Overcoming Therapeutic Resistance in HER2-Positive Breast Cancers with CDK4/6 Inhibitors

Abstract

Using transgenic mouse models, cell line-based functional studies, and clinical specimens, we show that cyclin D1/CDK4 mediate resistance to targeted therapy for HER2-positive breast cancer. This is overcome using CDK4/6 inhibitors. Inhibition of CDK4/6 not only suppresses Rb phosphorylation, but also reduces TSC2 phosphorylation and thus partially attenuates mTORC1 activity. This relieves feedback inhibition of upstream EGFR family kinases, resensitizing tumors to EGFR/HER2 blockade. Consequently, dual inhibition of EGFR/HER2 and CDK4/6 invokes a more potent suppression of TSC2 phosphorylation and hence mTORC1/S6K/S6RP activity. The suppression of both Rb and S6RP enhances G1 arrest and a phenotype resembling cellular senescence. In vivo, CDK4/6 inhibitors sensitize patient-derived xenograft tumors to HER2-targeted therapies and delay tumor recurrence in a transgenic model of HER2-positive breast cancer.

Figure 1. Characterization of the MMTV-rtTA/tetO-HER2 mouse model

(A) Top: breeding scheme to create MMTV-rtTA/tetO-HER2 mice. Bottom: 8-week old bitransgenic mice were fed a doxycycline diet for 48 hours and mammary gland lysates were analyzed with western blots. MMTV-rtTA mice serve as controls. (B) Mammary tumors (arrows) in a MMTV-rtTA/ tetO-HER2 mouse after 12 weeks of doxycycline. (C) Time to first palpable tumor in MMTV-rtTA/ tetO-HER2 mice. Doxycycline-naive bitransgenic mice and doxycycline-treated MMTV-rtTA mice serve as controls. (D) Representative staining of MMTV-rtTA/tetO-HER2 tumors. (E) Western blots on MMTV-rtTA/tetO-HER2 tumor lysates from mice on doxycycline or after doxycycline withdrawal. (F) Representative staining of tumors from the experiment in (E). (G) Percentage of cyclin D1 stained nuclei in tumors from experiment depicted in (F). (H) Percentage of cleaved caspase-3 stained nuclei in tumors from experiment depicted in (F) (For (G) and (H), n=6 per group. **p≤0.01; ****p≤0.0001 by one-way ANOVA [Fisher’s LSD test]) (I) Changes in MMTV-rtTA/ tetO-HER2 tumor volume after 10 days’ lapatinib treatment (*p≤0.05 by student’s t-test). (J) Western blots on tumor lysates from experiment in (I). (K) Representative micrographs of tumors from the experiment in (I). (L) Representative images of lung metastases in bitransgenic mice after 16 weeks of doxycycline. (M) Frequency of microscopic lung metastases in bitransgenic mice after 16 weeks doxycycline with or without 4 subsequent weeks of a doxycycline-free diet. All scale bars represent 100μm except upper panels in (K) where scale bars represent 500μm. All error bars represent SD. See also Figure S1.

Figure 2. MMTV-rtTA/tetO-HER2 tumors that recur after HER2 withdrawal exhibit CDK4/6 dependent proliferation

(A) Representative MMTV-rtTA/tetO-HER2 primary tumor growth curves during doxycycline induction and subsequent doxycycline withdrawal. (B) Time to development of recurrent tumors from time of doxycycline withdrawal. (C) Heat map shows clustering by transcriptome-wide gene expression in normal mammary tissue (n=6), primary tumors (n=9), and recurrent tumors (n=11) of bitransgenic mice. Columns represent genes and rows represent tumors. (D) Time to 5-fold increase in tumor volume for orthotopically implanted primary and recurrent tumors (****p≤0.0001 by Log-rank (Mantel-Cox) test). (E) Percentage of Ki67-stained nuclei in primary and recurrent tumors (n=6 per group, **p≤0.01 by student’s t-test). (F) CCNA2, CCNB1, and CCNB2 transcript levels in normal mammary glands, primary tumors and recurrent tumors. (G) Left: CDKN2A transcript levels in MMTV-rtTA/tetO-HER2 normal mammary glands, primary tumors and recurrent tumors. Right: CDKN2A genomic DNA levels in primary (n=2) and recurrent (n=2) tumors, normalized to TFRC reference gene. For (F) and (G) boxes show median, 25^th, and 75^th percentiles and whiskers show minimum and maximum values. (FPKM – fragments per kilobase of exon per million reads mapped). (H) CCND1, CCND2, CCND3, CDK4, and CDK6 transcript levels in MMTV-rtTA/tetO-HER2 normal mammary glands and recurrent tumors. (I) Representative staining of recurrent tumors for cyclin D1 and CDK4. (J) Tumor growth in orthotopically implanted MMTV-rtTA/tetO-HER2 recurrent tumors after treatment with abemaciclib (n=5 or 6 per group. *p≤0.05, **p≤0.01 by student’s t-test). (K) Western blot shows changes in Rb phosphorylation in B405 recurrent tumor allografts after abemaciclib treatment (5 days). All scale bars represent 100μm. Error bars represent SD unless otherwise described. See also Figure S2.

Figure 3. Tumor cells surviving HER2-pathway blockade retain cyclin D1 expression

(A) Timing of mammary gland harvesting to identify residual viable tumor after HER2-withdrawal. (B) Percentage of cyclin D1-stained nuclei in normal mammary ducts (n=3), MMTV-rtTA/ tetO-HER2 primary tumors before (n=6) and 72 hours after (n=5) doxycycline withdrawal, and residual carcinoma cells seen 1–2 months after doxycycline withdrawal (n=8) (*p≤0.05, ****p≤0.0001 by one way ANOVA). (C) Representative staining of tissues described in (B). (D) Representative staining of MMTV-rtTA/tetO-HER2 primary tumors treated with control or lapatinib. Box indicates viable DCIS, arrows indicate viable invasive carcinoma. (E) Percentage of cyclin D1-stained nuclei in MMTV-rtTA/tetO-HER2 primary tumor regions showing histologic response to lapatinib (n=6), no response to lapatinib (n=6), or after control treatment (n=6) (**p≤0.01 by one-way ANOVA). All scale bars represent 100μm except third column in (D) where scale bars represent 500μm. All error bars represent SD. See also Figure S3.

Figure 4. The cyclin D1-CDK4 axis mediates resistance to HER2-targeted therapy

(A) Fold change in CCND1 gene expression in cell lines after treatment with lapatinib 500nM for 6 hours. (B) Western blots on cell lysates after treatment with lapatinib 500nM or DMSO for 24 hours (NB: for cleaved PARP blots, upper band represents total PARP and lower band cleaved PARP). (C) Sensitivity to lapatinib or trastuzumab (IC₅₀) of SKBR3/BT474 cells constitutively overexpressing CCND1 or empty vector (EV). (**p≤0.01, ***p≤0.001 by one-way ANOVA). (D) Relative viability of MDA-MB-453 tet-shCCND1 and MDA-MB-361 tet-shCCND1 cells in response to trastuzumab in the presence or absence of doxycycline. (E) Design of clinical trial NCT00148668 (TCH – docetaxel, carboplatin, trastuzumab; VH – vinorelbine, trastuzumab) (F) CCND1 copy number (determined by a Global Parameter Hidden Markov Model) by pathological complete response (CR) status in the NCT00148668 trial. Each dot represents an individual patient and lines represent means (*p=0.016 by Wilcoxon signed rank test). All error bars represent SD. See also Figure S4.

Figure 5. The effects of combined CDK4/6-HER2 inhibition on cellular viability, proliferation, and apoptosis

(A) Isobolograms showing actual (heatmap, and solid line at 50 percent inhibition) and predicted additive (dashed line) effects of combined abemaciclib/lapatinib therapy on viability of MDA-MB-453, MDA-MB-361, and UACC-732 cells. (B) Viability of trastuzumab-resistant SKBR3 and BT474 cells after treatment with trastuzumab 300ng/mL and/or abemaciclib 300nM. (C) Top: viability of parental or T-DM1 resistant MDA-MB-361 or MD-MB-453 cells after T-DM1 treatment. Bottom: viability of the same cells after abemaciclib treatment. (D) Effect of lapatinib and/or abemaciclib on percentage of cells in S phase. Light grey bars indicate control values. “Multiplication” shows expected effect of combined treatment if single agent effects were multiplied; red arrow indicates actual effect of combination. (MDA-MB-361/MDA-MB-453: lapatinib 100nM, abemaciclib 25nM. BT474 lapatinib 25nM, abemaciclib 75nM. SKBR3 lapatinib 25nM, abemaciclib 500nM. 48h treatment). (E) Representative flow cytometry plots of MDA-MB-453 cells (from experiments in (D)) labeled with anti-BrdU and propidium iodide. (F) Representative SA-beta-galactosidase staining in MDA-MB-453 cells treated with lapatinib (100nM) and/or abemaciclib (25nM). Stained cells appear dark (black and white photographs). (G) Mean staining intensity for cells in (F). (H) Cleaved PARP levels after treating cells with DMSO, lapatinib and/or abemaciclib (BT474 and SKBR3: lapatinib 100nM, abemaciclib 300nM; MDA-MB-361: lapatinib 500nM, abemaciclib 300nM; MDA-MB-453: lapatinib 500nM, abemaciclib 25nM). (NB: for cleaved PARP blots, upper band represents total PARP and lower band cleaved PARP). (**p≤0.01, ***p≤0.001, ****p≤0.0001 by one-way ANOVA). All error bars represent SD. See also Figure S5.

Figure 6. Effects of CDK4/6 inhibition on signal transduction in treatment-resistant HER2-positive breast cancers

(A) MDA-MB-453 and MDA-MB-361 cells were treated with lapatinib and/or abemaciclib for 24 hours and cell lysates were probed with the antibodies shown (concentrations as in Figure 5H). (B) MDA-MB-361 and MDA-MB-453 xenografts were treated with control or abemaciclib for 3–4 days. Tumor lysates were probed with the antibodies shown. (C) Anti-HA immunoprecipitates from CCND1-HA or CDK4-HA transfected MDA-MB-453 cells were immunoblotted for TSC2. (D) MDA-MB-453 cells were treated with abemaciclib and cell lysates were probed with the antibodies shown. (E) MDA-MB-453 cells were treated as in (A) and cell lysates probed with the antibodies shown. See also Figure S6.

Figure 7. The effects of combined CDK4/6-HER2 inhibition in vivo

(A) Growth curves for MMTV-rtTA/tetO-HER2 primary tumors treated with control, trastuzumab and/or abemaciclib. (B) Time to 5-fold increase in tumor volume (analysis by log-rank [Mantel-Cox] test) from the experiment in (A). (C) BT474 tumor growth after treatment with control, trastuzumab and/or abemaciclib. (D) Tumor growth of PDX model 14-07 (patient’s prior treatments shown – OFS, ovarian function suppression) after treatment with control, trastuzumab and/or abemaciclib ((C) and (D) analysis by one-way ANOVA [Kruskal-Wallis test]) (E) Percentage of Ki-67 positive cells in MDA-MB-453 xenografts after 5 day treatment with lapatinib and/or abemaciclib, with representative micrographs. (F) Relative beta-galactosidase positive area/total area in MDA-MB-453 xenografts after 5-day treatment with lapatinib and/or abemaciclib with representative micrographs. (G) Time to development of recurrent tumors after doxycycline withdrawal in MMTV-rtTA/tetO-HER2 mice treated with abemaciclib vs control for 30 days at time of doxycycline withdrawal. Data shown to maximum follow-up of 135 days (analysis by log-rank [Mantel-Cox] test). (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Data analysis was by one-way ANOVA unless otherwise indicated). All scale bars represent 100μm. All error bars represent SD. See also Figure S7.

Figure 8. CDK4 inhibition re-sensitizes HER2-therapy resistant tumors to inhibition of EGFR-family kinases

A: Under basal conditions, signaling downstream of EGFR-family kinases stimulates both P70-S6K (via AKT/mTOR) and Rb phosphorylation (via cyclin D1/CDK4). S6K exerts negative feedback to partially suppress the RTK signaling. In addition cyclin D1/CDK4 bind to and regulate TSC2, further enhancing mTOR activity. B: After CDK4/6 inhibitor treatment (changes in red), Rb phosphorylation is potently suppressed inducing a partial G1 arrest. CDK4/6 inhibition also leads to a reduction in TSC2 phosphorylation, resulting in partial suppression of mTOR and thus S6K activity. The suppression of S6K activity relieves feedback inhibition on EGFR family kinases, evidenced by increased EGFR family and AKT phosphorylation. C: When CDK4/6 inhibitor treated cells are co-treated with anti-HER2 therapy, synergy is observed. First, the increased EGFR/HER2 activity “primes” cells to the effects of anti-HER2 therapy (in purple). Second, when both CDK4/6 and HER2 are inhibited, there is maximal suppression of TSC2 phosphorylation. Together these induce a more complete shutdown of S6RP phosphorylation. Thus combined therapy inhibits both Rb and S6RP phosphorylation, further reducing cellular proliferation.