And Long-Term Effects of CDK4/6 Inhibition on Early-Stage Breast Cancer William B

Published OnlineFirst August 26, 2019; DOI: 10.1158/1535-7163.MCT-19-0231

Small Molecule Therapeutics Molecular Cancer Therapeutics Short- and Long-Term Effects of CDK4/6 Inhibition on Early-Stage Breast Cancer William B. Kietzman1, Garrett T. Graham1, Virginie Ory1, Ghada M. Sharif1, Max H. Kushner1, Gregory T. Gallanis1, Bhaskar Kallakury2,3, Anton Wellstein1,3, and Anna T. Riegel1,3

Abstract

CDK4/6 inhibitors are used in the treatment of advanced tumor and stromal cell compartments showed that cell cycle estrogen receptor (ER)(þ) breast cancer. Their efﬁcacy in ER and senescence genes, and MUC16,anovariancancerbio- () and early-stage breast cancer is currently under inves- marker gene, were repressed during treatment. Knockdown tigation. Here, we show that palbociclib, a CDK4/6 inhib- of MUC16 in MCFDCIS cells inhibited proliferation of itor, can inhibit both progression of ductal carcinoma in situ invasive lesions but not progression of DCIS. After cessation (DCIS) and growth of invasive disease in both an ER() of palbociclib treatment genes associated with differentia- basal breast cancer model (MCFDCIS) and an ER(þ)lumi- tion, for example, P63,inﬂammation, IFNg response, and nalmodel(MCF7intraductalinjection).InMCFDCIScells, antigen processing and presentation remained suppressed palbociclib repressed cell-cycle gene expression, inhibited in the tumor and surrounding stroma. We conclude that proliferation, induced senescence, and normalized tumor- palbociclib can prevent progression of DCIS and is anti- spheres formed in Matrigel while the formation of acini by proliferative in ER() invasive disease mediated in part via normal mammary epithelial cells (MCF10A) was not affect- MUC16. Lasting effects of CDK4/6 inhibition after drug ed. Palbociclib treatment of mice with MCFDCIS tumors withdrawal on differentiation and the immune response inhibited their malignant progression and reduced prolif- couldimpacttheapproachtotreatmentofearly-stageER() eration of invasive lesions. Transcriptomic analysis of the breast cancer.

Introduction compared with hormone therapy alone in cases of advanced ER(þ)/HER2() breast cancer (8–10). Although approved use Entry into the cell cycle is mediated by a restriction point iscurrentlylimitedtoadvancedER(þ) breast cancer, these controlled by the CDK4/6-RB axis, which protects against aber- drugs have the potential for widespread application due to the rant proliferation (1–3). Cancer cells frequently subvert this central nature of cell-cycle escape in carcinogenesis (11). Clin- restriction point using a variety of signaling cascades that converge ical trials in a variety of other cancer types are underway such as on enhanced CDK4/6 activity and inhibition of RB to facilitate squamous cell lung cancer (NCT02785939), pancreatic neuro- cell-cycle entry (4–6). Consequently, the ability to take therapeu- endocrine tumors (NCT02806648), oligodendroglioma and tic control of the restriction point to block proliferation has been oligoastrocytoma (NCT02530320), along with others. long sought after. Though efficacious in ER(þ) breast cancer, the use of palbo- Palbociclib is a second-generation CDK inhibitor, and unlike ciclib in earlier stages of breast cancer and in ER() disease is previous inhibitors in this class, palbociclib demonstrates high currently under investigation. One lesion type that is frequently affinity to CDK4/6 with little influence on other CDK family diagnosed alongside invasive disease is ductal carcinoma in situ members and low toxicity (7). CDK4/6 inhibitors, when used (DCIS). DCIS is considered an in-obligate precursor to invasive in conjunction with standard-of-care hormone therapy, pro- ductal carcinoma (IDC) in that it has the potential to become vide substantial improvements in progression-free survival aggressive and potentially life-threatening but will not progress to IDC with 100% penetrance (12–16). Here we examine the effects of inhibition of CDK4/6 on DCIS and early-stage IDC. A challenge 1 Department of Oncology, Georgetown University, Washington, District of in these type of studies is that there are few models of early-stage Columbia. 2Department of Pathology, Georgetown University, Washington, District of Columbia. 3The Lombardi Comprehensive Cancer Center, George- breast cancer progression (17, 18). The best model of DCIS town University, Washington, District of Columbia. progression was derived from the MCF10A series of basal-like triple negative [ER(), PR(), HER2 non-amplified] cell lines Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). known as MCFDCIS (19, 20). These cells exhibit bi-polar pro- genitor properties and are able to give rise to luminal and Corresponding Author: Anna T. Riegel, Georgetown University, TRB E307, 3970 myoepithelial cell populations in xenografts, forming mammary Reservoir Road, Washington, DC 20057. Phone: 202-687-1479; Fax: 202-687- 4821; E-mail: [email protected] acinar structures that will progress through DCIS to IDC with a predictable time course (21, 22). Mol Cancer Ther 2019;18:2220–32 Here we demonstrate the palbociclib is able to inhibit prolif- doi: 10.1158/1535-7163.MCT-19-0231 eration and induce senescence in both normal MCF10A and 2019 American Association for Cancer Research. MCFDCIS cells mediated through the downregulation of cell-

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cycle driving genes and senescence genes. In 3D culture systems, qRT-PCR palbociclib was able to normalize the architecture of MCFDCIS Total RNA was extracted from cells using the RNeasy Kit spheres but interestingly has no effect on normal MCF10A mam- (Qiagen). RNA was extracted from tissues using the RNeasy Kit mary acini. In vivo treatment of MCFDCIS tumors inhibited DCIS as well but following a homogenization step using the MagNA progression and reduced proliferation of invasive lesions. Palbo- Lyser. qRT-PCR was performed on extracted RNA as described ciclib was also able to delay the invasive transition of a luminal previously (27). Primers were purchased from Integrated DNA model of DCIS, the mouse mammary intraductal (MIND) model, technologies, sequences can be found in Supplementary Table S2. in which MCF7 cells are injected intraductally. The inhibition of proliferation of invasive lesions was in part mediated by MUC16, Senescence assays a protein that also serves as an ovarian cancer biomarker. Drug To assess the senescence-related phenotype of increased cell withdrawal experiments demonstrate that palbociclib is predom- size, cells were treated for 72 hours with different concentrations inantly cytostatic in MCFDCIS, although lasting effects of CDK4/6 of palbociclib prior to collection and measurement of cell diam- inhibition on differentiation and the immune response after drug eter utilizing a Multisizer3 Coulter Counter. After 72 hours of withdrawal is of potential interest for rational design of thera- treatment with either vehicle or a dose of palbociclib, cells were peutic strategies targeting early-stage ER() breast cancer. stained for the senescence marker b-galactosidase (Cell Signaling Technologies: 9860) following manufacturer's instructions. Stain was allowed to develop for 48 hours. Images were acquired using Materials and Methods the Olympus IX-71 microscope as described above, and the Cell culture number of positive cells per field was quantified using the cell MCF10A and MCFDCIS cell lines were obtained from Dr. counting feature in ImageJ. Susette Mueller (Georgetown University) and were cultured as described previously (22, 23). HEK293 cells were obtained from 3D culture Dr. Rabindra Roy (Georgetown University) and were cultured in Matrigel sphere formation assay was conducted as described DMEM (GIBCO/Invitrogen) with 10% FBS. All cell lines were previously (23, 28) with a modification to the protocol to add 100 fingerprinted to confirm identity and mycoplasma tested regu- nmol/L palbociclib or sterile water control to the assay media in larly (most recent test 6/2018). Cells were used between passages which the tumor spheres were seeded. Spheres were photo- 4 and 15. The FOXM1-12D phospho-mimic plasmid was graphed at days 7 and 10 for MCF10A and MCFDCIS respectively, obtained from Dr. Peter Sicinski (Dana Farber; ref. 24) and was using an Olympus IX-71 Inverted epifluorescence microscope. used to transfect HEK293 cells alongside an empty-vector control Sphere sizes were quantified using Fiji software. Sphere isolation using the Fugene 6 reagent as recommended by the manufacturer and immunofluorescent staining were also performed as (Promega). Forty-eight hours after transfection, G418 selection described previously (23) using Laminin 5 (details in Supple- agent was added to cells at 800 mg/mL and maintained for 14 days mentary File S1; Table S1) and mounted with ProLong Gold generate stable HEK-12D and HEK-EV cell lines. shMUC16 Antifade Mountant with DAPI (Thermo Fisher Scientific). Images MCFDCIS lines were established using unique shRNA and non- were acquired using the Leica SP8 Confocal microscope and silencing control sequences inserted into the pGIPz (Dharmacon) fluorescent intensity was quantified using Fiji software. To isolate as described previously (25). RNA from spheres, Matrigel was dissolved by adding dispase 5 U/mL (Stem Cell Technologies) to chamber slides and incu- In vitro drug sensitivity assays bating at 37C for 45 minutes. Solubilized tumorspheres were Effects on cell phenotype were assessed after cells were treated collected, spun, and washed with sterile PBS prior to RNA extrac- for 72 hours with various concentrations of palbociclib (PALBO- tion using the Qiagen RNeasy Kit (Qiagen). CICLIB-0332991; Taizhou Crene Biotechnology—NMR was performed by Dr. Milton Brown at Georgetown University to confirm Animal models compound structure prior to use). Treatment was initiated Subcutaneous models: Six-week-old female athymic nude mice 24 hours after cells were seeded. Changes in cell morphology (average weight, 24 g) were purchased from Envigo. MIND were captured with an Olympus IX-71 inverted epifluorescence Model: Six-week-old female NOD-SCID mice (average weight, microscope. Cell proliferation was assessed via crystal violet as 25 g) were purchased from Jackson Labs. Animals were housed in described previously (25). Total protein and RNA were extracted a pathogen-free environment with controlled temperature and from cells for analysis via Western blot analysis and qRT-PCR. humidity. All animal experiments were conducted in accordance Cell-cycle analysis was done on one million cells per condition with Protocol Number ¼ 20161138. using the Vindelov method (26). Apoptosis was assessed in an aliquot of the cell-cycle cell sample suspension via an Annexin V Xenograft experiments staining and flow cytometry. For subcutaneous xenograft experiments, MCFDCIS cells were injected as described previously (22). Animals received sterile Western blot analysis water or 50 mg/kg palbociclib diluted in a water-based vehicle Cells were cold lysed in an NP40-based lysis buffer whereas containing 0.5% methylcellulose and 0.2% Tween 80 for 1 or frozen tumor samples were homogenized in lysis buffer using the 2 weeks total depending on cohort, as outlined in Figs. 3A and 5A. Roche MagNA Lyser instrument (Roche). Following protein Upon tumor collection, samples were divided for hematoxylin extraction, cellular and tumor lysates were prepared and separated and eosin (H&E) and IHC, qRT-PCR (see Materials and Methods) on an SDS-PAGE gel prior transfer and immunoblotting as and Western blot analysis (see Materials and Methods). Blood was described previously (25). Information on primary and secondary collected to assess the impact of treatment on white blood cell antibodies used can be found in Supplementary Table S1. counts using a Giemsa Stain Solution (Sigma-Aldrich) according

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to manufacturer's instructions. shMUC16 MCFDCIS cells were DCIS model cell line (20). Palbociclib, and a related CDK4/6 injected as previously described and tracked as outlined in inhibitor abemaciclib, inhibited cellular proliferation in a con- Supplementary Fig. S5A. Serum was taken from animals for centration-dependent manner with similar IC50 in MCF10A and circulating MUC16 expression using an ELISA for detection of MCFDCIS (Supplementary Fig. S1A and S1B), accompanied by the human protein following manufacturer's instructions (R&D diminished phosphorylation of the canonical CDK4/6 target RB Systems). For the mammary intraductal method (MIND mod- (Supplementary Fig. S1C) and reduced expression of known cell- el), MCF7-luc2-DSred cells (kindly donated by Dr. Cathryn cycle target genes (Fig. 1A; refs. 20, 31, 32). Palbociclib-treated Briskin—Swiss Federal Institute of Technology in Lausanne) cells accumulated in G1 with a reduction in both G2- and S-phase werepreparedandinjectedinto6-week-oldfemaleNOD-SCID (Supplementary Fig. S1D). mice purchased from Jackson Labs as described previously (29), CDK4/6 inhibition can also induce cellular senescence (Sup- outlined in Supplementary Fig. S4A. Mammary glands were plementary Fig. S1E), which likely contributes to the observed collected and H&E stained. increased cell size in dose–response assays and the measured concentration-dependent increases in median cell diameter (Sup- Histologic analysis plementary Fig. S1F). We also observed a dose-dependent accu- H&E and IHC analyses were performed on paraffin-embedded mulation of b-galactosidase positivity in both MCF10A and 5-mm sections using standard protocols described elsewhere (30). MCFDCIS cells with >20% stained for the senescence marker at Information on primary antibodies (Phospho-RB, RB, KI67, P63, IC50 concentrations of palbociclib (Fig. 1B; Supplementary MUC16 and cleaved-CASPASE-3) for IHC can be found in Sup- Fig. S1G) and significantly reduced expression of an early senes- plementary Table S1. Images were captured using an Olympus cence response gene, nuclear envelope factor Lamin B1 (LMNB1; BX40 microscope and quantification of histologic areas and ref. 33) in MCFDCIS cells (Fig. 1C). Notably, palbociclib was able staining positivity were performed using ImageJ. to significantly reduce phosphorylation and expression of FOXM1, a CDK4/6 target involved in the senescence response cDNA array and RNA-seq analysis (Supplementary Fig. S1H; Fig. 1D; ref. 24). These data indicate Total tumor RNA was extracted in triplicate, as described above. that palbociclib inhibits proliferation by regulating cell-cycle RNA with an integrity number greater than 8.7 were sent to the machinery and also induces senescence in both normal mammary UCLA Neuroscience Genomics Core (UNGC). Microarray anal- cells and early-stage breast cancer. ysis which was carried out by the UNGC using Illumina To determine the durability of palbociclib response, we HumanHT-12 v4 Expression BeadChip representing 47,000 designed reversal experiments in which MCFDCIS cells were well-annotated genes according to the manufacturer's instruc- treated with palbociclib for 72 hours followed by a recovery tions on RNA isolated from the xenograft described in Fig. 3A period of an additional 72 hours. In reversal experiments, cell (Illumina, Inc.). Data were analyzed in R using the Bioconductor proliferation rate did not recover to vehicle baseline following software. Greater than 2-fold difference in expression and a P- discontinuation of treatment despite the recovery of cell-cycle value <0.05 were set as the threshold for significance. Differential genes to pretreatment expression levels, with the exception of gene expression lists were generated by comparing palbociclib- MCM2 which remained significantly downregulated (Fig. 1E and treated tumor expression to vehicle-treated tumor expression and F). Consistent with this, we found that G1 arrest is transient and ranking genes by log2-fold change. RNA from the xenograft dependent on the continued presence of drug (Fig. 1G). However, described in Fig. 5A was used for RNA library construction and recovered cells experienced 2.5-fold higher levels of apoptosis sequencing (RNA-seq). Mouse genome GRCm38.p6 and human than control cells, which could explain the lag in proliferative rate genome GRCh38.p10 were concatenated into a meta-genome. A recovery (Fig. 1E and H). In terms of senescence during recovery, STAR index was generated using GTF annotation from GENCODE b-galactosidase staining returned to baseline, as did expression of for mouse and human genome features (version v18 and v27, LMNB1, although there was a significant induction of FOXM1 respectively). Samples were aligned using single sample two-pass following recovery (Fig. 1I and J). These suggested that palbociclib alignment, limiting multimapping reads to not more than one might influence both the proliferative and senescent responses unique location. Unique alignment rates were 80% to 85%, with through effects on FOXM1. To investigate this, we overexpressed, 10% to 15% reads mapping to more than once location. Only in HEK293 cells, a constitutively active FOXM1 "phospho-mimic" unique alignments were used to quantify expression at mouse and in which 12 CDK4/6 consensus sites have been altered to aspartic human genome features. Feature counts were used in downstream acid residues (Supplementary Fig. S1I; ref. 24). Expression of the comparisons. Data available on GEO: GSE130903. FOXM1-12D did not alter the palbociclib IC50 for proliferation (Supplementary Fig. S1J) but did significantly inhibit palbociclib- Statistical analysis induced senescence as measured by b-galactosidase staining RStudio and Prism 7 (Graphpad Inc.) were used for statistical (Fig. 1K). FOXM1-12D also attenuated the repression of several analysis and graphing. ANOVA was used for multiple compar- cell-cycle genes including CCNB1 and KIF20A (Supplementary isons and t tests or chi-square tests were used for paired compar- Fig. S1K). These suggest that FOXM1 may play an important role isons, with P < 0.05 as the threshold for statistical significance in in the senescence response to CDK4/6 inhibition in the context of all tests. early breast cancer.

Effects of palbociclib on MCFDCIS cells in 3D culture Results The effects of palbociclib on normal mammary cells and early Effects of palbociclib on normal breast and DCIS cells in vitro breast cancer could be modiﬁed by their stromal environment. To We compared the effects of palbociclib on MCF10A, an immor- investigate this, we examined palbociclib effects on both MCF10A talized normal breast epithelial cell line, and MCFDCIS cells, a and MCFDCIS cells grown on top of Matrigel and found that

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Figure 1. Palbociclib (PD) treatment in 2D. PD elicits canonical antiproliferation and senescence responses in both MCF10A and MCFDCIS that are reversible upon discontinuation of treatment. A, qPCR analysis of cell-cycle–regulated mRNA targets in MCF10A (left) and MCFDCIS (right) cells treated with PD as indicated. Mean fold change SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle). B, Quantification of b-galactosidase–stained MCF10A (left) and MCFDCIS (right) cells. Mean SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). C, qPCR analysis of Lamin B1 (LMNB1) expression in MCF10A (left) and MCFDCIS (right) treated as indicated. Mean fold change SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). D, qPCR analysis of FOXM1 expression in MCF10A (left) and MCFDCIS (right) treated as indicated. Mean fold change SEM (one-way ANOVA followed by Dunnett multiple comparisons test, relative to vehicle, 72-hour treatment). E, Crystal violet proliferation curves for MCFDCIS cells in vitro treated for 6 days with vehicle (constant vehicle), 6 days with 0.1 mmol/L palbociclib (constant palbociclib), or 3 days with 0.1 mmol/L palbociclib followed by 3 days with vehicle (reversal). F, qPCR analysis of cell-cycle–regulated mRNA targets in MCFDCIS cells treated with PD as indicated. Mean fold change SEM (Student T test, relative to vehicle). G, Cell-cycle analysis of MCFDCIS cells treated with PD as indicated. H, ANNEXIN-5 apoptosis assay on MCFDCIS cells treated as indicated; all apoptotic death is represented as a fold change from the normalized vehicle- treated baseline. I, Quantification of b-galactosidase–stained MCFDCIS cells in vitro treated with PD as indicated. Mean SEM (Student T test, relative to vehicle). J, qPCR analysis of LMNB1 (left) and FOXM1 (right) expression in MCFDCIS treated as indicated. Mean fold change SEM (Student T test, relative to vehicle). K, Quantification of PD senescence challenge in HEK293 cells transfected with constitutively active FOXM1-12D or empty vector. HEK293-EV and HEK293-12D were treated with indicated concentrations of PD for 72 hours prior to staining for b-galactosidase activity. Mean SEM (Student t test, relative to vehicle). For all: P < 0.05; P < 0.01; P < 0.001; P < 0.0001. n.s., not significant.

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MCF10A cells form normal mammospheres whereas MCFDCIS membrane marker (23), is deposited at the periphery of MCF10A cells form abnormal tumorspheres (22, 23, 28). The MCF10A mammospheres and remains unaltered after palbociclib treat- mammosphere phenotype is largely unchanged over 7 to 10 days ment (Fig. 2D and E). In contrast, LAMININ 5 shows a disorga- of treatment in the presence of palbociclib, whereas MCFDCIS nized expression across MCFDCIS tumorspheres, but reverted to a tumorspheres are signiﬁcantly smaller with a more normalized normal mammosphere pattern after palbociclib treatment phenotype when compared with vehicle controls (Supplementary (Fig. 2D and E). Together these results indicate potential selec- Fig. S2; Fig. 2A and B). MCFDCIS but not MCF10A spheres tivity for highly proliferative transformed cells in the 3D context as showed signiﬁcant reductions in the cell-cycle gene genes and differentiated MCF10A mammospheres were unaffected by the senescence marker, LMNB1 (Fig. 2C). LAMININ 5, a basement palbociclib.

Figure 2. Palbociclib (PD) treatment in 3D. PD selectively influences MCFDCIS spheres in 3D culture, normalizing their architecture. A, MCF10A (top) and MCFDCIS (bottom) spheres grown in 3D in the presence of vehicle or 0.1 mmol/L PD. 20 magnification, scale, 200 mm. B, Quantification of A; sphere areas calculated using Fiji software. Mean SEM (Student t test, P < 0.0001 relative to vehicle). C, qPCR analysis of cell-cycle/senescence-regulated mRNA targets in MCF10A (top) and MCFDCIS (bottom) spheres treated with PD as indicated. Mean fold change SEM (Student t test, P < 0.01, P < 0.001, P < 0.0001 relative to vehicle). D, Vehicle-treated MCF10A (top) and MCFDCIS (bottom) spheres stained for LAMININ 5 (red) and DAPI (blue) along with quantification of the distribution of LAMININ 5 signal intensity measured along the yellow line indicated in the photos. 63 magnification, scale, 25 mm. E, PD-treated MCF10A (top) and MCFDCIS (bottom) spheres stained for LAMININ 5 (red) and DAPI (blue) along with quantification of the distribution of LAMININ 5 signal intensity measured along the yellow line indicated. 63 magnification, scale, 25 mm.

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Palbociclib treatment reduces MCFDCIS xenograft growth and burden and bore fewer invasive lesions than their vehicle-treated malignant progression counterparts (Supplementary Fig. S4C–S4E). Palbociclib is there- The selective "normalization" effects of palbociclib on fore effective in postponing the invasive transition not only in MCFDCIS cells in Matrigel suggested that this CDK4/6 inhibitor basal-like DCIS but also in hormone-dependent luminal DCIS. could have beneficial effects on DCIS lesions in vivo. Therefore, we tested palbociclib efficacy over 2 weeks in vivo on xenograft tumors Role of MUC16 in the palbociclib response in athymic nude mice (Fig. 3A). We used a low dose of 50 mg/kg To assess global gene expression changes in DCIS following relative to previous preclinical studies (34–39) to minimize 2 weeks of palbociclib treatment, mRNA isolated from MCFDCIS potential toxicity such as neutropenia, which is a common tumors was analyzed using Illumina bead chip array. Overall, adverse event associated with palbociclib (8). We saw a small, 1,515 genes were found to be significantly (P < 0.05) up- or nonsignificant reduction in white blood cell counts following downregulated by at least 1.5-fold at 2 weeks on treatment with 2 weeks of treatment relative to control (Supplementary Fig. S3A) the majority of highly regulated genes playing a role in the cell and observed no difference in animal weight between groups over cycle (Fig. 4A and B). MUC16 (CA-125), a cancer antigen that has the course of our study indicating that this dose of palbociclib was been used as a biomarker and therapy response marker in ovarian well tolerated (Supplementary Fig. S3B). Tumors showed a sig- cancer (40–43), stood out in this analysis as it is not a cell-cycle nificantly reduced growth rate after palbociclib leading to stasis in regulatory gene. We verified by PCR that significant reductions in some cases but not to regression (Fig. 3B). MUC16 mRNA occurred after 1 and 2 weeks of treatment and this The MCFDCIS model is unique in that xenograft tumors was validated at the protein level by IHC (Fig. 4C and D). MUC16 transition from in situ carcinoma to invasive cancer between 4 expression appears to increase with MCFDCIS tumor progression and 6 weeks after injection (21). Thus, we were able to monitor as 2-week vehicle-treated tumor sections stained more intensely the impact of palbociclib on malignant progression of MCFDCIS than 1-week treated (Fig. 4D). To evaluate a functional role of lesions and found a significant delay of progression in palboci- MUC16 in MCFDCIS tumor growth and progression, we gener- clib-treated tumors. At 4 weeks, the tumor area of about half of the ated 3 independent shMUC16 MCFDCIS cell lines along with a control group showed invasive lesions, whereas about 90% in the nonsilencing control. Subcutaneous tumor growth of the palbociclib-treated group remained noninvasive DCIS (Fig. 3C, shMUC16 cell lines (Supplementary Fig. S5A) was reduced rel- first panel). Palbociclib treatment resulted in significantly dimin- ative to nonsilenced control tumors with both shMUC16_2 and ished expression of phosphorylated RB without significantly shMUC16_3 tumors demonstrating significantly reduced growth impacting total RB staining by IHC, although Western blot (Fig. 4E). Despite some differences in growth rate, all shMUC16 analysis demonstrates a reduction in treated tumor lysates MCFDCIS tumors had significantly less MUC16 expression than (Fig. 3C; Supplementary Fig. S3C). Palbociclib treatment also nonsilenced controls (Fig. 4F and G). Tumor-derived circulating significantly reduced KI67 proliferative index from >25% of MUC16 in serum was easily detectable in the controls but below nuclei stained to <10% within the tumor (Fig. 3C). P63 expres- detection threshold in the majority of shMUC16 tumors, with sion, which marks the invasive transition in the MCFDCIS mod- blood-borne expression correlating tightly with tumor growth el (21), was also significantly reduced (Fig. 3C), corroborating the rate across independent shMUC16 lines (Fig. 4H). Although above conclusion from the surgical pathology analysis. Despite shMUC16 tumors grew more slowly than their control counter- the smaller size of tumors and reduced proliferation, CASPASE-3 parts, loss of MUC16 did not delay the transition from DCIS to cleavage was unaltered within the tumors, which suggests that invasive lesions and tumor histology was indistinguishable palbociclib does not induce apoptosis in this context (Supple- between the groups (Fig. 4I). These suggest that MUC16 does mentary Fig. S3D). As expected, palbociclib-responsive cell-cycle not drive MCFDCIS to invasive transition but does contribute to genes also showed significant mRNA downregulation in tumors overall tumor growth. Of note is that shMUC16 tumors demon- (Fig. 3D) and FOXM1 expression was reduced at both the protein strate more widespread staining for cleaved CASPASE-3, suggest- and mRNA levels, mirroring in vitro assays (Fig. 3E and F). These ing that the reduction in tumor size relative to control tumors is observation, along with significant loss of LMNB1, suggest that due to an increased rate of cell death (Supplementary Fig. S5B). the DCIS tumors are undergoing both proliferative arrest and senescence induction following 2 weeks of palbociclib treatment Residual effects after cessation of treatment with palbociclib (Fig. 3E and F). Palbociclib's effects in vitro are largely but not entirely reversible as shown above (see Fig. 1E and F). Thus, we sought to determine Effect of palbociclib in the MIND model of DCIS if similar residual effects of CDK4/6 inhibition could be detected To determine if palbociclib effects on DCIS tumors were in vivo in either the tumor cells or stroma after discontinuation of dependent on the tumor microenvironment we utilized the therapy. To study this, we allowed mice an 11-day drug-recovery MIND model, in which tumor cells are directly injected into period following 2 weeks of palbociclib treatment (Fig. 5A). Upon the mammary duct. In this model, early-stage DCIS-like breast treatment termination, palbociclib-treated tumors resumed cancer lesions develop after injection of MCF7 luminal ER(þ) growth but final tumor sizes in the palbociclib recovery group breast cancer cells (Supplementary Fig. S4A; ref. 29). Following remained significantly smaller than vehicle-treated tumors injection of MCF7-luc2-DSred cells (kindly donated by Dr Cath- (Fig. 5B). Growth rates of tumors during the 11 days of recovery ryn Briskin—Swiss Federal Institute of Technology in Lausanne) paralleled those of the vehicle-treated tumors (Fig. 5B); notably, into mammary ducts, successful injection was verified by IVIS in tumors recovering from palbociclib treatment, almost 30% of imaging (Supplementary Fig. S4B). DCIS lesions were established tumor area remained DCIS whereas vehicle-treated tumors pro- over 2 months prior to treatment with palbociclib for 28 days by gressed to invasive disease with only 8.5% of tumor area remain- oral gavage. IVIS imaging and histology of the mammary duct ing DCIS (Fig. 5C). We observed partial re-expression of phos- lesions revealed that palbociclib-treated mice had reduced tumor phorylated RB, KI67, and P63 upon palbociclib withdrawal

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Figure 3. Palbociclib (PD) treatment in vivo. PD delays growth and progression of MCFDCIS xenograft tumors in nude mice. A, Schematic of experimental design and time points. B, Tumor growth curves for vehicle- or PD-treated MCFDCIS tumors following subcutaneous injection of cells, represented as the change (delta) in tumor size normalized to initial tumor size (2-way ANOVA and Bonferroni posttests, P < 0.05, P < 0.01, P < 0.001, P < 0.0001 relative to vehicle). C, Cohort 2 tumor sections stained with hematoxylin and eosin as well as IHC for phosphorylated RB (S807/811), total RB, KI67, and P63, treated with PD as indicated and associated quantiﬁcations (chi-square tests, P < 0.0001, 20 magnication, scale, 100 mm). D, qPCR analysis of cell-cycle regulated mRNA targets in MCFDCIS tumors treated with PD as indicated. Mean fold change SEM (Student t test, P < 0.01, P < 0.001, P < 0.0001 relative to vehicle). E, Western blot analysis for expression of FOXM1 and actin in tumor lysates at both 1 and 2 weeks of treatment with PD as indicated. F, qPCR analysis of senescence-regulated mRNA targets in MCFDCIS tumors treated with PD as indicated. Mean fold change SEM (Student t test, P < 0.0001 relative to vehicle). n.s., not signiﬁcant.

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Figure 4. MUC16's role in palbociclib (PD) response. MUC16 can serve as a biomarker of palbociclib treatment in MCFDCIS tumors and may play a functional role in tumor growth. A, Cigar plot of Log2 expression of cDNA array data generated from RNA isolated from vehicle- and palbociclib-treated tumors of cohort 2. Points highlighted in red are upregulated by palbociclib, whereas those in blue are downregulated by drug. B, Top 15 most PD-regulated genes from the cDNA array at 2 weeks of treatment, ranked by Log2-fold change and significance of P < 0.05. Upregulated genes are depicted in red, and downregulated genes in blue. C, qPCR analysis of MUC16 expression in MCFDCIS tumors treated as indicated. Mean fold change SEM (Student t test, P < 0.001, P < 0.0001 relative to vehicle). D, Representative cohorts 1 and 2 tumor sections stained by IHC for MUC16, treated with PD as indicated. 10 magnification, scale, 200 mm. E, Tumor growth curves for nonsilenced (NS) or shMUC16 MCFDCIS tumors following subcutaneous injection of cells, represented as the delta in tumor size normalized to initial tumor size (two-way ANOVA and Bonferroni posttests, P < 0.05, P < 0.01 relative to vehicle). F, Representative NS and shMUC16 MCFDCIS tumor sections stained by IHC for MUC16. 10 magnification, scale, 200 mm. G, Quantification of MUC16 IHC staining as a percent of tumor area. Mean SEM (1-way ANOVA followed by Dunnett multiple comparisons test, P < 0.001, relative to vehicle). H, ELISA for circulating MCFDCIS tumor-derived MUC16 in serum taken at day 28 from mice bearing NS or shMUC16 MCFDCIS tumors. I, Representative NS and shMUC16 MCFDCIS tumor sections stained using the PAS method. 10 magnification, scale, 200 mm. n.s., no significant.

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Figure 5. Palbociclib (PD) treatment in vivo is reversible. Most, but not all of, PD's effects on tumor growth and progression are reversible upon discontinuation of drug treatment. A, Schematic of experimental design, time points, and comparisons made using RNA isolated from tumors. B, Tumor growth curves for vehicle and PD-treated MCFDCIS tumors, represented as the change (delta) in tumor size normalized to initial day of treatment (two-way ANOVA and Bonferroni posttests, P < 0.05, P < 0.01, P < 0.001, P < 0.0001 relative to vehicle). C, Cohort 4 tumor sections stained with hematoxylin and eosin as well as IHC for phosphorylated RB (S807/811), total RB, KI67, and P63, treated with PD as indicated and associated quantifications (chi-squared tests, P < 0.05, P < 0.001, P < 0.0001, 20 magnification, scale, 100 mm). D, Fold change values of the cell-cycle gene panel in palbociclib-treated (cohort 3) and -recovered (cohort 4) tumors relative to a vehicle baseline, represented by the dotted line. E, Fold change values of genes significantly upregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. F, Fold change values of genes significantly downregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. D–F are based on values derived from RNA-seq. All values were filtered for minimum expression (CPM > 2) and significance (P < 0.05). G, ELISA for circulating MCFDCIS tumor-derived MUC16 in serum taken from cohort 4 mice. n.s., not significant.

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(Fig. 5C). P63 expression remained lower in palbociclib-treated both in the human tumor and the surrounding stroma (Fig. 6F). and recovered tumors than in vehicle-treated tumors (Figs. 5C Although both the tumor and stroma experience a downregula- and 3C), suggesting that palbociclib has some lasting effects on tion of cell-cycle driver pathways including E2F Targets, G2M tumor differentiation status and progression to invasive lesions. Checkpoint, and the Mitotic Spindle, the stroma is less impacted Interestingly, the panel of cell-cycle genes completely returned to and better able to recover following treatment cessation, as baseline as did the senescence markers LMNB1 and FOXM1 indicated by upregulation of these pathways (Fig. 6F). However, (Fig. 5D). unlike the cell-cycle pathways, a more dramatic regulation of To better understand lasting versus reversible effects of CDK4/6 hallmark inflammatory and IFNg signaling pathways is seen in inhibition, we performed RNA-seq on RNA isolated from treated the stroma compared with the tumor cell compartment. These and recovered MCFDCIS tumors. We confirmed that the majority pathways remain significantly downregulated in the tumor micro- of regulated genes are involved in cell-cycle regulation and that environment even after cessation of palbociclib treatment, reflect- nearly all of them recover to a baseline level of expression ing a lasting gene signature in the mouse stroma (Fig. 6E and F). following treatment cessation (Supplementary Fig. S6A). Inter- estingly, several genes uninvolved with the cell cycle broke this pattern. Some genes were upregulated >1.5-fold by palbociclib Discussion and remained upregulated following drug withdrawal such as the In this study we show that CDK4/6 inhibition delays malignant differentiation factor NELL2, which experienced even further progression to invasive disease in both basal MCFDCIS subcuta- upregulation in recovery (Fig. 5E). High expression of NELL2 is neous xenograft and luminal MCF7 intraductal injection associated with improved recurrence-free survival in basal breast models of DCIS. Even after cessation of palbociclib treatment, cancers (Supplementary Fig. S6B). Other factors initially upregu- the residual effects of the drug result in less aggressive tumors and lated by palbociclib were significantly downregulated following reduced overall recurrence. In vitro, palbociclib caused canonical palbociclib withdrawal (Fig. 5E) and some genes that experienced cell-cycle arrest in normal and transformed breast cells and in significant downregulation during palbociclib treatment did not parallel also displayed classic features of senescence with no recover to baseline following discontinuation of treatment, discernible increase in apoptosis. In 3D assays, which are more including several RNA processing factors, matrix metalloprotei- faithful predictors of cell behavior and drug responsiveness nases, and inflammatory factors (Fig. 5F). In the RNA-seq anal- in vivo (44), there was differential sensitivity of MCF10A and ysis, we also saw that MUC16 was significantly downregulated by MCFDCIS spheres to palbociclib. This is likely explained by the treatment; its mRNA levels increased somewhat following drug fact that MCF10A cells grown as spheres undergo a well-described withdrawal but did not return to baseline. Consistent with this, process of differentiation, quickly enter a maintenance phase MUC16 protein in the circulation remained significantly reduced marked by long-term quiescence (23), and are relatively insen- even after treatment cessation (Fig. 5G). sitive to the effects of CDK4/6 inhibition. Conversely, MCFDCIS spheres do not experience differentiation or quiescence as Palbociclib has residual effects on stromal immune and IFN- they grow in 3D; palbociclib is able to significantly reduce related genes expression of E2F target genes while simultaneously inducing To gain insight into the effects of palbociclib on the tumor host, senescence as evidenced by loss of LMNB1. Consistent with these we simultaneously aligned the RNA-seq reads from tumors to observations, palbociclib treatment had no discernable effects on both the human and mouse genomes (see Materials and Meth- normal, differentiated mammary gland architecture over the ods). Mouse stromal cells contributed 15% to 25% of the mRNA course of therapy in both subcutaneous xenografts and in MIND isolated from tumor samples that were harvested at different model experiments. Cell-cycle inhibition and G1 arrest following points during treatment (Fig. 6A and B). The cell cycle and palbociclib is mainly cytostatic as the majority of MCFDCIS senescence gene panel regulated in tumor cells was also impacted tumor cells were able to re-express cell-cycle driving genes and in the mouse stroma, although the downregulation of these genes reinitiate growth following treatment cessation, similar to other was smaller than seen in the tumor cell compartment. Following studies (34, 37, 45). recovery from treatment, several genes exceeded the expression To our knowledge, this is the first interrogation of early-stage observed at baseline (Fig. 6C). Using the same method described breast cancer cells in parallel with the immediate mouse stromal above to identify lasting influence of human tumor genes, we cell transcriptomes during palbociclib treatment and after recov- identified mouse stromal genes that experienced a lasting impact ery from treatment. The majority of the 97 genes that recover of palbociclib (Fig. 6D and E). Interestingly, the genes that expression after cessation of palbociclib treatment contribute to showed significant downregulation during palbociclib treatment hallmark pathways related to the cell cycle. The tight regulation of that persists after treatment cessation are almost entirely immune- cell-cycle genes on and after treatment highlights the specificity of related, specifically those related to interferon signaling with roles CDK4/6 inhibition in targeting proliferation. Many genes iden- in transcriptional regulation and antigen processing and presen- tified in our signature overlap with those previously identified as tation (Fig. 6E). Xenografts of human cells do impact stromal gene responding to CDK4/6 inhibition (35). This work suggests that expression and signaling in the host, but the repression of these there is a core set of targets that are influenced by palbociclib immunity-related genes was only noted in palbociclib-treated treatment across different contexts that may be useful indicators animals indicating it is a drug-specific effect. Quite strikingly, when assessing drug responsiveness in patients. The mouse stro- reduced expression of these genes is associated with a highly ma also experienced similar changes in canonical cell-cycle driver significant reduction in recurrence-free survival of patients with genes but to a lesser magnitude, accompanied by a more complete basal breast cancers (Supplementary Fig. S7A). expression recovery following treatment cessation. Stromal tissue Gene set enrichment analysis (GSEA) revealed that similar is not as proliferative as the tumor, which likely provides some hallmark pathways were significantly regulated by palbociclib resistance to palbociclib's primary effects.

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Figure 6. Palbociclib (PD)'s influence on the tumor microenvironment. PD affects gene expression in mouse stromal cells with a residual effect after treatment cessation on immune-related signaling pathways. A, Percent of RNA-seq reads for each tumor that aligned to the murine or human genome. Plotted as mean SEM with individual tumors plotted as separate symbols. B, Schematic of experimental design, time points, and comparisons made using mouse RNA isolated from tumors. C, Fold change values of the cell-cycle gene panel in palbociclib-treated (cohort 3) and palbociclib recovered–treated (cohort 4) tumor stroma relative to a vehicle baseline, represented by the dotted line. D, Fold change values of genes significantly upregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. E, Fold change values of genes significantly downregulated during treatment (palbo) and their expression following recovery (palbo-recovered) relative to a vehicle baseline indicated by the dotted line. C–E are based on values derived from RNA-seq. All values were filtered for minimum expression (CPM > 2) and significance (P < 0.05). F, Normalized enrichment scores for hallmark pathways identified in both the human tumor and mouse stromal RNA, and the dotted line indicates a significance cutoff based on P values < 0.05 and FDR Q values < 0.25.

The discovery that a non–cell-cycle regulatory gene, the cancer- human basal breast cancers, MUC16 expression could indicate specific antigen MUC16 (also known as CA-125), is one of the patient response to palbociclib therapy. It is of note that estab- most significantly downregulated genes by palbociclib is surpris- lishment of predictive biomarkers of response to CDK4/6 inhi- ing. MUC16 is well-known in the ovarian cancer field where it has bitors in advanced stages of disease has been challenging with been used clinically as a biomarker of disease in tracking the many parameters related to cell cycle such as Cyclin D1 or efficacy of therapy (40–42). Interestingly, it has been reported that p16INK4A expression levels demonstrating no correlation with ovarian cancer and basal breast cancer have significant genomic response (48). similarities, and MUC16 is frequently mutated in breast cancer Other genes of interest revealed in the transcriptomic analyses cases (46). In our model, MUC16 is not only a biomarker of of palbociclib-treated and recovered MCFDCIS tumors were those palbociclib responsiveness but also contributes to the growth of that did not return to baseline following cessation of treatment. It invasive lesions. MUC16 has been linked to cancer progression is notable that in both the human tumor cells and the surrounding and aggressive behaviors in both ovarian and breast cancers (43), mouse stroma the majority of these genes have no known role in although the mechanism MUC16 utilizes to increase proliferation cell-cycle regulation. In the tumor cells, both NELL2 and KLK11 of cancer cells is not clear. Nevertheless, it appears that CDK4/6- were upregulated by palbociclib that was further increased fol- mediated repression of MUC16 is partially responsible for the lowing termination of drug treatment, which may suggest that efficacy of palbociclib in our MCFDCIS model. MUC16 has been treatment selects for populations of tumor cells expressing high previously implicated in triple-negative breast cancer as a poten- levels of these genes. KLK11 is a serine protease and NELL2 is a tial driver of progression and may play a key role in early glycoprotein with several epidermal growth factor-like domains. disease (47). It will be interesting to determine if, in a subset of Although the former has no known role basal breast cancer

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survival, high expression of NELL2 is associated with significantly after treatment cessation may result in less aggressive tumors and prolonged recurrence-free survival in patients with basal breast reduced overall recurrence. Based on these findings, palbociclib cancers (Supplementary Fig. S6B). Therefore, treatment with might be best applied as a neoadjuvant therapy following diag- palbociclib could generate lasting benefits. Many genes that were nosis with DCIS and prior to surgical removal of lesions as a downregulated within the tumor cells by palbociclib did not means of preventing further disease progression and potential return to baseline levels after cessation of treatment are involved dissemination. An ongoing phase II clinical trial at Georgetown in processes such as extracellular reorganization, RNA processing, University titled "Preoperative Palbociclib in Patients with DCIS and inflammatory signaling. The contributions of these genes to of the Breast that are Candidates for Surgery (WI223281)" long-term CDK4/6 inhibitor response is worthy of further study. (NCT03535506) will investigate this potential. The results of this Genes with lasting downregulation in the mouse stroma were study and other future studies should address whether different more homogeneous and comprised almost entirely of factors doses, increased duration of treatment, or combination therapies involved in IFN-mediated immune signaling. CDK4/6 inhibition with palbociclib could contribute to a more lasting impact of and its relationship to immune regulation has been a topic of both CDK4/6 in the treatment of earlier stage breast cancer. interest and controversy, as groups have reported both pro- and anti-immune responses which may impact the overall outcome of Disclosure of Potential Conflicts of Interest treatment with this class of drugs (49–52). One proposed mech- No potential conflicts of interest were disclosed. anism of antitumor immune regulation driven by CDK4/6 inhibition is the induction of an IFN-driven viral mimicry response Authors' Contributions initiated by cell-intrinsic factors, mediated through repression of Conception and design: W.B. Kietzman, V. Ory, G. Sharif, A. Wellstein, the E2F target gene DMNT1 (50). However, in our study we did A.T. Riegel Development of methodology: W.B. Kietzman, V. Ory, G. Sharif, A. Wellstein, not observe significant DMNT1 repression in either the human A.T. Riegel tumor or the mouse stroma in response to palbociclib. Acquisition of data (provided animals, acquired and managed patients, Furthermore, several of the genes involved in antigen proces- provided facilities, etc.): W.B. Kietzman, B. Kallakury, A.T. Riegel sing and presentation such as Tap1, Ifit2, Oas2, Oasl2, and Stat1 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, that were previously reported to be upregulated in tumor cells by computational analysis): W.B. Kietzman, G.T. Graham, M.H. Kushner, CDK4/6 inhibition (50) actually showed lasting downregulation B. Kallakury, A. Wellstein, A.T. Riegel Writing, review, and/or revision of the manuscript: W.B. Kietzman, in the stromal analysis in our mouse model (Fig. 6E). These G.T. Graham, V. Ory, M.H. Kushner, G.T. Gallanis, B. Kallakury, differences could be due to the cell types examined, treatment A. Wellstein, A.T. Riegel duration or the breast cancer models used. Our xenograft experi- Administrative, technical, or material support (i.e., reporting or organizing ments were performed in athymic nude mice that lack functional data, constructing databases): W.B. Kietzman, G.T. Graham, G.T. Gallanis T-cell populations and it is difficult to assess the full impact of Study supervision: A.T. Riegel palbociclib on antitumor immunity in this model. The suppression of antigen-processing genes is associated with a significant Acknowledgments reduction in recurrence-free survival of patients with basal breast We thank Drs. Deb Berry, Elena Tassi, and Marcel Schmidt for their valuable discussions. We thank Maria Idalia Cruz for her technical assistance with in vivo cancer and indicates that palbociclib in some contexts may have experiments. We thank Dr. Paula Pohlmann, lead investigator of long-term and potentially detrimental immunosuppressive NCT03535506, for her collaboration and insight on this project. T32 Training effects (Supplementary Fig. S7). Future studies could address this Grant in Tumor Biology CA009686, F31 CA232664 (to M. Kushner), R01 CA question in early stage breast cancer models from syngeneic 231291 (to A. Wellstein), R01 CA 205632 (to A. Riegel), and P30CA051008 (PI: immune-competent animals. Weiner): usage of the following shared resources: microscopy and imaging, fl Three CDK4/6 inhibitors are now FDA approved for the treat- tissue culture, ow cytometry, histopathology, and animal models. ment of advanced stage breast cancer either alone or in combi- – The costs of publication of this article were defrayed in part by the payment of nation with aromatase inhibitor treatment (8 10). Our study page charges. This article must therefore be hereby marked advertisement in demonstrates that short-term treatment with palbociclib is able to accordance with 18 U.S.C. Section 1734 solely to indicate this fact. slow the growth and progression of both basal DCIS and early invasive tumor models, generating tumor stasis without signifi- Received March 14, 2019; revised June 5, 2019; accepted August 14, 2019; cant side effects, and that the persistent influence of the drug even published first August 26, 2019.

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2232 Mol Cancer Ther; 18(12) December 2019 Molecular Cancer Therapeutics

Short- and Long-Term Effects of CDK4/6 Inhibition on Early-Stage Breast Cancer

William B. Kietzman, Garrett T. Graham, Virginie Ory, et al.

Mol Cancer Ther 2019;18:2220-2232. Published OnlineFirst August 26, 2019.

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