Translational strategy using multiple nuclear imaging biomarkers to evaluate target engagement and early therapeutic efficacy of SAR439859, a novel selective estrogen receptor degrader

Both endogenous and exogenous steroid hormones such as estrogen and progesterone have been implicated in the pathogenesis of breast cancer. Clinical treatment decisions are driven by the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) and the classification of breast tumors according to their receptor status into HER2-positive, ER-positive/HER2-negative, and triple-negative clinical subtypes. About 70–75% of breast cancers express ERα which is a hormone regulator transcription factor [1]. ERα-positive breast cancers respond well to therapy targeting ERα signaling either through competitive binding of ER antagonists such as tamoxifen or by blocking the production of estrogen by aromatase inhibitors [2]. However, despite the benefits of those therapies, de novo and acquired resistance remain an issue in many patients [3].

Several strategies have been used in the clinic to further target the ER pathway including the development of oral selective estrogen receptor downregulators/degraders (SERDs). Given the efficacy of fulvestrant, the first-in-class intramuscular SERD compound, oral SERDs are likely to play an important role in therapeutic management of ER+ tumors. This class of antagonists induces a degradation of the ERα receptor; their potential to block endocrine- and non-endocrine-dependent signaling has been recognized to offer a therapeutic approach in early-stage disease and improves outcomes for those with advanced ER+-resistant breast cancer [4, 5]. Fulvestrant exhibits both poor physicochemical and pharmacokinetic properties and the intramuscular route of administration has also limited its use. A new generation of SERDs with improved properties is being currently developed [6‐8].

SAR439859 is an oral ER antagonist and selective ER degrader (SERD) with potent anti-tumor activity regardless of ESR1 mutation status, and this compound is proposed for the treatment of locally advanced or metastatic ER-positive breast cancer. SAR439859 binds with high affinity to human wild-type ERα as well as to mutants ERα (ERα-Y537S and ERα-D538G). SAR439859 not only antagonizes the binding of estradiol to ER but also promotes the transition of ERα to an inactive conformation that leads to as up to 98% receptor degradation at nanomolar concentrations in cellular assays. These dual properties of SAR439859 translate in a robust inhibition of ERα pathways and a more effective anti-proliferative activity in ERα-dependent breast cancer cell lines driven by mutant or wild-type ERα compared to other ERα inhibitors [9]. SAR439859 induces ERα degradation and inhibits in a dose-dependent manner the cell proliferation of MCF7 cells harboring wild-type ERα or mutant ERα. Cell growth inhibition was also shown in a larger panel of ERα-positive breast cancer cell lines. SAR439859 has no effect on the growth of ERα-negative cell line confirming the selective effect of SAR439859 to ERα. In nude mice bearing the ERα-positive MCF-Y537S tumor model, a single oral administration of SAR439859 over 2.5–25 mg/kg dose range induced a dose-dependent intra-tumoral degradation of ERα. At 12.5 mg/kg, SAR439859 decreased ERα by 90% for at least 8 h [9]. In vivo efficacy data obtained against MCF7-Y537S demonstrated that the tumor growth inhibition induced by SAR439859 was correlated with ERα intra-tumoral degradation. Of note SAR439859 induced better tumor regression at 12.5 mg/kg twice a day regimen when compared to the 25 mg/kg daily regimen over 3 weeks (internal data). Since SAR439859 administration at 12.5 mg/kg was shown to maintain ERα decrease for 8 h, this suggests that continuous target occupation/inhibition is required to achieve tumor regression.

Positron emission tomography (PET) is described as the most valuable technique to measure non-invasively the target engagement in preclinical and clinical settings [10]. Using [¹⁸F]-FluoroEstradiol (FES) as a PET tracer enables evaluation of ER expression/function in patients with breast cancer. FES-PET may thus emerge as a valuable tool to predict which patients with primary, recurrent, or metastatic breast cancer will respond to hormone therapy [11]. Quantification of target engagement is a prerequisite for receptor occupancy studies and would provide a means to relate dosage of drug to the occupancy of target receptor by the drug candidate. A good correlation exists between [¹⁸F]-FES uptake and ERα expression measured by immunohistochemistry (IHC) [12]. However, the question remains whether FES-PET can predict the response to anti-estrogen therapy in patients. In a study published in 2001, Mortimer et al. [13] concluded that the level of [¹⁸F]-FES uptake at baseline predicts the response to tamoxifen treatment. Linden et al. [14] confirmed that the baseline measurement of [¹⁸F]-FES uptake (SUV < 1.5–2.0) would predict responses to targeted hormonal therapy. More recently, Chae et al. [15] in a neoadjuvant therapy showed that ER-rich patients with low [¹⁸F]-FES uptake (SUVmax < 7.3) are more susceptible to better respond to neoadjuvant chemotherapy compared to neoadjuvant endocrine therapy. In a recent review, van Kruchten [16] described the potential of FES-PET imaging in providing information about ER binding of endocrine drugs. Gong et al. [17] conducted a preliminary study to monitor the change in [¹⁸F]-FES uptake post-therapy and concluded that [¹⁸F]-FES could be a predictive indicator in patients receiving docetaxel combined or not with fulvestrant. Of note, to date only a few clinical trials support FES-PET imaging as a tool for selecting the therapeutic dose of selective ERα inhibitors [18, 19]. Clinical investigations with SAR439859 are currently under way; FES-PET imaging was retained to select the dose in a cohort of breast cancer patients (clinical trial #NCT03284957). In this respect, FES-PET as direct imaging technique can provide robust and valuable datasets for assessment of ERα modulation by SAR439859.

Recently, Christofanilli et al. [4] published data of a clinical trial aiming at comparing the effect of fulvestrant combined to a cyclin-dependent kinase (CDK) 4/6 inhibitor (palbociclib) versus fulvestrant plus placebo; patients treated with the combined regimen had a significant improvement in progression-free survival. Numerous clinical trials in cancer breast patients are currently being performed with CDK 4/6 inhibitors (for review see [20]); their favorable characteristics in terms of toxicity and route of administration make them of great interest for combination with endocrine therapy [21]. In nude mice bearing MCF7-Y537S tumor model, combining SAR439859 with palbociclib led to further growth-inhibitory effects compared with monotherapy alone [22] suggesting that the combination of SAR439859 with palbociclib could bring therapeutic benefit to ERα-positive breast cancer patients.

The preclinical work reported herein presents the use of FES-PET for monitoring response to SAR439859 endocrine therapy in SCID mice bearing a mouse xenograft model of ERα+ breast cancer. We assessed whether the target occupancy measured with FES-PET imaging could help select the best regimen to maximize the drug’s efficacy. To assess the feasibility of measuring therapeutic efficacy early in the course of treatment with SAR439859 and palbociclib, tumor metabolism and tumor proliferation were also investigated using [¹⁸F]FluoroDeoxyGlucose (FDG) PET and [¹⁸F]FluoroThymidine (FLT) PET.

In these preclinical experiments performed on an ERα-positive tumor model (MCF7-Y537S), we show that FES-PET can be used as a pharmacodynamic imaging biomarker to demonstrate ER target engagement and map impact of SAR439859 SERD. The study also shows that FLT-PET is useful to measure impact of palbociclib CDK 4/6 inhibitor on proliferation while suggesting that FDG-PET is inadequate to predict therapy efficacy in this experimental context.

Based on ER degradation kinetic profile (in vitro data not shown), we first assessed in vivo quantitative changes of SAR439859-induced ER occupation/degradation following a single administration and imaging at 4 h time-point (in vitro, the maximum effect appeared at 4 to 8 h in mice bearing MCF7-Y537S xenografts). We found a dose-dependent reduction of FES-PET signal with SAR439859 treatment with a maximum [¹⁸F]-FES uptake reduction of 50% at the highest dose tested of 12.5 mg/kg compared to the control group. A limitation of our study is that the global level of [¹⁸F]-FES uptake is weak in the MCF7-Y537S xenograft model; The mean SUV is between 0.30 and 0.42, compared to clinical cases where SUV is greater than 1.5–2.0 for ER-positive tumors [14]. Consequently, the dose-response effect might be hampered due to the limit of quantification of our PET scanner. However, in our experimental paradigm, the analysis of estrogen receptor expression levels by immunohistochemistry corroborated non-invasive FES-PET imaging to evaluate the effects of SAR439859. These results correlate with efficacy data where MCF7-Y537S tumor regression was observed when treated over 3 weeks at 12.5 mg/kg dose. The in vivo efficacy data also confirmed that the dose of 12.5 mg/kg administered twice daily can be considered the optimal dose since upper dosages did not significantly improve the anti-tumor activity [9].

In order to investigate [¹⁸F]-FDG and [¹⁸F]-FLT as pharmacodynamic biomarkers and support their potential use in phase 1 clinical trial, we explored the effectiveness of each radiotracer to measure the early functional response to SAR439859. FDG-PET imaging revealed no significant reduction in [¹⁸F]-FDG uptake for SAR439859 treatment groups at any dose tested. Kurland et al. [25] in a clinical study showed that the combination of FES-PET and FDG-PET could be effective in predicting the progression-free survival of ER+ patients, the follow-up with FDG-PET imaging only was not informative enough to guide therapy selection and dosing. In our study, the utility of FDG-PET imaging in measuring therapeutic efficacy under SAR439859 was not demonstrated; Heidari et al. [26] also concluded that the metabolic impact of fulvestrant was not detectable with [¹⁸F]-FDG after 3 days of treatment. Although FES-PET imaging provides early evidence that SAR439859 is effective in triggering ERα receptor modifications, a longer drug exposure period would be required to monitor downstream functional impacts (glycolytic or proliferative). In a different tumor model, He et al. [27] demonstrated lack of early significant outcomes with [¹⁸F]-FDG in animals administered with fulvestrant over 3 weeks. These preclinical data do not support the use of [¹⁸F]-FDG as an early sensitive pharmacodynamic biomarker for selective estrogen receptor degrader compounds such as SAR439859.

Our studies demonstrate that dual targeting of ERα and CDK 4/6 inhibitor can induce marked inhibition of [¹⁸F]-FLT uptake; this observation is associated with an enhanced inhibition of tumor growth compared to that observed with either single agent. The PET radiotracer [¹⁸F]-FLT thus represents a promising proof-of-concept surrogate biomarker for SERD therapy in combination with palbociclib.

The [¹⁸F]-FLT uptake decreases early after initiating the combination treatment with the CDK 4/6 inhibitor palbociclib and SAR439859; FLT-PET imaging appears as the most relevant imaging biomarker for early efficacy assessment in our experimental conditions. However, SAR439859 when injected alone did not show any impact upon [¹⁸F]-FLT uptake at any dose tested; the limited treatment duration is probably the reason for such observation. When measured with the caliper (data not shown), oral administration of SAR439859 induced significant tumor regression at 12.5 mg/kg (bid) starting 7 days after first administration, this indicates that longer exposure time to the drug is necessary to trigger downstream anti-proliferative effect on the MCF7-Y537S tumor model. The anti-proliferative effect measured in our study has been correlated with other biomarkers of proliferation namely Ki-67 immunohistochemistry. This robust proliferation index is significantly decreased in groups of animals treated with palbociclib alone or in combination. The difference in FLT-PET imaging between combination and palbociclib alone did not reach statistical significance; however, Ki-67 IHC revealed significant differences between those 2 groups, the intensity of staining decreasing more in the combination group. The reason of this discrepancy between FLT-PET imaging and IHC results might be due to the way data are collected; FLT-PET imaging data are expressed as SUVmean, i.e., the average uptake across the whole tumor, while for Ki-67, information is obtained from a limited number of tumor slices which might not be representative of the whole tumor and could bias the quality of quantification [28]. In their study, Pio et al. [29] concluded that FLT-PET imaging would be a valuable tool for predicting long-term clinical outcomes for women with breast cancer treated with endocrine therapy. Moreover, the authors noticed that [¹⁸F]-FLT changes were more closely correlated with the later changes in other pharmacodynamic biomarkers or morphometric analysis than were early [¹⁸F]-FDG changes.

Numerous preclinical and clinical data suggest that combination of cyclin inhibitors with SERD would improve the anti-tumoral efficacy especially in advanced breast cancer expressing ESR1 mutations [30]. Therefore, FLT-PET imaging appears as a valuable nuclear imaging modality for early visualization and monitoring of response to a combined targeted therapy [31]. Incorporating PET imaging into the development process of a new SERD therapeutic is highly relevant; recently Wang et al. [19] were able to determine the dosage of GDC-0810 to achieve 90% of ER+ receptors occupancy using FES-PET imaging; the authors concluded that patient dose selection could be carried out with FES-PET imaging. However, in this paper the authors did not present data of drug efficacy; a predictive biomarker such as FLT-PET imaging would have been of major interest for parallel assessment of treatment response.

As another illustration of proof-of-concept studies for investigation of compound efficacy with nuclear molecular imaging, it further suggests that preclinical imaging can provide important information on drug dosing and regimen which is relevant for clinical design [32]. The paradigm of implementing several PET tracers in order to monitor the tumor characteristics of each patient is of great promise; this remains a challenging objective, the achievement of which would improve the therapeutic management of patients [33, 34].

In our preclinical experiments, target engagement measured with [¹⁸F]-FES-PET showed that the magnitude of uptake reduction is correlated with the administered dose of SAR439859. Moreover, [¹⁸F]-FES uptake correlated well with immunohistochemical scoring for ERα. As a biomarker of efficacy [¹⁸F]-FDG-PET was not adequate for early prediction and monitoring of therapy response since glucose metabolism may not be directly affected by endocrine therapy. In mice bearing subcutaneous MCF7-Y537S mutant ERα+ breast cancer model, [¹⁸F]-FLT effectively measures proliferation changes after the administration of the CDK 4/6 palbociclib inhibitor alone or in combination with SAR439859. In our study, [¹⁸F]-FES-PET is a valuable tool to non-invasively assess target engagement of SAR439859, allowing a quantitative assessment of ERα degradation.