abemaciclib (cat# cs-1230)  (ChemScene llc)

Journal: Science Advances

Article Title: A noncanonical function of the tyrosine degradation enzyme FAH drives CDK4/6 inhibitor resistance in breast cancer

doi: 10.1126/sciadv.aef1145

Figure Lengend Snippet: ( A and B ) IC 50 curve (A) and IC 50 value (B) of empty vector (EV)– and FAH -overexpressing MCF7 cells treated with abemaciclib for 72 hours. ( C ) IC 50 value in T47D gAAVS and gFAH cells treated with abemaciclib for 72 hours. Three guide RNAs (#g1-3) were used to knock out FAH by CRISPR-Cas9. ( D ) Representative images of FAH immunohistochemistry in patients #8, #10, #26, and #27 PDOs. ( E ) Representative images of EdU and FAH staining in patient #10 gAAVS and gFAH#3 PDOs treated with vehicle or 300 nM abemaciclib. ( F and G ) Quantification of EdU+ cells in patient #8 gAAVS and gFAH PDOs treated with vehicle (F) or 300 nM abemaciclib (G). ( H and I ) Quantification of EdU+ cells in patient #10 gAAV and gFAH PDOs treated with vehicle (H) or 300 nM abemaciclib (I). ( J ) Representative images of EdU and FAH staining in patient #26 EV- and FAH-overexpressing PDOs treated with vehicle or 300 nM abemaciclib. ( K and L ) Quantification of EdU+ cells in patient #26 EV- and FAH -overexpressing PDOs treated with vehicle (K) or 300 nM abemaciclib (L). ( M and N ) Quantification of EdU+ cells in patient #27 EV- and FAH -overexpressing PDOs treated with vehicle (M) or 300 nM abemaciclib (N). PDOs were treated with 300 nM abemaciclib for 7 days, whereafter proliferation was assessed by pulsing PDOs with EdU for 4 hours and quantifying the ratio of EdU+ cells per total (DAPI+) number of cells in 10 to 20 PDOs per biological replicate. Error bars indicate mean + SD, and red line indicates median. Biological replicates: n = 5 (B), n = 2 (N), n = 3 [(C), (G), (I), and (L)], and n = 4 [(C), (F), (H), (K), and (M)]. Student’s unpaired t test [(B) and (K) to (N)], one-way ANOVA with Dunnett’s test [(C) and (H)], or Kruskal-Wallis with Dunn’s test [(F), (G), and (I)] was used to assess significance. Scale bars: 100 μm (D) and 40 μm [(E) and (J)]. ns, not significant.

Article Snippet: The following day, the media was replaced with DMEM containing 1% FBS, and cells were treated with increasing concentrations of abemaciclib (SelleckChem, #S5716) ranging from 0.05 to 10 μM.

Techniques: Plasmid Preparation, Knock-Out, CRISPR, Immunohistochemistry, Staining

Journal: Science Advances

Article Title: A noncanonical function of the tyrosine degradation enzyme FAH drives CDK4/6 inhibitor resistance in breast cancer

doi: 10.1126/sciadv.aef1145

Figure Lengend Snippet: ( A and B ) Percentage of 13 C labeling and abundance of intracellular tyrosine (A) and intracellular fumarate (B) after 4 hours of labeling measured by liquid chromatography–mass spectrometry (LC-MS). ( C and D ) Percentage of 13 C labeling and abundance of intracellular tyrosine (C) and intracellular fumarate (D) after 24 hours of labeling. Tyrosine labeling was performed by growing MCF7 EV control and FAH -overexpressing cells in media supplemented with 5% dialyzed fetal bovine serum (FBS) and 13 C9 15 N tyrosine (0.12 g/liter) for 4 hours [(A) and (B)] or 24 hours [(C) and (D)]. ( E and F ) Quantification of EdU+ cells in MCF7 EV, FAH WT, and FAH H133A mutant cells (E) and FAH A134D mutant cells (F) treated with 200 nM abemaciclib for 72 hours. ( G and H ) Volcano plot showing enriched and suppressed transcripts in MCF7 FAH -overexpressing cells treated with 200 nM abemaciclib for 72 hours (G) and T47D FAH KO (gFAH #3) cells treated with 500 nM abemaciclib for 72 hours (H). Proliferation-related transcripts are highlighted in red. Gene set enrichment analysis of MCF7 FAH -overexpressing cells ( I ) and T47D FAH KO cells treated with abemaciclib ( J ). The data are presented as normalized enrichment scores and FDR. Error bars indicate mean + SD. Biological replicates: n = 3 (E) and n = 4 (F). Student’s unpaired t test [(A) to (D)] or one-way ANOVA with Dunnett’s test [(E) and (F)] was used to assess significance. h, hours.

Article Snippet: The following day, the media was replaced with DMEM containing 1% FBS, and cells were treated with increasing concentrations of abemaciclib (SelleckChem, #S5716) ranging from 0.05 to 10 μM.

Techniques: Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Mutagenesis, Analysis

Journal: Science Advances

Article Title: A noncanonical function of the tyrosine degradation enzyme FAH drives CDK4/6 inhibitor resistance in breast cancer

doi: 10.1126/sciadv.aef1145

Figure Lengend Snippet: ( A and B ) Representative confocal images of FAH immunofluorescence staining (A) and quantification (B) of nuclear FAH in MCF7 FAH -overexpressing cells treated with 200 nM abemaciclib for 72 hours. ( C and D ) Representative confocal images (C) of FAH immunofluorescence in MCF7 NLS (FAH-NLS) and FAH nuclear export signal (FAH-NES) cells and (D) quantification of EdU+ cells in MCF7 EV control, FAH-WT, FAH-NLS, and FAH-NES cells treated with 200 nM abemaciclib for 72 hours. Proliferation was assessed by pulsing cells for 2 hours with EdU. ( E and F ) Representative confocal images EdU and FAH staining (E) and quantification (F) of EdU+ cells in patient #26 EV, FAH-WT, FAH-NLS, and FAH-NES expressing PDOs treated with 300 nM abemaciclib for 7 days. Proliferation was assessed by pulsing PDOs with EdU for 4 hours and quantifying the ratio of EdU+ cells per total (DAPI+) number of cells in 15 to 20 PDOs from each biological replicate. ( G and H ) Representative images of FAH immunohistochemistry (G) and scoring of nuclear and cytoplasmic FAH (H) of patient tumors pre- and post-CDK4/6 inhibitor treatment. Student’s unpaired t test (B), one-way ANOVA with Dunnett’s test [(D) and (F)], or Wilcoxon rank sum test, using the Holm-Bonferroni correction method (H), was used to assess significance. Error bars indicate mean + SD, and red line indicates median. Biological replicates: n = 5 [(B) and (D)] and n = 2 (F). Scale bars, 20 μm [(A) and (C)] and 40 μm [(E) and (G)].

Article Snippet: The following day, the media was replaced with DMEM containing 1% FBS, and cells were treated with increasing concentrations of abemaciclib (SelleckChem, #S5716) ranging from 0.05 to 10 μM.

Techniques: Confocal, Immunofluorescence, Staining, Control, Expressing, Immunohistochemistry

Journal: Science Advances

Article Title: A noncanonical function of the tyrosine degradation enzyme FAH drives CDK4/6 inhibitor resistance in breast cancer

doi: 10.1126/sciadv.aef1145

Figure Lengend Snippet: ( A ) Table with top 10 potential FAH interactors in MCF7 FAH-NLS BioID cells treated with 200 nM abemaciclib. Table shows average peptide counts from triplicate samples analyzed by mass spectrometry. CTRL BioID and FAH-NES BioID peptide counts are shown as controls. ( B ) Bar chart of GO molecular function pathway enrichment of proteins interacting with FAH-NLS under abemaciclib treatment. Data are plotted as fold enrichment of GO terms that are overrepresented in the dataset compared to the background set and FDR of the enrichment. snRNA, small nuclear RNA. ( C and D ) Representative images (C) and quantification (D) of nuclear PLA foci in vehicle- and abemaciclib-treated MCF7 FAH-V5 cells. PLA was used to assess FAH (V5-tag) and CDK9 interaction in 100 cells. ( E and F ) Enrichment score of MYC targets and B-MYB targets in MCF7 EV and FAH-OE cells (E) and T47D gAAVS and gFAH#3 KO cells (F) treated with abemaciclib for 72 hours. Yu MYC targets up and Shepard BMYB targets through the mSigDB were used for the analysis. ( G to I ) Western blot analysis of pRB, cMYC, FAH, and vinculin (VCL) protein in vehicle- and abemaciclib-treated MCF7 EV control and FAH -expressing cells (G), patient #26 EV and FAH PDOs (H) and T47D gAAVS and gFAH #3 KO cells (I). Biological replicates: n = 2 (D). Red line indicates median. Mann-Whitney test was used to assess significance. Scale bar, 10 μm.

Article Snippet: The following day, the media was replaced with DMEM containing 1% FBS, and cells were treated with increasing concentrations of abemaciclib (SelleckChem, #S5716) ranging from 0.05 to 10 μM.

Techniques: Mass Spectrometry, Analysis, Western Blot, Control, Expressing, MANN-WHITNEY

Journal: Science Advances

Article Title: A noncanonical function of the tyrosine degradation enzyme FAH drives CDK4/6 inhibitor resistance in breast cancer

doi: 10.1126/sciadv.aef1145

Figure Lengend Snippet: ( A ) AlphaFold prediction of FAH-CDK9 interaction. AlphaFold multimer prediction was performed using the Cosmic 2 platform. ( B ) Quantification of nuclear FAH-D143K in MCF7 cells treated with vehicle or 200 nm abemaciclib for 72 hours. ( C and D ) Representative images (C) and quantification (D) of PLA foci in vehicle- and abemaciclib-treated MCF7 FAH-WT-V5 and FAH D143K-V5 cells. PLA was used to assess V5-tag and CDK9 interaction in 100 cells. ( E ) Abemaciclib IC 50 of MCF7 EV, FAH-WT, and FAH D143K cells treated with increasing doses of abemaciclib for 72 hours. PrestoBlue cell viability reagent was used to assess cell viability. ( F ) Western blot analysis of pRB, cMYC, FAH, and vinculin (VCL) protein in vehicle- and abemaciclib-treated MCF7 EV control, FAH-WT, and FAH-D143K cells. ( G ) Representative confocal images of cleaved caspase 3 and EdU staining, ( H ) quantification of EdU+ cells, and ( I ) cleaved caspase 3+ PDOs in patient #26 PDOs treated with vehicle, 300 nM abemaciclib, 15 nM AZD4573 (CDK9 inhibitor), or the combination of abemaciclib and AZD4573 for 96 hours. Proliferation was assessed by pulsing PDOs with EdU for 4 hours and quantifying the ratio of EdU+ cells per total (DAPI+) number of cells. Apoptosis was quantified by staining for cleaved caspase 3–positive PDOs. Ten to 15 PDOs were analyzed per each individual experiment. Biological replicates: n = 3 (D), n = 5 (E), and n = 3 [(H) and (I)]. Error bars indicate mean + SD, and red line indicates median. Student’s unpaired t test [(H) and (I)], one-way ANOVA with Dunnetts’ test (E), or Kruskal-Wallis with Dunn’s test (D) was used to assess significance. Scale bars, 10 μm (C) and 40 μm (H).

Article Snippet: The following day, the media was replaced with DMEM containing 1% FBS, and cells were treated with increasing concentrations of abemaciclib (SelleckChem, #S5716) ranging from 0.05 to 10 μM.

Techniques: Western Blot, Analysis, Control, Confocal, Staining