Journal: eBioMedicine

Article Title: A bedside-to-bench translational analysis of NF1 alterations and CDK4/6 inhibitor resistance in hormone receptor-positive metastatic breast cancer

doi: 10.1016/j.ebiom.2025.105828

Figure Lengend Snippet: NF1 deletion is causal to CDK4/6i resistance in HR + breast cancer cell lines. a. Immunoblot analysis of NF1 WT and NF1 KO MCF7 and T47D cells cultured in oestrogen-free media (0% CSS). Lysates were probed with the indicates antibodies. b. Cell proliferation of NF1 WT and NF1 KO MCF7 and T47D cells grown in oestrogen-free media (10% CSS) for 6 days was measured by Incucyte live-cell analysis. c-f. NF1 WT and NF1 KO MCF7 and T47D cells were treated with 9 concentrations of palbociclib in full media (c, d) or in oestrogen-depleted media (10% CSS) (e, f) for 6 days. The number of cells per well at the end of treatment was measured by Incucyte live-cell analysis. Data represent mean ± SD (n = 3 technical replicates).

Article Snippet: Palbociclib (#S1579), abemaciclib (#S7158), fulvestrant (#S1191), capivasertib (#S8019), trametinib (#S2673), ulixertinib (#S7854) were purchased from Selleck Chemicals.

Techniques: Western Blot, Cell Culture, Cell Analysis

Journal: eBioMedicine

Article Title: A bedside-to-bench translational analysis of NF1 alterations and CDK4/6 inhibitor resistance in hormone receptor-positive metastatic breast cancer

doi: 10.1016/j.ebiom.2025.105828

Figure Lengend Snippet: NF1 -deficient breast cancer cells are sensitive to RAS or AKT inhibition in combination with palbociclib. a-d. NF1 KO MCF7 (a and b) and T47D (c and d) cells were treated for 6 days with increasing concentrations (3-fold) of palbociclib, capivasertib, RMC-6236 or trametinib up to 10 μM. For combination assays, all cells were treated with 250 nM palbociclib and increasing concentrations of the second drug. The number of cells per well at the end of treatment was measured by Incucyte live-cell analysis. Data represent mean ± SD (n = 3 technical replicates). Data from palbociclib vs. palbociclib in combination with capivasertib or RMC-6236, capivasertib vs. capivasertib + palbociclib 250 nM, and RMC-6236 vs. RMC-6236+palbociclib 250 nM were analysed with ANOVA multiple comparisons. e-h. Growth of a patient-derived organoid (PDO) 33682 under different treatment conditions. PDOs from an NF1-mutant, CDK4/6 inhibitor-resistant patient were cultured for 22 days in the presence of vehicle (DMSO), palbociclib 250 nM or 1000 nM, and the combination of palbociclib 250 nM with increasing concentrations of capivasertib, RMC-6236, or trametinib. Organoid proliferation in 3D was assessed using the CellTiter-Glo 3D assay. Bar graph represents mean organoid growth relative to vehicle control (e) or to palbociclib 250 nM (f–h), with error bars indicating ±SD. Data correspond to four technical replicates. ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001; ∗∗∗∗, p < .0001.

Article Snippet: Palbociclib (#S1579), abemaciclib (#S7158), fulvestrant (#S1191), capivasertib (#S8019), trametinib (#S2673), ulixertinib (#S7854) were purchased from Selleck Chemicals.

Techniques: Inhibition, Cell Analysis, Derivative Assay, Mutagenesis, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma

doi: 10.3390/ijms222313087

Figure Lengend Snippet: Efficacy of cell cycle inhibitors. ( A ) Inhibition of cell proliferation due to the incubation with cell cycle inhibitors. An MTT viability assay was used for IC 50 determination. Here, PCI 1 was chosen as the representative cell line with nocodazole (G2/M phase) treatment. Photo documentation after four days of inhibitor treatment confirmed the inhibition of cell proliferation. PCI 13 was chosen as the representative cell line with aphidicolin (S phase) treatment. Images are shown at four- and 20-fold magnification. ( B ) Cell cycle analysis via flow cytometry after inhibitor treatment. Flow cytometry confirmed that palbociclib treatment resulted in G1 phase, aphidicolin in S phase, and nocodazole in G2/M phase arrest. Here, PCI 13 was also chosen as the representative cell line. Incubation with DMSO instead of a specific inhibitor were used as controls. To quantify the DNA content, cells were incubated with DAPI. For analysis 5 × 10 4 cells were used per sample. The proportion of cells in a particular cell cycle phase is expressed as a percentage (%). ( C ) Effective concentrations of cell cycle inhibitors. Concentrations for palbociclib, aphidicolin, and nocodazole are listed in nanomolar (nM). High concentrations are highlighted in dark, whereas low concentrations are highlighted in light colors. Cell lines were subdivided into their epithelial and mesenchymal differentiation status and ranked according to their PD-L1 basal expression.

Article Snippet: Palbociclib (PD 0332991 isethionate), a potent cyclin-dependent CDK4 and CDK6 inhibitor that induces G1 cell cycle arrest, was obtained from Tocris Bioscience (Bristol, UK).

Techniques: Inhibition, Incubation, MTT Viability Assay, Cell Cycle Assay, Flow Cytometry, Expressing

Journal: Oncogenesis

Article Title: CDK4/6 inhibitors and the pRB-E2F1 axis suppress PVR and PD-L1 expression in triple-negative breast cancer

doi: 10.1038/s41389-023-00475-1

Figure Lengend Snippet: A Immunoblot analysis showing the effect of 2-day palbociclib treatment on ngPVR and gPVR expression. B , C Immunoblot analysis of PD-L1 in TNBC lines treated with increasing ( B ) or high ( C ) concentrations of palbociclib for 2 days. D Immunoblot analysis of PD-L1 in TNBC lines with stable knockdown of RB1 , treated with/without palbociclib for 2 days. E Immunoblot analysis of PD-L1 in livers of mice on mixed background treated with palbociclib (140 mg/kg; gavage; 5 consecutive days/week) for 28 days and probed with AF1019 antibody (left) or in livers of C57BL/6 mice treated with palbociclib (140 mg/kg; gavage) for 7 consecutive days, probed with MAB90781 antibody (right). Bottom, PD-L1 quantification. Pure palbociclib (FA65120) was suspended in sodium lactate (50 nM, PH 4.0), which was used in the vehicle control mice. F Heatmap analysis of RNA-seq data (GSE177054) of MDA231 treated with ribociclib or ribociclib/D4476 for 2 days. White boxes denote out-of-range intensity. G Immunoblot analysis of PD-L1 in indicated tumor cells following knockdown of SPOP by RNAi over 2 (D2) or 3 (D3) days. H Immunoblot analysis of PD-L1 in indicated TNBC lines with/without SPOP knockdown after 2 days (D2) and treated with/without palbociclib for 24 h. I Immunoblot analysis of PD-L1 and SPOP in indicated TNBC lines after palbociclib treatment for 48 h. Numbers above immunoblots denote band intensity normalized to loading control.

Article Snippet: In vitro: pure palbociclib (Carbosynth, cat. FA65120) was solubilized in HCl in equal molar ratio.

Techniques: Western Blot, Expressing, Knockdown, Control, RNA Sequencing

Journal: Oncogenesis

Article Title: CDK4/6 inhibitors and the pRB-E2F1 axis suppress PVR and PD-L1 expression in triple-negative breast cancer

doi: 10.1038/s41389-023-00475-1

Figure Lengend Snippet: A Immunoblot analysis of PD-L1 in TNBC lines seeded at various densities in 6-cm plates and treated with 1 µM palbociclib for ~2 days. Bottom, PD-L1 quantification of ( A ) and Supplementary Fig. . Statistics calculated by pairwise analysis of seeding densities and treatment durations. B Immunoblot analysis of TNBC lines treated with various concentrations of hydrochloric acid (HCl) for 2 days. Right, PD-L1 quantification and analysis by Welch’s t -test. C Immunoblot analysis of MDA231 treated with/without palbociclib and/or with/without indicated solvents. D Immunoblot analysis of PD-L1 and PVR in different TNBC lines treated with physiological concentrations of lactic acid for 2 days. Numbers above immunoblots denote band intensity normalized to loading control. E A model suggesting that in response to mitogenic signals, CDK4/6 controls PD-L1 turnover by inducing its degradation via Culin3 SPOP and its transcription via pRB-E2F1. PD-L1 in red and green depicts old or newly synthesized glycosylated protein, respectively. F A model suggesting that the CDK4/6-RB-E2F axis modulates immune surveillance by transcriptionally regulating PD-L1, PVR and potentially other IC modulator genes. Solid and segmented arrows indicate validated (herein) and unvalidated targets, respectively; single arrows denote pRB and E2F1 binding sites as well as strong E2F1 recruitment by ChIP-seq analysis, whereas double arrows denote no or weak E2F1 recruitment.

Article Snippet: In vitro: pure palbociclib (Carbosynth, cat. FA65120) was solubilized in HCl in equal molar ratio.

Techniques: Western Blot, Control, Synthesized, Binding Assay, ChIP-sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: The combination of AKT and CDK inhibitors is effective against HCC cells (A) HepG2 cells were treated with palbociclib (PB) (10 μM) or MK-2206 (MK) (5 μM) as single agents or with the combination of the two (PB 5 μM + MK 5 μM). A fourth group of cells received no treatment (NT). Viability and apoptosis were evaluated 72 h after the beginning of treatment. Data are represented as mean ± SD. ∗p value ≤0.05; ∗∗p value ≤0.01; ∗∗∗p value ≤0.001; ∗∗∗∗p value ≤0.0001. (B) Western blot analysis and quantification of RB1, AKT proteins and their phosphorylated forms in HepG2-treated cells. The values are normalized on the GAPDH protein levels. (C) Tumor nodule volumes in DEN-treated TG221 mice of the following groups: (1) palbociclib (PB) (n = 6); (2) MK-2206 (MK) (n = 6); (3) palbociclib + MK-2206 (PB + MK) (n = 5); (4) vehicle (CTRL) (n = 13). Experimental therapies started at 6 months, when all mice presented one or more tumor nodules in their livers. Single tumor nodules were monitored by ultrasound at the beginning and end of treatment. (D) Mice weight was monitored during treatment to check drug toxicity. Data are represented as mean ± SD.

Article Snippet: For all in vivo experiments, drugs were administered daily by oral gavage as indicated by the manufacturer’s instructions: sorafenib (S-8599, LC Laboratories, Woburn, MA, USA) was dissolved in a 50:50 Cremophor EL and ethanol solution; MK2206 (MK2206 dihydrochloride, Medchem Express, HY-10358-0002, NJ, USA) was dissolved in 15% Captisol (SBE-b-CD, Medchem Express, HY-17031-0731); palbociclib (palbociclib Isethionate Salt, CAS No. 827,022-33-3, BOC sciences, Shirley, NY, USA) was dissolved in sodium lactate buffer (50 mM, pH 4.0)

Techniques: Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: miR-199a-3p increased palbociclib efficacy on human HCC cells in vitro and in vivo (A) Hep3B cells were transduced with an Adeno Associated Virus expressing miR-199a-3p (AAVV-199) or with a control AAVV (AAVV-CTRL) at MOI = 100. A group of cells received no treatment (NT). Palbociclib (PB) (20 μM) was added to cell culture 72 h before cell collection. The levels of miR-199a-3p were measured 120 h after transduction. (B) Viability and apoptosis were evaluated 120 h after transduction. Data are represented as mean +SD. (C) Western blot analysis and quantification of RB1, AKT, FOXM1, and their phosphorylated forms in Hep3B-treated cells. The values were normalized on the GAPDH expression. Because Hep3B cells exhibit a low level of full-length protein, digital images of RB1 and p-RB1 were acquired with an exposure time of 300 s instead of 30 s. (D) Tumor nodule volumes in DEN-treated TG221 mice of the following groups: (1) palbociclib (PB) (n = 11); (2) miR-199a-3p mimics (miR-199) (n = 9); (3) palbociclib + miR-199a-3p mimics (PB + miR-199) (n = 11); (4) sorafenib (SF) (n = 9); (5) vehicle/scramble oligonucleotides (CTRL) (n = 9). Experimental therapies started at 6 months, when all mice presented one or more tumor nodules in their livers. Single tumor nodules were monitored by ultrasound at the beginning and end of treatment. (E) Distribution of tumor nodule size measured at the time of euthanization. (F) Mice weight was monitored during treatment to check drug toxicity. Data are represented as mean ± SD. ∗p value ≤0.05; ∗∗p value ≤0.01; ∗∗∗p value ≤0.001; ∗∗∗∗p value ≤0.0001.

Article Snippet: For all in vivo experiments, drugs were administered daily by oral gavage as indicated by the manufacturer’s instructions: sorafenib (S-8599, LC Laboratories, Woburn, MA, USA) was dissolved in a 50:50 Cremophor EL and ethanol solution; MK2206 (MK2206 dihydrochloride, Medchem Express, HY-10358-0002, NJ, USA) was dissolved in 15% Captisol (SBE-b-CD, Medchem Express, HY-17031-0731); palbociclib (palbociclib Isethionate Salt, CAS No. 827,022-33-3, BOC sciences, Shirley, NY, USA) was dissolved in sodium lactate buffer (50 mM, pH 4.0)

Techniques: In Vitro, In Vivo, Transduction, Virus, Expressing, Control, Cell Culture, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: In vivo anti-cancer activity of palbociclib and mir-199a-3p

Article Snippet: For all in vivo experiments, drugs were administered daily by oral gavage as indicated by the manufacturer’s instructions: sorafenib (S-8599, LC Laboratories, Woburn, MA, USA) was dissolved in a 50:50 Cremophor EL and ethanol solution; MK2206 (MK2206 dihydrochloride, Medchem Express, HY-10358-0002, NJ, USA) was dissolved in 15% Captisol (SBE-b-CD, Medchem Express, HY-17031-0731); palbociclib (palbociclib Isethionate Salt, CAS No. 827,022-33-3, BOC sciences, Shirley, NY, USA) was dissolved in sodium lactate buffer (50 mM, pH 4.0)

Techniques: In Vivo, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: miR-199a-3p enhanced palbociclib therapeutic effect on xenografts of sorafenib-resistant cells (A) AAVV-199 transduced SF-resistant cells were implanted into the right side, while AAVV-CTRL transduced cells were implanted into the left side of each mouse. (B and C) When tumor reached a volume of ∼50 mm 3 (10 days after injection of cells), mice received the following treatments: (1) not treated (NT) (n = 5); (2) palbociclib (PB) (n = 10); (3) sorafenib (SF) (n = 10). Size of tumor nodules was measured with a caliper every 2 days until euthanization. (D and E) Endpoint mean tumor volume; (F and G) miR-199a-3p expression levels in tumors at time of euthanization. Data are represented as mean ± SD. ∗p value ≤0.05; ∗∗p value ≤0.01.

Article Snippet: For all in vivo experiments, drugs were administered daily by oral gavage as indicated by the manufacturer’s instructions: sorafenib (S-8599, LC Laboratories, Woburn, MA, USA) was dissolved in a 50:50 Cremophor EL and ethanol solution; MK2206 (MK2206 dihydrochloride, Medchem Express, HY-10358-0002, NJ, USA) was dissolved in 15% Captisol (SBE-b-CD, Medchem Express, HY-17031-0731); palbociclib (palbociclib Isethionate Salt, CAS No. 827,022-33-3, BOC sciences, Shirley, NY, USA) was dissolved in sodium lactate buffer (50 mM, pH 4.0)

Techniques: Injection, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: Palbociclib in combination with miR-199a-3p represents an effective and better tolerated therapy after sorafenib treatment (A) At 6 months of age, all DEN-treated mice were treated with sorafenib (two cycles of 21 days each) as a first-line treatment. Then, mice were split into three groups and treated for an additional 21 days as follows: (1) no further treatment (n = 3); (2) sorafenib (SF) (n = 3); (3) combination of palbociclib + miR-199a-3p mimics (PB + miR199) (n = 3). Tumor growth was monitored with ultrasound (usg) at the beginning and at the end of each treatment. Results indicated that the suspension of sorafenib treatment led to a reduction of toxicity effects (E) but also tumors grew back (B). Continuation of sorafenib led to a reduction of tumor growth (C) but also to a critical weight loss. (D) PB + miR-199 combination led to a reduction of tumor nodule sizes and was well tolerated (G).

Article Snippet: For all in vivo experiments, drugs were administered daily by oral gavage as indicated by the manufacturer’s instructions: sorafenib (S-8599, LC Laboratories, Woburn, MA, USA) was dissolved in a 50:50 Cremophor EL and ethanol solution; MK2206 (MK2206 dihydrochloride, Medchem Express, HY-10358-0002, NJ, USA) was dissolved in 15% Captisol (SBE-b-CD, Medchem Express, HY-17031-0731); palbociclib (palbociclib Isethionate Salt, CAS No. 827,022-33-3, BOC sciences, Shirley, NY, USA) was dissolved in sodium lactate buffer (50 mM, pH 4.0)

Techniques: Suspension