, n = 6; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 5), and D MMTV-T1p, a transgenic model overexpressing LMW-E (vehicle, n = 5; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4). Palbociclib: 50 mg/kg, orally, once daily; BLU-222: low-dose 50 mg/kg and high-dose 100 mg/kg, orally, twice daily. Error bars represent the SEM from n = biological replicates. E - T IHC quantification of tumors collected from treated mice, with percentage of cells with positive staining for Ki67, γH2AX, p21, and p27 in models. E, I, M, Q XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), ( F, J, N, R ) BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), ( G, K, O, S ) BCX017 (vehicle, n = 6; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 5), and ( H, L, P, T ) transgenic T1p (vehicle, n = 5; palbociclib, n = 5; BLU-222 50 mg/kg, n = 5; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4). Each tumor piece was quantified by the average percentage of positivity of cells in 2-3 different areas. Tumor growth curves were analyzed using a two-way ANOVA with Tukey’s multiple comparisons test to compare treatment groups across time. Survival analyses were performed using the Kaplan–Meier method, and statistical significance among treatment groups was assessed with the log-rank (Mantel–Cox) test. Median survival times are reported in the figure. IHC quantification was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Transgenic Assay, Staining

Journal: Nature Communications

Article Title: CDK2 inhibitor BLU-222 synergizes with CDK4/6 inhibitors in drug resistant breast cancers through p21/p27 induction

doi: 10.1038/s41467-025-67865-4

Figure Lengend Snippet: A Dot plot showing normalized enrichment scores (NES) of Hallmark gene sets from gene set enrichment analysis (GSEA) for the combination BLU-222 and palbociclib treatment arm across HR+/HER2− (PR1, n = 3 and PR4, n = 4) and TNBC (BCX070, n = 5 and XC5172013, n = 4) PDX models. B Venn diagram showing common and unique upregulated genes associated with pathways upon combination treatment with BLU-222 and palbociclib across 4 PDX models. C – F Heatmaps showing the standardized single-sample GSEA (ssGSEA) scores for each sample of interferon-α and -γ pathways, TNF-α, and inflammation response pathways across 4 PDX models. Bar plots on top of each heatmap indicate the average value of the standardized ssGSEA scores for each sample. G , H Enrichment plots of the interferon-γ gene set from GSEA performed on XC5172013 and PR1 tumors for the comparison of the treatment combination of BLU-222 and palbociclib versus vehicle treatment. I , J Heatmaps of fold-change values of genes associated with interferon-α and -γ pathways in treated tumors compared to vehicle-treated tumors across all PDX models. K – N Heatmaps showing the standardized ssGSEA scores for each sample of gene sets related to senescence across 4 PDX models. O , P Representative images of Sudan Black B (SBB)-stained lipofuscin (far-red, Cy5), indicative of therapy-induced senescence, in O BCX070 and P PR4 tumors from vehicle-treated and combination-treated (BLU-222+palbociclib) mice. Scale bars, 100 μm. Q Quantification of SBB-positive lipofuscin area (%) in BCX070 (vehicle, n = 5; palbociclib, n = 3; BLU-222 100 mg/kg, n = 5; BLU-222 + palbociclib, n = 4), XC5172013 (vehicle, n = 7; palbociclib, n = 2; BLU-222 100 mg/kg, n = 8; BLU-222 + palbociclib, n = 5), PR1 (vehicle, n = 5; BLU-222, n = 5; palbociclib, n = 4; BLU-222 + palbociclib + fulvestrant, n = 5), and PR4 tumors (vehicle, n = 4; BLU-222, n = 4; palbociclib, n = 6; BLU-222 + palbociclib + fulvestrant, n = 4) across indicated treatment groups. Data represent the mea n ± SEM of the percentage of positively stained cells from the above-mentioned study animals per group (n), with each sample quantified from 2–3 randomly selected high-power fields. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: In vitro treatments with single agents palbociclib (HY-50767, MCE), abemaciclib (HY-16297A, MCE), ribociclib (HY-15777, MCE), upadacitinib (HY-19569, MCE), abrocitinib (HY-107429, MCE), or BLU-222 were administered for 6 consecutive days, followed by 6 days without treatment.

Techniques: Comparison, Staining