Journal: bioRxiv

Article Title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

doi: 10.1101/2025.01.08.631992

Figure Lengend Snippet: (A) A positive selection CRISPR/Cas9 screen was conducted in ER+ MCF-7-Cas9 expressing cells using the human Brunello library. Cells were treated with the combination of 500 nM tamoxifen and 250 nM palbociclib (early: 6 week and late: 10 week timepoint), or 500 nM monotherapy palbociclib (early: 2 week and late: 4 week timepoint) or vehicle (tetrahydrofuran) before genomic DNA was collected and sequenced. (B) Venn diagram showing single guide RNAs (sgRNAs) that increased following treatment with palbociclib and tamoxifen + palbociclib. sgRNAs selected with a false discovery rate <0.5, and occurrence in ≥3 screens, where at least one screen was with combination therapy. Selected sgRNAs indicated with dashed white line. (C) Gene set enrichment analysis of commonly downregulated genes with ≥2.5 aggregate β-score. (D) Top sgRNAs enriched following treatment with palbociclib and tamoxifen + palbociclib, and corresponding β-scores (sgRNAs with aggregate β-score ≥5 are shown). Orange * indicates sgRNAs in the JNK pathway. Black * indicates sgRNAs described in the literature to drive CDK4/6 inhibitor resistance. (E) Schematic of the JNK signalling pathway and enriched sgRNAs, as well as aggregate β- score scale. P indicates phosphorylation events.

Article Snippet: Cells were treated with the following: tamoxifen (Sigma-Aldrich; resuspended in tetrahydrofuran), fulvestrant (Sigma-Aldrich; resuspended in EtOH), palbociclib (Selleckchem; resuspended in water), anisomycin (Sigma-Aldrich; resuspended in dimethyl sulfoxide), and puromycin (Sigma-Aldrich; resuspended in water).

Techniques: Selection, CRISPR, Expressing

Journal: bioRxiv

Article Title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

doi: 10.1101/2025.01.08.631992

Figure Lengend Snippet: (A) Representative Western blot of ERα, MKK7, pJNK T183/Y185 , JNK and GAPDH in MCF-7 pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (Absolute ethanol (EtOH) and tetrahydrofuran (THF)) for 48 hours (full length blots in Supplementary Figure 3B). (B) Quantitation of MKK7 expression by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (C) Quantitation of pJNK T183/Y185 activity by densitometry and normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (D) Representative Western blot of ERα, MKK7, pJNK T183/Y185 , JNK and GAPDH in T-47D pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) for 48 hours (full length blots in Supplementary Figure 3D). (E) Quantitation of MKK7 expression by densitometry and normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (F) Quantitation of pJNK T183/Y185 activity by densitometry and normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (G) MCF-7 pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) and analysed by time-lapse microscopy using an IncuCyte ZOOM over 7 days. Cell count was determined by red fluorescent count, and data analysed by two-way ANOVA with Sidak’s multiple comparisons test. Experiment was performed in triplicate. (H) T-47D pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) and analysed by time-lapse microscopy using an IncuCyte ZOOM over 7 days. Cell count was determined by red fluorescent count, and data analysed by two-way ANOVA with Sidak’s multiple comparisons test. Experiment was performed in triplicate. (I) Representative colony formations from MCF-7 pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) for 3 weeks, with colony-formation detected with 0.1 – 0.5% crystal violet stain. (J) Colony formation was quantitated using ImageJ. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (K) Representative brightfield images of MCF-7 pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) for 72h and stained with senescence-associated β-galactosidase. (L) Quantification of MCF-7 pLenti and MAP2K7 -/- cells staining positive for senescence-associated β-galactosidase from (K). Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. Scale bar = 100 µm. (M) Representative brightfield images of T-47D pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) for 72h and stained with senescence-associated β-galactosidase. (N) Quantification of T-47D pLenti and MAP2K7 -/- cells staining positive for senescence-associated β-galactosidase from (M). Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. Scale bar = 100 µm.

Article Snippet: Cells were treated with the following: tamoxifen (Sigma-Aldrich; resuspended in tetrahydrofuran), fulvestrant (Sigma-Aldrich; resuspended in EtOH), palbociclib (Selleckchem; resuspended in water), anisomycin (Sigma-Aldrich; resuspended in dimethyl sulfoxide), and puromycin (Sigma-Aldrich; resuspended in water).

Techniques: Western Blot, Quantitation Assay, Expressing, Activity Assay, Time-lapse Microscopy, Cell Counting, Staining

Journal: bioRxiv

Article Title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

doi: 10.1101/2025.01.08.631992

Figure Lengend Snippet: (A) Experimental schematic of pLenti and MKK7_3 cells (MCF-7 or T-47D) treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (Absolute ethanol (EtOH) and tetrahydrofuran (THF)) for 48 hours. RNA was collected and analysed by RNAseq. (B) Principal components analysis of RNAseq analysis of MCF-7 cells +/- treatments, and T-47D cells +/- treatments. (C) Relative change in gene expression between vehicle and treatment (tamoxifen + palbociclib or fulvestrant + palbociclib) of MAP2K7 -/- vs pLenti cells. Boxed regions are genes that are less downregulated, or less upregulated, in MKK7_3 cells compared to pLenti cells. Pearson’s correlation coefficient shown. (D) Analysis of gene set enrichment of hallmark gene sets from genes that are less downregulated (purple plots) or less upregulated (orange plots) with treatment in MAP2K7 -/- cells, in either MCF-7 or T-47D cell lines. Size of dot is fold enrichment of the signature, with enriched signatures with false discovery rate (FDR) of 0.05 shown. FDR values shown in Supplementary Table 3. (E) (i) Venn spider plots of transcription factors (TFs) commonly deregulated in MAP2K7 -/- MCF-7 and MAP2K7 -/- T- 47D cells that are untreated; Veh = vehicle; boxed region contains significantly altered TFs. (ii) Venn spider plots of TFs commonly deregulated in MAP2K7 -/- MCF-7 and T-47D cells that are treated with combination therapy; tam + palb = 500 nM tamoxifen + 250 nM palbociclib; or fas+palb = 25 nM fulvestrant + 125 nM palbociclib; boxed region contains significantly altered TFs.

Article Snippet: Cells were treated with the following: tamoxifen (Sigma-Aldrich; resuspended in tetrahydrofuran), fulvestrant (Sigma-Aldrich; resuspended in EtOH), palbociclib (Selleckchem; resuspended in water), anisomycin (Sigma-Aldrich; resuspended in dimethyl sulfoxide), and puromycin (Sigma-Aldrich; resuspended in water).

Techniques: Expressing

Journal: bioRxiv

Article Title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

doi: 10.1101/2025.01.08.631992

Figure Lengend Snippet: (A) Representative Western blot of p-cJUN Ser63 , cJUN, JUND and GAPDH in MCF-7 pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (Absolute ethanol (EtOH) and tetrahydrofuran (THF)) for 48 hours (full length blots in Supplementary Figure 5D). (B) Quantitation of p-cJUN Ser63 activity by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (C) Quantitation of cJUN expression by densitometry and normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (D) Representative Western blot of p-cJUN Ser63 , cJUN, JUND and GAPDH in T-47D pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with 500 nM tamoxifen + 250 nM palbociclib (tam+palb), 25 nM fulvestrant + 125 nM palbociclib (fas+palb) or vehicle (EtOH and THF) for 48 hours (full length blots in Supplementary Figure 5F). (E) Quantitation of p-cJUN Ser63 activity by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (F) Quantitation of cJUN expression by densitometry and normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (G) Schematic of treatment to examine short-term fulvestrant-mediated arrest and release. MCF-7 and T-47D cells were treated with 10 nM fulvestrant for 48 hours to induce a cell cycle arrest. Cells were then stimulated to re-enter the cell cycle with 100 nM 17β-estradiol for 12 hours and 24 hours. (H) Representative Western blot of ERα, MKK7, p-cJUN Ser63 , cJUN, pJNK T183/Y185 , JNK and GAPDH in MCF-7 pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with fulvestrant and estradiol (full length blots in Supplementary Figure 5I). (I) Quantitation of pJNK T183/Y185 activity by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (J) Quantitation of p-cJUN Ser63 activity by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (K) Quantitation of cJUN expression by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (L) Representative Western blot of ERα, MKK7, p-cJUN Ser63 , cJUN, pJNK T183/Y185 , JNK and GAPDH in T-47D pLenti and MAP2K7 -/- (MKK7_1 and MKK7_3) cells treated with fulvestrant and estradiol (full length blots in Supplementary Figure 5L). (M) Quantitation of pJNK T183/Y185 activity by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (N) Quantitation of p-cJUN Ser63 activity by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (O) Quantitation of cJUN expression by densitometry. Band intensity was normalised to GAPDH. Data analysed by two-way ANOVA with Tukey’s multiple comparisons test. (P) Expression of JUN and JUND in primary ER+ breast cancers from the TCGA cohort compared to MAP2K7 expression. Pearson’s correlation coefficient shown.

Article Snippet: Cells were treated with the following: tamoxifen (Sigma-Aldrich; resuspended in tetrahydrofuran), fulvestrant (Sigma-Aldrich; resuspended in EtOH), palbociclib (Selleckchem; resuspended in water), anisomycin (Sigma-Aldrich; resuspended in dimethyl sulfoxide), and puromycin (Sigma-Aldrich; resuspended in water).

Techniques: Western Blot, Quantitation Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

doi: 10.1101/2025.01.08.631992

Figure Lengend Snippet: (A) JNK1, JNK2, pJNK1 Y185 (NP_001265477.1), pJNK2 Y185 (NP_001128516.1) Clinical Proteomic Tumour Analysis Consortium data from the TCGA cohort, showing expression in normal breast tissue (n=18) and luminal breast cancers (n=64). Data analysed by unpaired two-tailed t-test. (B) Copy number status of MAP3K11 , MAP2K4 , MAP2K7 , MAPK8 , MAPK9, JUN and ESR1 in primary (TCGA; n=808 and METABRIC; n=1817) and metastatic (Metastatic Breast Cancer Project; n=77) cohorts. Data analysed by unpaired two- tailed t-test. (C) Kaplan-Meier curves of the probability of overall survival and relapse-free survival in ER+ breast cancers comparing high, medium, and low tertiles of JNK pathway expression, where JNK pathway is MAP2K7 , MAPK8 and MAPK9 . Kaplan-Meier analysis was performed on pooled breast cancer datasets using KMPlotter . P-value calculated by log-rank (Mantel-Cox) test. (D) Correlation of JNK pathway with the anti-proliferative response of pre-operative palbociclib (POP) trial ER+ breast cancer patients . Anti- proliferative response was determined by change in TYMS mRNA expression between initial biopsy and post-treatment (Δ TYMS ) and correlated with JNK signalling ( MAP2K7 , MAPK8 and MAPK9 ). Pearson’s correlation coefficient shown. (E) Immunohistochemistry analysis of advanced metastatic ER+ breast cancer cohort treated with endocrine therapy + CDK4/6 inhibition. Images of FFPE sections with high, medium, and low pJNK T183/Y185 activity. Scale bar = 100 µm. (F) Kaplan-Meier curves of survival in the endocrine + CDK4/6 inhibitor- treated cohort comparing pJNK T183/Y185 tertiles or low, medium and high pJNK T183/Y185 . Significance determined by log-rank (Mantel-Cox) test. (G) mRNA expression of MAP3K11 , MAP2K4 , MAP2K7 , MAPK8 , MAPK9 and JUN stratified into patients who did not develop metastatic disease (no metastasis; n=107) and patients who did (metastasis; n=48). Comparisons between metastatic and non-metastatic by Mann-Whitney unpaired two-tailed t- test. (H) Anti-proliferative response of POP trial ER+ breast cancer patients and association with individual JNK pathways genes. Patients with high (n=64) and low (n=8) JNK pathway gene expression ( MAP3K11 , MAP2K4 , MAP2K7 , MAPK8 , MAPK9 and JUN ) were stratified based on TYMS anti-proliferative response. Comparisons between high and low by unpaired two-tailed t-test.

Article Snippet: Cells were treated with the following: tamoxifen (Sigma-Aldrich; resuspended in tetrahydrofuran), fulvestrant (Sigma-Aldrich; resuspended in EtOH), palbociclib (Selleckchem; resuspended in water), anisomycin (Sigma-Aldrich; resuspended in dimethyl sulfoxide), and puromycin (Sigma-Aldrich; resuspended in water).

Techniques: Expressing, Two Tailed Test, Immunohistochemistry, Inhibition, Activity Assay, MANN-WHITNEY

Journal: Molecular cancer therapeutics

Article Title: Hypomorphic mTOR downregulates CDK6 and delays thymic Pre-T LBL tumorigenesis

doi: 10.1158/1535-7163.MCT-19-0671

Figure Lengend Snippet: (A) Upstream regulator analysis (Ingenuity) of genes differentially expressed (Table S1) between WT (n=4) and KD (n=3) thymic lymphomas (green designates downregulated in KD mice and pink designates upregulated, numbers indicate fold change in KD mice relative to WT). Upstream regulators, miR-21 and let-7a, were increased in (B) KD tumors vs. WT, and in (C) rapamycin treated tumors vs. untreated. Values for the target genes were normalized to endogenous control (SnoRNA-234) with the WT or WT-untreated values standardized to 1. (D) Levels of Cdk6 transcripts were evaluated in WT and KD tumor cells by RT-PCR (p=0.05). (E) CDK6 protein levels were compared between pre-tumor thymocytes and tumors from representative WT and KD mice (representative mice). (F) CDK6 and pS6 levels in untreated vs. rapamycin treated mTOR WT tumor cell lines (3 independent lines)(see Supplemental Fig. 2B for CDK6 and CDK4 levels in additional untreated tumors). (G) Cell viability of WT and KD tumor cells (n=3/group) treated in vitro for 48 hours with 1 nM rapamycin (mTORC1 inhibitor) relative to untreated cells. Error bars indicate the standard error. (H) Cell cycle analysis (propidium idodide (PI) staining of untreated and treated (1nM rapamycin, 48 hours) mTOR WT cells. (I) Cell cycle analysis (PI staining) of WT, KD and KD cells transfected with CDK6 (from a representative experiment). (J) Cell cycle analysis (PI staining) of KD tumor cells and KD tumor cells transfected with a let-7 sponge (representative experiment). (K) Low passage WT and KD tumor cell lines (n = 3 each) treated for 24 hours with a CDK4/6 inhibitor palbociclib over a range of doses. IC50 values between WT and KD tumor cell lines were significantly different (p <0.03). (L) Low passage WT cells treated with single agents, palbociclib (P) (500nM) and rapamycin (R) (1nM), or the combination of the two drugs (R+P).

Article Snippet: Mice were randomized into four treatment groups of 8 mice each, and then received once-daily treatments (Monday-Friday) beginning 4 days post transplantation of either vehicle (PO and IP), palbociclib (LC Labs, gavage at 50 mg/kg in 1% Tween-80 in sterile water), rapamycin (LC labs, IP at 2.5 mg/kg in 5% Tween-80;5%PEG in sterile saline), or a combination of palbociclib and rapamycin.

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vitro, Cell Cycle Assay, Staining, Transfection

Journal: Molecular cancer therapeutics

Article Title: Hypomorphic mTOR downregulates CDK6 and delays thymic Pre-T LBL tumorigenesis

doi: 10.1158/1535-7163.MCT-19-0671

Figure Lengend Snippet: Hypothetical signaling model for upregulation of let-7 and miR21 leading to the downregulation of Cdk6 after genetic or pharmacological downregulation of mTOR in AKT induced tumors. Genetic knockdown of mTOR expression in transgenic mice via expression of Mtortm1. Lgm (mTOR KD) instead of wild type Mtor or by treatment with the mTOR inhibitor rapamycin (Rapa), leads to upregulation of let-7 and miR-21 and downregulation of Cdk6. Genetic or pharmacological downregulation of mTOR signaling combined with inhibition of CDK6 activity by palbociclib (Palb) results in decreased tumor cell proliferation compared to treatment with Rapa or Palb alone.

Article Snippet: Mice were randomized into four treatment groups of 8 mice each, and then received once-daily treatments (Monday-Friday) beginning 4 days post transplantation of either vehicle (PO and IP), palbociclib (LC Labs, gavage at 50 mg/kg in 1% Tween-80 in sterile water), rapamycin (LC labs, IP at 2.5 mg/kg in 5% Tween-80;5%PEG in sterile saline), or a combination of palbociclib and rapamycin.

Techniques: Expressing, Transgenic Assay, Inhibition, Activity Assay

Journal: Molecular cancer therapeutics

Article Title: Hypomorphic mTOR downregulates CDK6 and delays thymic Pre-T LBL tumorigenesis

doi: 10.1158/1535-7163.MCT-19-0671

Figure Lengend Snippet: (A) The average weight of tumors generated from Lck-Akt2;mTOR WT tumor cells injected into the flanks of nude mice and treated individually or with the combination of mTOR (rapamycin, R) and CDK4/6 (palbociclib, P) inhibitors for 5 days. Error bars represent standard error of the mean for the tumors from the 4 mice/group. (B) Tumors from 3 mice/treatment group were labeled for Ki67 (proliferation marker) and percent positive Ki67 nuclei in each tumor group is depicted. (C) Percent cells in G1 in tumor transplants treated with single agents or the combination. (D) Immunoblots from representative WT flank tumor transplants after 5-day treatment with vehicle, palbociclib, rapamycin, and the combination. Proteins designated with a “p-“ are the phosphorylated forms of the protein. (E) Survival curves of mice (N=8/group) tail vein injected with 5×106 WT pre-T LBL tumor cells and treated with vehicle, either single agent, or the combination. Red arrows indicate the cycle of drug treatment. Mice were off drug treatment for one week between cycles 1 and 2. Statistical analysis was done using Mantel-Cox log-rank testing with Bonferroni correction. (F) Graphs show the Excess over Highest Single Agent (EOHSA) for T-ALL/LBL cell lines treated for 48h with increasing doses of palbociclib (CDK6i, 0 nm-1 μM, starting at 62.5nM and increasing 2 fold for each dose) and rapamycin (mTORi, 0 nM to 100 nM, increasing 10-fold for each dose), representing the difference in viability between cells treated by the single agent and the combination at each dose. Values are averaged results from 4 replicate experiments. See Supplemental Fig. 4A,B for dose matrix and synergy scores.

Article Snippet: Mice were randomized into four treatment groups of 8 mice each, and then received once-daily treatments (Monday-Friday) beginning 4 days post transplantation of either vehicle (PO and IP), palbociclib (LC Labs, gavage at 50 mg/kg in 1% Tween-80 in sterile water), rapamycin (LC labs, IP at 2.5 mg/kg in 5% Tween-80;5%PEG in sterile saline), or a combination of palbociclib and rapamycin.

Techniques: Generated, Injection, Labeling, Marker, Western Blot