PTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and ... · Mari Mino-Kenudson 2, James R. Stone 1, Nicholas J. Dyson 1, Leif W. Ellisen 1 ... 5 , Cyril H. Benes 1, Jeffrey

72 | CANCER DISCOVERY JANUARY 2020 aacrjournals.org

PTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and PI3K` Inhibitors in Breast Cancer Carlotta Costa 1 , Ye Wang 1 , Amy Ly 2 , Yasuyuki Hosono 3 , Ellen Murchie 1 , Charlotte S. Walmsley 1 , Tiffany Huynh 2 , Christopher Healy 1 , Rachel Peterson 1 , Shogo Yanase 3 , Charles T. Jakubik 1 , Laura E. Henderson 1 , Leah J. Damon 1 , Daria Timonina 1 , Ioannis Sanidas 1 , Christopher J. Pinto 1 , Mari Mino-Kenudson 2 , James R. Stone 1 , Nicholas J. Dyson 1 , Leif W. Ellisen 1 , Aditya Bardia 1 , Hiromichi Ebi 3 , 4 , 5 , Cyril H. Benes 1 , Jeffrey A. Engelman 1 , and Dejan Juric 1

RESEARCH ARTICLE

ABSTRACT The combination of CDK4/6 inhibitors with antiestrogen therapies signifi cantly improves clinical outcomes in ER-positive advanced breast cancer. To identify

mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN , through increased AKT activation, was suffi cient to promote resistance to CDK4/6 inhibition in vitro and in vivo . Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these fi ndings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies.

SIGNIFICANCE: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as fi rst-line treatment for ER-positive advanced breast cancer. Importantly, these fi ndings have near-term clinical relevance because PTEN loss also limits the effi cacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting.

1 Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts. 2 Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. 3 Division of Molecular Therapeutics, Aichi Cancer Center Research Insti-tute, Nagoya, Japan. 4 Precision Medicine Center, Aichi Cancer Center, Nagoya, Japan. 5 Division of Advanced Cancer Therapeutics, Nagoya Uni-versity Graduate School of Medicine, Nagoya, Japan. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). C. Costa and Y. Wang contributed equally to this article. Current affi liation for C. Costa, Y. Wang, and J.A. Engelman: Novartis Insti-tutes for BioMedical Research.

Corresponding Authors: Jeffrey A. Engelman, Novartis Institutes for Bio-Medical Research, Cambridge, MA 02139. Phone: 617-871-7530; E-mail: [email protected] ; Carlotta Costa, Novartis Institutes for BioMedical Research, Klybeckstrasse 141, Basel CH-4002, Switzerland. Phone: 416-1324-4110; E-mail: [email protected] ; and Dejan Juric, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114. Phone 617-726-6500; E-mail: [email protected] Cancer Discov 2020;10:72–85 doi: 10.1158/2159-8290.CD-18-0830 ©2019 American Association for Cancer Research.

Research. on February 4, 2021. © 2020 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst October 8, 2019; DOI: 10.1158/2159-8290.CD-18-0830

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JANUARY 2020 CANCER DISCOVERY | 73

INTRODUCTIONThree different CDK4/6 inhibitors in combination with

endocrine therapy are approved by the FDA as the stand-ard of care for women with estrogen receptor (ER)–positive, HER2-negative advanced breast cancer (1–3). CDK4/6 forms a complex with D-cyclins and phosphorylates RB, thereby inactivating it and releasing cells to progress through the G1 checkpoint of the cell cycle. ER modulates cyclin D1 expres-sion, and cyclin D1 in turn increases ER transcriptional activ-ity, likely reinforcing the unique dependence of ER-positive cancer cells on cyclin D1 to initiate the G1 to S-phase transi-tion (4). Despite markedly improved clinical outcomes with this combination, acquired resistance emerges similarly to other targeted therapies (5, 6).

RESULTSAcquired Loss of RB and PTEN Expression in Patients Resistant to Letrozole and Ribociclib

The combination of CDK4/6 inhibitors and antiestrogen therapy has been tested in a phase Ib trial using ribociclib and

the aromatase inhibitor letrozole in postmenopausal women with ER-positive, HER2-negative advanced breast cancer (NCT01872260; ref. 7). Of the 7 patients treated on this protocol at our institution, we were able to collect paired pre-treatment and progression biopsies from 5 patients (Table 1). To understand reasons for acquired resistance to ribociclib, all the collected samples were analyzed for RB expression, because RB loss can confer resistance to CDK4/6 inhibition in preclinical models (8). Loss of RB expression at the time of progression was observed in patients 1, 2, and 3, whereas RB expression was preserved in patients 4 and 5 (Table 1; Fig. 1A–C; Supplementary Fig. S1). Thus, in agreement with preclinical models (8) and circulating tumor DNA analyses (9, 10), RB loss does appear to drive resistance to CDK4/6 inhibitors in the clinic.

Notably, patient 1 was subsequently treated with the combi-nation of selective ER degrader fulvestrant with a beta-sparing PI3K inhibitor taselisib on the clinical trial NCT01296555, but developed rapidly progressive disease after only one cycle of therapy (Fig. 1A). Because PTEN loss mediates clinical resistance to PI3Kα inhibitors (6), we tested whether PTEN expression was also lost during the treatment with ribociclib to explain the




Costa et al.RESEARCH ARTICLE


primary resistance to PI3Kα inhibition. Of note, to analyze PTEN and RB expression, we utilized IHC because multiple studies have shown that their loss can occur through epigenetic mechanisms. To our surprise, tumor biopsies from patient 1 (acquired at the time of resistance to ribociclib but prior to the treatment with taselisib) revealed acquired concomitant loss of PTEN and RB, raising the possibility that both alterations may contribute to acquired resistance to CDK4/6 inhibition ( Fig. 1A ). In support of this hypothesis, analysis of the progressing lesion in patient 4 revealed discrete PTEN loss ( Fig. 1C ), sug-gesting that PTEN loss alone confers resistance to CDK4/6 inhibition. Patients 2, 3, and 5 had preserved PTEN expression in their biopsies at the time of progression ( Fig. 1B ; Supplemen-tary Fig. S1A and S1B). Only patient 5 showed loss of PTEN at baseline in the lymph node lesion (Supplementary Fig. S1B). Consistent with our hypothesis, this lesion, which responded to the prior line of therapy with taselisib (Supplementary Fig. S1B, CT images on the top left), did not reduce in size dur-ing the ribociclib-based therapy (Supplementary Fig. S1B, CT images on the top right), suggesting that PTEN loss can drive both primary and acquired resistance to CDK4/6 inhibitors. In contrast, this patient had a liver lesion that initially responded to a much higher extent (and was the primary reason for the patient to be deemed to have a partial response per RECIST of −45.3%), but ultimately progressed and was sampled at the end of treatment (Supplementary Fig. S1B, CT images on the bottom). At the time of progression of this lesion, both PTEN and RB were intact, indicating that other mechanisms can con-tribute to resistance to CDK4/6 inhibitors. Overall, we observed acquired loss of either RB or PTEN or both as potential drivers of resistance to ribociclib in 4 of 5 analyzed cases ( Table 1 ). In addition, for patients 3 and 4, rapid autopsies were performed

in an effort to understand intratumoral heterogeneity of their refractory disease. This revealed RB loss in 17 of 18 sampled lesions for patient 3 ( Fig. 2A ) and PTEN loss in 7 of 10 collected lesions for patient 4 ( Fig. 2B ). Thus, multiple lesions revealed loss of PTEN as a potential mechanism of resistance. High prevalence of lesions that lack PTEN or RB expression among all progressive lesions is consistent with the preexisting nature of RB- or PTEN-defi cient cells with their rapid selection under treatment pressure.

Importantly, although RB and PTEN loss appeared in the context of combination therapy with ribociclib and letrozole, all studied patients had previously progressed on an aro-matase inhibitor ( Table 1 ), suggesting, although not proving, that selection for cells harboring the loss of RB or PTEN was primarily driven by ribociclib.

PTEN Loss Induces Resistance to CDK4/6 Inhibitors in ER-Positive Breast Cancer Models

We aimed to determine whether PTEN loss is a bona fi demechanism of resistance to CDK4/6 inhibitors, because the effect of RB loss has already been evaluated in other preclini-cal studies ( 8 ). We generated CRISPR-based PTEN knockout T47D cells, an ER-positive breast cancer model. Multiple clones for each guide (named A and B) showed complete loss of PTEN expression (Supplementary Fig. S2A). As expected, PTEN -defi cient cells had increased levels of phosphorylated AKT (Supplementary Fig. S2A) and were resistant to the p110α-selective inhibitor BYL719 (Supplementary Fig. S2B; ref. 6 ). Of note, because different clones displayed a slightly different proliferation rate (Supplementary Fig. S2C) and this could potentially affect their sensitivity to cell-cycle inhibitors in viability assays ( 11 ), the duration of cell-growth

Table 1. Patient information

Patient ID Age

Receptor status

PIK3CA/AKT1 status pretreatment

Prior systemic therapy Letrozole/ ribociclibPretreatment

biopsyProgression

biopsyAdjuvant Metastatic Best response DOT RB PTEN RB PTEN

1 53 ER + /PR − /HER2 −

PIK3CA E545K, AKT1 WT

Tamoxifen, anastrozole None PR (−32%) 8.4 + + − −

2 61 ER + /PR − /HER2 −

PIK3CA WT, AKT1 E17K

None ( de novo metastatic)

Letrozole SD (+13.5%) 1.8 + + − +

3 a 60 ER + /PR − /HER2 −

PIK3CA E545K, AKT1 WT

Docetaxel/ cyclophosphamide, anastrozole

Carboplatin/paclitaxel/veliparib

SD (−8.1%) 8.3 + + − +

4 a 68 ER + /PR + /HER2 −

Not available Tamoxifen Letrozole, fulvestrant, capecitabine

Non-CR/non-PD b 8.2 + + + −

5 45 ER + /PR + /HER2 −

PIK3CA H1047R, AKT1 WT

Doxorubicin/ cyclophosphamide/paclitaxel, tamoxifen/ leuprolide

Letrozole, fulvestrant, taselisib

PR (−45.3%) 5.5 + − + +

Abbreviations: CR, complete response; DOT, duration of treatment; PD, progressive disease; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; WT, wild-type. aExamined also by autopsy. b Evaluable but not measurable per RECIST 1.1.




Mechanisms of Resistance to CDK4/6 Inhibitors RESEARCH ARTICLE


Patient 1

APretreatment

(lesion I)

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Progression(lesion II)

Pretreatment(lesion I)

Progression(lesion I)

Pretreatment(lesion I)

Progression(lesion II)

C

RB

PTEN

NegativePositive

NegativePositive

Ribociclib + letrozole Taselisib + fulvestrant

8.4 months 1 month

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Nadir

RB

PTEN

Positive Positive

NegativePositive

Ribociclib + letrozole8.3 months

Autopsy

Pretreatment (lesion I)

End of treatment (lesion I)

RB

PTEN

Positive

Positive Positive

Negative

Patient 4 Ribociclib + letrozole

8.2 months

Autopsy

End of treatment(Liver lesion: lesion II)

Nadir Pretreatment

(Bone lesion: lesion I)End of treatment

Nadir Pretreatment

Figure 1. Loss of RB and PTEN expression in patients with acquired resistance to letrozole and ribociclib. A–C, Left, CT images of patients 1 (A), 3 (B), and 4 (C, additional bone scan) demonstrating initial response lesions that subsequently progressed. Inset shows an image detail. Arrows indicate the lesion that was biopsied. Above the CT images, treatment and biopsy history of the patient are depicted. Right, IHC staining of RB and PTEN of baseline and progression biopsies. Inset shows an image detail.






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Patient 3

Patient 4

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Left skin

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Right middle lobe

Pericardium

Right lower lobe superior

Right lateral skin

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Right medial abdominal wall

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Positive

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Positive

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Negative

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Negative

Negative

Positive

Negative

Negative

Negative

Positive

Figure 2. IHC analysis of RB and PTEN expression in autopsy cases. Left, CT scan showing a summary of the lesions collected during the autopsy of patients 3 (A) and 4 (B). Right, a summary of the IHC staining results for RB and PTEN.

assays were performed so that each of the untreated clones (and untreated parental control) underwent the same num-ber of cell doublings. All PTEN-null clones showed decreased sensitivity to two distinct CDK4/6 inhibitors (ribociclib and palbociclib) in comparison with CAS9 control cells (Fig. 3A; Supplementary Fig. S2C). Results were confirmed in a second ER-positive breast cancer model, MCF7 (Fig. 3B; Supplementary Fig. S2A and S2C). In addition, ectopic expression of the wild-type (WT) form of PTEN, but not of a catalytically inactive version, increased the sensitivity to CDK4/6 inhibitors of a PTEN-deficient breast cancer model (Fig. 3C; Supplementary Fig. S2A). Moreover, T47D PTEN-null cells were also more resistant to the combination of CDK4/6 inhibitors with fulvestrant (Fig. 3D) or when treated with CDK4/6 inhibitors in the absence of estro-gen (Supplementary Fig. S2D). In accordance with in vitro results, PTEN-deficient T47D xenograft models were resist-ant to ribociclib in vivo (Fig. 3E). Overall, these data suggest

that loss of PTEN expression alone reduces sensitivity to multiple CDK4/6 inhibitors.

We next investigated the mechanism by which PTEN loss promotes resistance to CDK 4/6 inhibitors. Cell-cycle analysis revealed that CAS9 control in both T47D and MCF7 cells underwent a dramatic arrest in G1 induced after 24 hours of treatment with CDK4/6 inhibitors (Fig. 3F; Supplementary Fig. S2E–S2G). For T47D, a modest increase in the number of cycling cells was observed at 72 hours, as previously reported (8). The isogenic PTEN-deficient cells initially showed a simi-lar strong arrest in G1 after 24 hours of treatment; however, unlike the control cells, a large fraction of the PTEN-deficient cells reentered S-phase by 48 hours (Fig. 3F; Supplementary Fig. S2F and S2G). In CAS9 control cells, CDK4/6 inhibition resulted in decreased RB phosphorylation and expression of multiple cyclins (such as cyclin D3 and E2) that are normally regulated by RB. In contrast, CDK4/6 inhibition was cor-related with increased expression of cyclin D1 and E1, likely






Figure 3. Loss of PTEN drives resistance to CDK4/6 inhibitors. A and B, T47D (A) and MCF7 (B) clones were treated with increasing concentrations of ribociclib or palbociclib. Mean ± SEM of one representative experiment carried out in triplicate is shown (n = 3). The cell-growth assay was normalized on cell doubling; P value was calculated by two-way ANOVA. C, EVSA-T cells expressing PTEN WT or the phosphatase-dead mutant PTEN C124S were induced or not induced with doxycycline (200 ng/mL) for 48 hours, and cells were treated with various concentrations of ribociclib or palbociclib. Data are presented as the mean ± SEM of three independent experiments each carried out in triplicate. The cell-growth assay was normalized on cell doubling; P value was calculated by two-way ANOVA. D, T47D cells were treated with ribociclib (RIBO), fulvestrant (FUL), or the combination and mean ± SEM of one representative experiment carried out in triplicate is shown (n = 3). The cell-growth assay was normalized on cell doubling. E, In vivo tumor-growth meas-urement (relative to initial volume) of T47D xenografts treated with either vehicle or ribociclib for 45 days. Data are presented as the mean ± SEM; P value was calculated by two-way ANOVA. F, T47D cells were treated with ribociclib or palbociclib for the indicated period of time, after which cell-cycle analysis was performed. The percentage of cells in S-phase is depicted. Data are presented as the mean ± SEM (n = 3). G, T47D cells were treated with ribociclib for the indicated period of time. Lysates were prepared and immunoblot performed using the indicated antibodies. ****, P < 0.0001; *, P < 0.05.

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reflecting a stabilization of these proteins in cells arrested in G1 (8, 12; Fig. 3G; Supplementary Fig. S2E–S2G). In contrast, PTEN-null cells initially showed a decrease in pRB (24 hours) but rephosphorylation of RB was clearly apparent after 48 hours (Fig. 3G; Supplementary Fig. S2E–S2G). To dif-ferentiate whether the increased phosphorylation of RB was either a cause or a consequence of PTEN-deficient cells rapidly reentering the cell cycle, a dose–response experiment with ribociclib was performed (Fig. 4A). In PTEN-deficient T47D cells, a substantially higher dose of ribociclib was necessary to suppress RB phosphorylation. Interestingly, 6 μmol/L of ribo-ciclib reduced pRB in PTEN-deficient cells to the same extent as 1 μmol/L in control cells (Fig. 4A). This higher dose also completely blocked these PTEN-deficient cells from reenter-ing the cell cycle (Fig. 4B). The same concentration of drug (6 μmol/L) was unable to induce cell-cycle arrest in T47D cells in which RB was knocked down (Fig. 4C), indicating that at this concentration the effect of ribociclib is still mediated by on-target suppression of RB. This finding also underscores that PTEN loss leads to a relative decrease in potency of ribociclib, but RB loss causes complete resistance. However, at clinically relevant doses of ribociclib (1 μmol/L; ref. 13), PTEN-null (Fig. 3A) and RB knockdown (Fig. 4D) cells showed a comparable level of resistance to ribociclib. Altogether, these data suggest that the loss of PTEN expression is sufficient to reduce sensitivity to CDK4/6 inhibitors by maintaining RB phosphorylation and thereby alleviating cell-cycle arrest.

Inhibition of AKT Restores Sensitivity to CDK4/6 Inhibitors

In PTEN-deficient cells, the addition of MK2206, an allosteric AKT inhibitor, restored the capacity of CDK4/6 inhibitors to reduce RB phosphorylation (Supplementary Fig. S3A), dem-onstrating that increased AKT activity is a critical mediator of the resistance induced by PTEN loss. Accordingly, the combi-nation of CDK4/6 and two distinct AKT inhibitors resulted in cell-cycle block in G1 (Fig. 4E; Supplementary Fig. S3B and S3C) and decreased cell proliferation (Fig. 4F; Supplementary Fig. S3D–S3F). The effect of the combination was comparable to that achieved with single-agent CDK4/6 inhibitor in the Cas9 control cells. In addition, the combination of MK2206 and ribociclib induced tumor regression in the PTEN-deficient T47D xenograft model (Fig. 4G). As observed previously, the combination of CDK4/6 and fulvestrant was less effective at inhibiting the proliferation of PTEN-deficient cells (Fig. 3D; Supplementary Fig. S3G). However, the triple combination of CDK4/6 and AKT inhibitors with fulvestrant was sufficient

to suppress proliferation of PTEN-deficient cells. Importantly, both the CDK4/6 and AKT inhibitors’ doses could be lowered and still yield efficacy in the triple combination, compared with the doses of single-agent CDK4/6 or AKT inhibitors required for efficacy in combination with fulvestrant (Supple-mentary Fig. S3G). Overall, inhibiting AKT effectively reverses the effect of PTEN loss on resistance to CDK4/6 inhibitors and restores their capacity to suppress RB phosphorylation and cell-cycle progression.

PTEN Loss Drives Resistance to CDK4/6 Inhibitors by Increasing CDK2 and CDK4 Activity through Delocalization of p27 Outside of the Nucleus

We next aimed to understand how increased AKT activa-tion leads to resistance to CDK4/6 inhibitors. Previous stud-ies in breast cancer have shown that AKT can induce cell-cycle progression by activating CDK2, which in turn phosphoryl-ates RB during the G1 to S-phase transition (4). AKT directly phosphorylates threonine 157 of p27, an inhibitor of CDK2. Phosphorylation of p27 impairs its nuclear import, thereby blocking access to CDK2 and resulting in increased CDK2 activation (14–16). We therefore initially tested whether p27 localization was affected by PTEN loss in the paired biopsies (Fig. 5A). Although loss of RB did not alter p27 localization (patient 2), a significant decrease in p27 nuclear staining was observed in the lesions that did not express PTEN (patients 4 and 5). These results are consistent with the hypothesis of PTEN loss causing decreased p27 expression in the nucleus.

To explore whether modulation of p27 localization could affect the resistance to CDK4/6 inhibitors, both T47D and MCF7 cells were transduced with either WT or a mutant form of p27 (T157A) that is unable to be phosphorylated by AKT (Supplementary Fig. S4A). Consistent with increased levels of AKT, PTEN-deficient cells showed decreased locali-zation of WT p27 in the nucleus in comparison with con-trol cells (Supplementary Fig. S4B). However, the T157A mutant p27 displayed a more prominent nuclear localiza-tion in PTEN-deficient cells, comparable to WT p27 in con-trol cells ( Supplementary Fig. S4C). The expression of the T157A-p27 in PTEN-deficient cells restored the capacity of ribociclib to suppress RB phosphorylation and cell growth (Fig. 5B and C; Supplementary Fig. S4D–S4F), indicating that relocalization of p27 in the nucleus is sufficient to partially resensitize PTEN-deficient cells to CDK4/6 inhi-bition. In support of the hypothesis of PTEN loss causing

Figure 4. AKT inhibition restores sensitivity to CDK4/6 inhibitors. A, T47D cells were treated with the indicated concentrations of ribociclib for 48 hours. Lysates were prepared and proteins detected with the indicated antibodies. B, T47D cells were treated with different concentrations of ribociclib (RIB) for 48 hours. Left, cell-cycle analysis was performed and the percentage of cells in S-phase is depicted. Data are presented as the mean ± SEM (n = 3). Right, a portion of cells was analyzed by Western blot with the indicated antibodies. C, Top, T47D CAS9 cells expressing inducible shRNA for RB were treated or not treated with doxycycline (500 ng/mL) for 48 hours and subsequently treated with ribociclib for 48 hours. Cells were analyzed for cell cycle with the percentage of cells in S-phase depicted. Data are presented as the mean ± SEM (n = 3). Bottom, a portion of cells was analyzed by Western blot with the indicated antibodies. D, T47D CAS9 cells expressing inducible shRNA for RB were induced or not induced with doxycycline and treated with increasing concentrations of BYL719 (top) or ribociclib (bottom). Mean ± SEM of one representative experiment carried out in triplicate is shown. P value was calculated by two-way ANOVA. E, Cells were treated with ribociclib, MK2206, or their combination, after which cell-cycle analysis was performed. Left, the percentage of cells in S-phase is depicted. Data are presented as the mean ± SEM (n = 3). Right, a portion of cells was analyzed by Western blot with the indicated antibodies. F, Cells were treated with ribociclib, MK2206, or the combination. Data are presented as the mean ± SEM of three independent experiments each carried out in triplicate. The cell-growth assay was normalized on cell doubling. G, In vivo tumor growth measurement (relative to initial volume) of PTEN-null clone A7 T47D xenografts treated with either vehicle, MK2206, ribociclib, or combination. Data are presented as the mean ± SEM; P value was calculated by two-way ANOVA. ****, P < 0.0001; ***, P < 0.001.






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p27-mediated increased activity of CDK2, in T47D cells we observed a decreased association of p27 with CDK2 in the absence of PTEN (Fig. 5D), also in line with previously reported findings (17). Moreover, PTEN-deficient T47D cells demonstrated an enhanced association of CDK2 with cyclin D3 and cyclin E1 before and after 24-hour treatment with ribociclib (Fig. 5E; Supplementary Fig. S4G). This was likely due to an upregulation in the total levels of expression of cyclins D3 and E1, in line with previous reports showing that AKT activation can increase cyclin D3 protein stabil-ity and cyclin E1 expression (17, 18). Of note, we compared cells after 24 hours of treatment, when all cells were equally arrested in G1 (Fig. 3F), and thus the expression levels of cyclins were not a mere consequence of the cells being in a different phase of the cell cycle. Consistently, although only approximately 50% suppression of CDK2 via RNAi was achieved, this was sufficient to partially resensitize the PTEN-null T47D and MCF7 cells to ribociclib with respect to RB phosphorylation and progression into the S-phase of the cell cycle (Supplementary Fig. S4H–S4M).

Although p27 was initially identified as a CDK2 inhibitor, studies have shown that it also promotes assembly of the cyc-lin D–CDK4 complex (19). When AKT phosphorylates p27, this leads to increased cytoplasmic localization and bind-ing to cyclin D1–CDK4 (but not CDK2) and promotes the assembly and/or stabilization of this ternary complex (20). Consistently, an increased association of CDK4 with p27 in the absence of PTEN was observed (Supplementary Fig. S5A). To determine whether PTEN loss affects cyclin D1–CDK4 complex formation, immunoprecipitation experiments were performed. In PTEN-deficient cells, we observed an increase in association between CDK4 and cyclin D1 (after 24 hours of treatment with ribociclib) and D3 (at baseline; Fig. 5F; Sup-plementary Fig. S5B). Although increased complex formation with cyclin D3 may simply be a consequence of increased cyc-lin D3 expression levels, cyclin D1 levels were not increased in PTEN-deficient cells, further supporting the notion that PTEN loss promotes the formation and/or stabilization of the CDK4/cyclin D1 complex. Notably, whereas downregu-lation of cyclin D1 resensitized PTEN-deficient T47D and MCF7 cells to ribociclib (Fig. 5G; Supplementary Fig. S5C and S5D), downregulation of cyclin D3 showed a more mod-est and consistent effect only in MCF7 cells (Fig. 5H; Sup-plementary Fig. S5E and S5F), suggesting that the relative contribution of the cyclins D in mediating CDK4 activity might be different among breast cancer cell models. In addi-tion, siRNA was used to decrease the expression of CDK4/6, and although only a modest decrease in their expression lev-els was achieved, this partially resensitized the PTEN-deficient

cells to ribociclib (Supplementary Fig. S6A–S6C). These data suggest that in PTEN-null cells, CDK4/6 enzymes are still partially active despite the presence of clinically relevant doses of ribociclib, and contribute to RB phosphorylation and progression through the cell cycle. This is also consistent with the finding that higher doses of ribociclib were necessary to fully suppress RB phosphorylation in the PTEN-deficient cancer cells (Fig. 4A). Overall, these mechanistic data indicate that loss of PTEN, through increased AKT activity, induces delocalization of p27 outside the nucleus and therefore causes increased activity of both CDK4/6 and CDK2, which together contribute to overcome the blockade in G1 induced by CDK4/6 inhibitors.

DISCUSSIONIn this study, we observed that RB and PTEN loss are both

mechanisms of resistance to CDK4/6 inhibitors in ER- positive metastatic breast cancer. Although CDK4/6 inhibitors are commonly used in patients with advanced ER-positive breast cancer, the PI3Kα-selective inhibitor alpelisib was only recently approved as a second line of treatment for PIK3CA-mutated ER-positive breast cancer (21). Significantly, previ-ous studies established that PTEN loss also causes resistance to alpelisib (6). These partially overlapping resistance mecha-nisms (i.e., PTEN loss for both CDK4/6 and PI3Kα inhibitors; RB loss just for CDK4/6 inhibitors) have potential clinical implications, as these drugs are sequentially deployed in the management of patients with advanced breast cancer. Indeed, because PTEN loss is a mechanism of resistance to both PI3Kα and CDK4/6 inhibitors, a subset of patients progress-ing on CDK4/6 inhibitors harboring this alteration may have diminished response to PI3Kα inhibitors (see patient 1). In contrast, this study (Fig. 4D) and others (22) have shown that RB loss does not confer resistance to PI3Kα inhibitors in breast cancer models in vitro. Thus, cancers with this mecha-nism of resistance to CDK4/6 inhibitors may derive similar benefit from PI3Kα inhibitors as cancers that had not been exposed to CDK4/6 inhibitors. Notably, RB or PTEN expres-sion levels are currently not being used for patient selection in second-line clinical trials with PI3Kα inhibitors, especially because both RB and PTEN loss are rare in treatment-naïve ER-positive tumors (23, 24). However, the findings in this study suggest that the prevalence of this alteration is likely to increase under selective pressure with CDK4/6 inhibitors and highlight the importance of investigating RB and PTEN expression in prospective clinical trials in the post-CDK4/6 setting. Of note, with a relatively small number of samples, we cannot determine the true prevalence of each of these

Figure 5. Loss of PTEN impairs p27 nuclear localization and increases CDK2 and CDK4 activity. A, IHC staining of p27 of baseline and progression biopsies; inset shows % of p27 nuclear staining. B and C, T47D cells were transduced to express p27 WT or p27 T157A. Cells were treated with ribociclib for 48 hours. Lysates were prepared and blotted with the indicated antibodies (B). Cells were treated with various concentrations of ribociclib. Data are presented as the mean ± SEM of three independent experiments each carried out in triplicate. The cell-growth assay was normalized on cell doubling; P value was calculated by two-way ANOVA (C). D, T47D cells were treated for 24 hours with ribociclib and then cell lysates were immunoprecipitated with p27 antibody. Precipitates and whole-cell lysates were analyzed by Western blot with the indicated antibodies. E and F, T47D cells were treated with ribociclib for indicated period of time and lysates were immunoprecipitated with CDK2 antibody (E) or CDK4 antibody (F). Precipitates were analyzed by Western blot with the indicated antibodies. G and H, T47D cells were transfected with control (CT) or cyclin D1 (G) or D3 (H) targeted siRNA for 24 hours, followed by treatment with ribociclib (RIBO) for an additional 48 hours, after which cell-cycle analysis was performed. Left, the percentage of cells in S-phase is depicted. Data are presented as the mean ± SEM (n = 3). Right, a portion of cells was analyzed by Western blot with the indicated antibodies. ****, P < 0.0001; *, P < 0.05.






alterations, and larger studies are needed to assess the gener-alizability of these findings. Nevertheless, for one of the five patients, the lesion at the time of progression retained both RB and PTEN expression, indicating that other mechanisms contribute to resistance to CDK4/6 inhibitors, as recent pub-lications have demonstrated (10, 25–27).

To understand how loss of PTEN promotes resistance, we used gene-editing approaches to develop isogenic ER-positive breast cancer models. In PTEN-deficient cells, increased AKT activation mediates resistance to CDK4/6 inhibitors, and selective AKT inhibitors can restore sensitivity in vitro and in vivo. This mechanistic link to resistance is consistent with a recent study showing that increased AKT activation, in that case mediated by PDK1, is sufficient to drive resistance to ribociclib in breast cancer cells (28). Notably, four of five pre-treatment biopsies (for one, patient data were not available) showed that these tumors harbor additional PI3K pathway alterations (either in PIK3CA or AKT1; Table 1), similar to the cell lines we utilized for in vitro studies. It is conceivable that clinical resistance to CDK4/6 inhibitors is achieved only through substantial activation of the PI3K–AKT axis, medi-ated by multiple alterations, such as PIK3CA or AKT1 and concomitant PTEN loss. However, future studies should clar-ify whether PTEN loss can cause resistance to CDK4/6 inhibi-tors also in tumors that do not carry additional mutations in the PI3K pathway. AKT activation itself may drive resistance by many potential mechanisms. Several groups have reported that AKT-dependent phosphorylation of p27 increases p27 cytoplasmic localization (14–16). This cytoplasmic retention of p27 may help drive resistance by at least two mechanisms. First, the AKT-dependent delocalization of p27 may reduce nuclear p27 availability to bind and inhibit cyclin E/CDK2, thus contributing to cyclin E/CDK2 activation (29). Accord-ingly, in our paired biopsies, we observed that loss of PTEN expression correlates with exclusion of p27 from the nucleus. Notably, PTEN-deficient cells also had a modest but consist-ent increased expression of cyclin E1, which may also partially contribute to increased CDK2 activity (25). Second, p27 can increase the assembly/stability of cyclin D1–CDK4 com-plexes in the cytoplasm (19). Indeed, we observed increased complex formation in the PTEN-deficient cells. It is possible, however, that the PTEN-mediated effect on CDK4–cyclin D complex might also be partially p27-independent. Of note, in the paired biopsies, despite observing loss of nuclear PTEN staining, we were not able to observe an accumulation of p27 in the cytoplasm when PTEN was lost. This might be due to multiple reasons, including a technical artifact related to the specific antibody we used. Indeed, this antibody has been generated by immunization with an epitope that contains the first 200 amino acids of p27, which includes the residue phosphorylated by AKT. It is thus conceivable that the phos-phorylated form of p27 that accumulates in the cytoplasm in the absence of PTEN is not efficiently recognized by this antibody. Alternatively, we cannot exclude the possibility that the cytoplasmic fraction of p27 is less stable than the nuclear fraction in these needle biopsies.

The findings in this article suggest that PTEN loss causes diminished sensitivity to clinically relevant doses of CDK4/6 inhibitors by initiating signaling cascades that induce hyper-activation of cyclins/CDKs. This suggests that these resistant

cancers still require RB inactivation for G1-to-S progression. The fact that PTEN-null cells are still dependent on inacti-vating RB to progress through the cell cycle suggests that these cells could gain additional proliferative advantage by concomitant loss of RB, as observed in patient 1.

Previous preclinical studies have shown that PTEN loss also affects the activity of BRAF (30), EGFR (31), and immune checkpoint inhibitors (32) in other settings, suggesting that PTEN loss may emerge as a resistance mechanism in many therapeutic paradigms. Furthermore, this study provides a proof of concept of how a single genetic event can cause cross-resistance to multiple targeted therapies and affect sub-sequent lines of treatment in the same patient population.

METHODSDNA Constructs

For SpCas9 expression and generation of single guide RNA for human PTEN, two 20-nt target sequences were selected. A: CCAAA TTTAATTGCAGAGGT; B: AGAGGCCCTAGATTTCTATG. Oli-gonucleotides were annealed and cloned into the BsmbI-BsmBI sites downstream from the human U6 promoter in LentiCRISPR v2 plasmid (Addgene, # 52961). The doxycycline-inducible sys-tem pINDUCER11 was used to express shRNA that targets RB1 at the sequence 5′-CAGAGATCGTGTATTGAGATT-3′. Stable T47D cell lines were generated by lentiviral transduction with the pINDUCER11-shRB1, followed by FACS for GFP expression. Plasmid expressing p27 cDNA with N-terminus–fused eGFP was purchased from GeneCopoeia (# EX-C0696-Lv182). Mutation of Threonine 157 to Alanine was introduced by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, #E0554S). The following oligonucleotide primers were used for mutagene-sis: T157A-FW: 5′-GCGACCTGCAgCCGACGATTC-3′; T157A-RV: 5′-TTCCTTATTCCTGCGCATTGC-3′. PTEN-WT and PTEN-C124S constructs (Addgene, #10785 and #10931) were cloned in pTREX doxycycline-inducible vector kindly provided by Novartis.

Cell Lines and ReagentsT47D cells were cultured in RPMI-1640 with 10% FBS. EVSA-T

and MCF7 cells were cultured in DMEM with 10% FBS. Cancer cell lines were obtained from the Center for Molecular Therapeu-tics at the Massachusetts General Hospital Cancer Center, which performs routine SNP and short tandem repeat authentication. In addition, cell lines underwent SNP validation at the completion of the project. Cell lines were routinely tested for Mycoplasma during experimental use. For in vitro experiments, the following drugs were used at the following concentrations (unless otherwise specified): ribociclib (1 μmol/L), palbociclib (250 nmol/L), MK2206 (1 μmol/L), GDC0068 (500 nmol/L), fulvestrant (10 nmol/L), BYL719 (1 μmol/L), all obtained from Selleckchem.

Immunoblotting and siRNA KnockdownAntibodies against RB (#9309), pRB (Ser608; #8147), pRB

(Ser780; #9307), pRB (Ser807/811; #8516), pAKT (T308; #4056), pAKT (S473; #4060), cyclin A2 (#4656), cyclin E1 (#4129), cyclin E2 (#4132), cyclin D1 (#2922), cyclin D3 (#2936), PTEN (#9552), CDK6 (#13331), CDK4 (#12790), CDK2 (#2546), p27 (#3686), Vinculin (#13901), and Actin (#4967) were purchased from Cell Signaling Technology. GAPDH antibody was purchased from Millipore. Cells were seeded into 6-well plates; 24 hours later, cells were transfected with ON-TARGETplus SMARTpool siRNA against CDK2, CDK4, CDK6, cyclin D1, and cyclin D3, or ON-TARGET plus Non-Targeting Pool as negative control (Dharmacon) using HiPerFect Transfection






Reagent (Qiagen) according to the manufacturer’s instructions. Transfected cells were cultured for 24 hours before drug treatment.

ImmunoprecipitationCells were seeded and treated the following day with the indicated

drugs for the specified period of time. After treatment, cells were washed twice with ice-cold PBS and homogenized in immunoprecipitation (IP) lysis buffer. Soluble cell lysates were collected after 13,500 rpm centri-fuge at 4°C for 15 minutes. An equal amount of total protein was incu-bated with anti-CDK2 (#07-631, EMD Millipore; 1 μg antibody/200 μg lysate) or anti-p27 (#3686, Cell Signaling Technology; 0.4 μg anti-body/200 μg lysate) rabbit antibody plus protein A-Sepharose beads (#17-0963-03, GE Healthcare), or anti-CDK4 mouse antibody (#sc-23896, Santa Cruz Biotechnology; 1 μg antibody/200 μg lysate) plus protein G-Sepharose beads (#17-0618-01, GE Healthcare) for more than 3 hours at 4°C. Beads were washed at least three times with IP lysis buffer after incubation and centrifuged at 4,000 rpm at 4°C for 3 minutes to remove supernatant. After washing, SDS loading buffer was added and boiled at 95°C for 10 minutes.

Viability StudiesViability assays were performed by plating cells in 96-well plates and

treating them the following day with the indicated drug(s) for 5 to 8 days, according to the cell doubling. At the end of all experiments, cells were fixed and stained with Hoechst (1 μg/mL). Ninety-six–well plates were imaged with ImageXpress Micro (Molecular Devices) and nuclei counts were performed with MetaXpress program. For experi-ment with estrogen depletion, cells were cultured with Phenol red-free RPMI-1640 and 10% charcoal-stripped FBS for two days, after cells were seeded in the same medium with or without 0.2 nmol/L β-estradiol.

Cell CycleAfter drug treatment, cells were incubated with 20 μmol/L

5-ethynyl-2′-deoxyuridine (Life Technologies) for 2 hours, fixed with 3.7% formaldehyde for 15 minutes, blocked with 3% BSA for 5 min-utes, permeabilized with 0.5% Triton X-100 in PBS for 30 minutes, and stained with ClickIT reaction kit (100 mmol/L Tris-HCl pH 7.5, 3 mmol/L CuSO4, 50 mmol/L ascorbic acid, 2.5 μmol/L Alexa-647 azide, Life Technologies) for 30 minutes and 3 μmol/L DAPI (Life Technologies) in staining buffer (100 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L CaCl2, 0.5 mmol/L MgCl2, 0.1% NP-40) for 15 minutes. FACS analysis was performed with LSR II Flow Cytometer (BD Biosciences).

Xenograft Mouse StudiesAll mouse studies were conducted through Institutional Animal

Care and Use Committee–approved animal protocols in accordance with institutional guidelines. For xenograft experiments, female nude mice were implanted with estrogen (0.72 mg) of 17β-estradiol pellets (Innovative Research of America). A suspension of 2 × 107 cells was inoculated subcutaneously into the left flank of 6- to 8-week-old female athymic nude mice. Tumors were monitored until they reached an average size of 100 mm3, after roughly 6 to 8 weeks, at which point mice were randomized into the different groups (n = 4 mice/group) and treatments were begun. Ribociclib, kindly provided by Novartis, was dissolved in 0.5% methylcellulose and administered at 75 mg/kg once a day by oral gavage. Xenografts derived from PTEN-null T47D cells were treated with vehicle, ribo-ciclib (60 mg/kg daily), MK-2206 (60 mg/kg twice a week, dissolved in 30% captisol), or both inhibitors in combination. Tumors were measured twice weekly using calipers.

ImmunofluorescenceCells were seeded into 96-well plates with a density of 3,000 cells

per well and treated the following day with the indicated drugs for

the specified period of time. After treatment, cells were fixed with 2.25% parafo rmaldehyde in 0.1% PBS-Tween at room temperature for 30 minutes followed by permeabilization with 0.1% Triton X-100 in PBS at room temperature for 10 minutes. The cells were then washed with PBS twice and blocked with 2% BSA at room tempera-ture for 1 hour. After blocking, cells were subjected to immunofluo-rescence staining with anti-GFP antibody (#A11122, Thermo Fisher Scientific) overnight at 4°C. Then, the cells were rinsed with PBS with 0.1% Tween-20 twice and incubated with Alexa 488–labeled anti-rabbit secondary antibody (#A21206, Life Technologies) at room temperature for 1 hour. After that cells were incubated with Alexa 568–labeled Phalloidin to stain the cytoplasm (#A12380, Thermo Fisher Scientific) at room temperature for 20 minutes fol-lowed by staining with Hoechst (1 μg/mL) for 30 minutes. Images were acquired with Opera Phenix (PerkinElmer) and analyzed with Harmony software.

PatientsPatients were enrolled in the phase I clinical study of ribociclib in

combination with letrozole (NCT01872260), and the response was assessed using routine cross-sectional imaging as per RECIST v1.1 criteria. All clinical data and tumor specimens were collected and analyzed in accordance with Institutional Review Board–approved pro-tocols, to which patients provided written informed consent, and all studies were conducted in accordance with the Declaration of Helsinki.

IHC on Patient SamplesIHC was performed on a 5-μm section using an automated stainer

(Bond Rx, Leica Microsystems). Following deparaffinization and rehy-dration of the slides, antigen retrieval was performed using citrate buffer (pH 6.0) for 20 minutes. After endogenous peroxidase activity was blocked with 3% to 4% hydrogen peroxide, samples were incubated at room temperature with RB mouse mAb (clone 1F8, Bio SB), PTEN mouse mAb (Biocare Medical), and p27 mouse antibody (#610242, BD Biosciences). Horseradish peroxidase polymer for detection was subsequently applied onto the slides. Sections were then incubated with DAB refine (10 minutes) for visualization and counterstained with hematoxylin (5 minutes). As defined previously (33), RB protein expression was considered lost if >95% of tumor nuclei showed lack of staining (0+). Staining of endothelial cells served as internal posi-tive control; cases without this endothelial cell staining were excluded. PTEN protein expression was considered lost if the staining intensity of the tumor cell cytoplasm was markedly decreased or absent across all tumor cells, compared with adjacent benign glands/stroma (inter-nal positive control). Cases were excluded if benign tissue lacked PTEN staining.

Statistical AnalysesUnless otherwise specified, statistical comparison among groups

was carried out with one-way ANOVA, with Sidak multiple group comparison test (GraphPad Prism; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Disclosure of Potential Conflicts of InterestC. Costa is an employee of Novartis. Y. Wang is an employee

of Novartis. A. Bardia is a consultant/advisory board member at Novartis, Radius Pharma, Pfizer, Daiichi, Sanofi, Genentech, Merck, Immunomedics, Spectrum, and Taiho. C.H. Benes reports receiving a commercial research grant from Novartis and other commercial research support from Amgen. J.A. Engelman is a global head, oncol-ogy, and has ownership interest (including patents) in Novartis. D. Juric is a scientific advisory board member for Novartis, Eisai, Genentech, Petra Pharma, EMD Serono, Ipsen, Syros, and Guardant and reports receiving commercial research support from Novartis, Eisai, Genentech, EMD Serono, Syros, Takeda, Placon Therapeutics,






Celgene, and Amgen. No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: C. Costa, A. Bardia, J.A. Engelman, D. JuricDevelopment of methodology: Y. Wang, C. Healy, J.R. Stone, H. Ebi, C.H. Benes, D. JuricAcquisition of data (provided animals, acquired and man-aged patients, provided facilities, etc.): C. Costa, Y. Wang, A. Ly, Y. Hosono, E. Murchie, T. Huynh, C. Healy, R. Peterson, S. Yanase, L.E. Henderson, L.J. Damon, D. Timonina, I. Sanidas, J.R. Stone, N.J. Dyson, A. Bardia, H. Ebi, C.H. Benes, D. JuricAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Costa, Y. Wang, A. Ly, Y. Hosono, C. Healy, S. Yanase, L.J. Damon, M. Mino-Kenudson, H. Ebi, J.A. Engelman, D. JuricWriting, review, and/or revision of the manuscript: C. Costa, Y. Wang, A. Ly, M. Mino-Kenudson, J.R. Stone, L.W. Ellisen, A. Bardia, H. Ebi, J.A. Engelman, D. JuricAdministrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Wang, C.S. Walmsley, R. Peterson, C.T. Jakubik, L.E. Henderson, C.J. Pinto, H. Ebi, D. JuricStudy supervision: C. Costa, C.H. Benes, J.A. Engelman, D. Juric

AcknowledgmentsWe would like to thank Silvia Goldoni, Tamara Gilbert, and Lesley

Griner from Novartis for the help with image acquisition and analy-sis with the Opera Phenix. This work was funded by the HMS Labo-ratory for Systems Pharmacology Grant (P50GM107618), Susan Eid Tumor Heterogeneity Initiative, Dr. Jerry Younger Grant for Clinical and Translational Breast Cancer Research, the Jonathan Kraft Trans-lational Award for Innovation in Cancer Research, and generous philanthropic support from Andrew Hertneky, and Stephen and Kathleen Chubb. Y. Hosono, S. Yanase, and H. Ebi’s contribution was supported by the Fund for the Promotion of Joint International Research (15KK0303) from the Japan Society for the Promotion of Science (to H. Ebi).

Received July 23, 2018; revised July 12, 2019; accepted October 3, 2019; published first October 8, 2019.

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2020;10:72-85. Published OnlineFirst October 8, 2019.Cancer Discov Carlotta Costa, Ye Wang, Amy Ly, et al.

Inhibitors in Breast CancerαPI3KPTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and

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