PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer

Abstract

Endocrine therapies (ET) with cyclin-dependent kinase 4/6 (CDK4/6) inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of patient-derived xenografts (PDXs) from patients with 22 ER+ breast cancer demonstrated that protein kinase, membrane-associated tyrosine/threonine one (PKMYT1), a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX-derived organoids and PDXs, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

Figure 1.

PKMYT1 mRNA and protein levels are E2-regulated in ER⁺ breast cancer PDXs and cell lines. A, Volcano plot showing the effect of E2-deprivation on kinase levels in 22 ER⁺ PDXs whose tumor growth was either E2-dependent or E2 independent. All mice were ovariectomized before exogenous E2 was supplied or not. Levels were determined by KIPA. Differentially expressed kinases with P-values < 0.05 are labeled. B, Absolute PKMYT1 protein levels in individual PDX lines were measured by KIPA-SureQuant. C,PKMYT1 mRNA levels in individual PDX lines were determined by RNA-seq. D, RT-qPCR of PKMYT1 mRNA in hormone-deprived MCF7 and T47D cells treated with vehicle, 10 nmol/L E2, and E2 with 100 nmol/L fulvestrant for 2 days. E, Immunoblotting of protein lysates from hormone-deprived MCF7 and T47D cells treated with vehicle, 10 nmol/L E2, and E2 with 100 nmol/L fulvestrant for 2 days. ERα was decreased by fulvestrant as expected. GAPDH serves as a loading control. The figure is a representative image of three independent biological replicates. In (A), P-values were calculated by paired t test. In (B) and (C), Wilcoxon signed-rank test P-values were shown after the median difference; paired t test P-values were shown after the mean difference. Wilcoxon rank sum test and t test were used to compare E2-deprived samples. In (D), bars show the means of individual biological repeats indicated by the dots. P-values were calculated with one-way ANOVA and Tukey HSD.

Figure 2.

PKMYT1 mRNA levels are significantly associated with patient outcome and endocrine therapy response. A, Kaplan–Meier survival curves of disease-specific survival of patients with Luminal A and B (ER⁺) breast cancer in the METABRIC cohort, stratified by the median of PKMYT1 mRNA. Numbers of patients with high or low PKMYT1 mRNA are shown at the bottom. B,PKMYT1 mRNA level of pre- and post- AI treatment tumors of the patients from the ACOSOG Z1031B trial, grouped by AI clinical response (AI sensitive or AI resistant). C,PKMYT1 mRNA level of pre- and post-anastrozole treatment tumors of the patients from the NeoPalAna trial, grouped by anastrozole clinical response. In (A), the P-value and hazard ratio were calculated by the Cox Proportional-Hazards model. In (B and C), Wilcoxon signed-rank test P-values were shown after the median difference; paired t test P-values were shown after the mean difference. Wilcoxon rank sum test and t test were used to compare E2 post-treatment samples.

Figure 3.

Expression of PKMYT1 is negatively regulated by palbociclib treatment in sensitive, but not resistant, ER⁺ breast cancer cell lines and patients. A, Volcano plot of the correlation of PKMYT1 protein levels and Hallmark ssGSEA scores in 22 ER⁺ breast cancer in ovariectomized PDX mice given exogenous E2. Hallmark pathways with P-values < 0.05 are labeled. B, RT-qPCR of relative PKMYT1 mRNA level after 1 μmol/L palbociclib treatment for 2 days, adjusted by GAPDH mRNA and normalized by the vehicle-treated cells. C, Luciferase assays were performed to determine PKMYT1 promoter activities in cells transduced with a lentivirus-expressing Gaussia luciferase (GLuc) under the control of a ∼1.3 kb PKMYT1 promoter (see “Methods”). Cells were treated with 1 μmol/L palbociclib for 2 days, and GLuc values were normalized by the vehicle-treated cells. D, Immunoblotting of protein lysates made from cells treated with 1 μmol/L palbociclib or vehicle for 2 days. GAPDH serves as a loading control. The figure is a representative image of three independent biological replicates. E, Scatterplots of PKMYT1 mRNA and Hallmark pathway “E2F Targets” score of NeoPalAna patient samples collected at three stages of treatments [baseline, cycle 1 day 1 (4 weeks treatment with anastrozole) and cycle 1 day 15 (2 weeks treatment with anastrozole plus palbociclib)]. F,PKMYT1 mRNA level of pre- and post-anastrozole and palbociclib (A+P) treatment tumors of the patients from the NeoPalAna trial who did not initially respond to anastrozole alone, grouped by A+P response. In (B and C), bars show the means of three individual biological repeats. P-values were calculated by t test. In (E), the trend line was calculated by a linear regression model and P-values were calculated by Pearson and Spearman correlation. In (F), Wilcoxon signed-rank test P-values are shown after the median difference; paired t test P-values are shown after the mean difference. Wilcoxon rank sum test and t test were used to compare E2 post-treatment samples.

Figure 4.

A clinical-grade PKMYT1 inhibitor (RP-6036) and nucleoside analog (gemcitabine) synergistically and significantly reduce the viability of palbociclib-resistant ER⁺ breast cancer cells that lack functional p53 protein. A, Dose–response curves of RP-6306 effect on the viability of T47D parental and Palbo-R cells, with and without 1 nmol/L gemcitabine cotreatment. B and C, Loewe synergy scores (B) and combination viability scores (C) of T47D parental and Palbo-R cells treated with different concentrations of RP-6306 and gemcitabine. D and E, Loewe synergy scores (D) and combination viability scores (E) of MCF7 EDR and MCF7 EDR Palbo-R cells stably transduced with lentiviruses expressing nontargeting shRNA (shNC) or two different TP53-targeting shRNAs, treated with different concentrations of RP-6306 and gemcitabine. In (A), curves and IC₅₀s were derived by the three-parameter log-logistic model. Vertical error bars show the standard errors of the mean of the viabilities. Horizontal error bars show the standard errors of the IC₅₀s. In (B and D), P-values were calculated by Wilcoxon signed-rank test, adjusted by the Holms method if more than one comparison was performed. The (arrows) display whether the value is higher or lower than the baseline. In (C and E), error bars show the SEM, the approximate P-values were calculated by Z-test using the SEM of the model, adjusted by the Holms method if more than one comparison was performed. In all experiments, data were analyzed from three independent biological replicates.

Figure 5.

The combination of RP-6306 and gemcitabine increases apoptosis and activates DNA damage signaling in palbociclib-resistant T47D cells. A, The relative abundance of dead cells after 3 days drug treatment, measured by a CellTox Green assay, is shown relative to the vehicle treatment. Bars show the means of individual biological repeats indicated by the dots. B, Quantification of immunoblotting of protein lysates made from cells treated with 100 nmol/L RP-6306 and/or 2 nmol/L gemcitabine for 3 days. Cleaved PARP1 and caspase 3 are known markers of apoptosis. GAPDH serves as a loading control. The figure is a representative image of three independent biological replicates. C, Quantification of immunoblotting of protein lysates made from cells treated with 100 nmol/L RP-6306 and/or 2 nmol/L gemcitabine for 1 day. PKMYT1 phosphorylation of CDK1 at pT14 was assayed. DNA damage was assayed by induction of phosphorylation of ATR (pY1989) and histone variant H2AX pS139 (also known as γH2AX). Total CDK1 and ATR serve as normalizing controls of the corresponding phosphorylated proteins, while GAPDH serves as a loading control. The figure is a representative image of four independent biological replicates. Supplementary Fig. S18 shows representative immunoblots for quantified data in (B and C). In all data panels, P-values were calculated by Dunnett’s test (using the vehicle as the baseline) within each cell line.

Figure 6.

The combination of RP-6306 and gemcitabine reduces the growth of TP53-mutant, palbociclib-resistant ER⁺ breast cancer PDX organoids (PDxO) and PDX tumors. A, Heatmap depicts the ERα IHC status, PAM50 gene expression, E2 dependence, TP53 mutational status, an RNA-based MGPS, and RB1 and cyclin E1 copy number variation, mRNA, and protein levels in our collection of 22 ER⁺ breast cancer PDXs. PDXs were clustered by hierarchical clustering with one minus Pearson correlation. B, Two-week growth assay of four different PDxOs treated with 1 μmol/L palbociclib. Viability was assayed with a Cell Titer Glo 3D assay from three biological replicates. C, Two-week growth assay of four different PDxOs after treatment with vehicle, 0.5 nmol/L gemcitabine, 30 nmol/L RP-6306, or the combination of gemcitabine and RP-6306. In (B and C), bars show the means of three individual biological repeats indicated by the dots, and P-values of one-way ANOVA and Tukey HSD are reported in Supplementary Table S5. D, Tumor volumes of BCM-7441 PDX mice treated with vehicle (n = 8), RP-6306 (n = 8), gemcitabine (n = 7), and the combination of RP-6306 and gemcitabine (n = 7). Arrows indicate the start and the end of the 5-week treatment. Error bars reflect the standard errors of the mean. P-values were calculated by Dunnett’s test (day 11, using the vehicle as the baseline) and t test (days 35 and 56, comparing the gemcitabine vs. combination groups).