ER+ breast cancers depend on ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents, providing a rationale for further optimization and development of ER-targeting agents. Fulvestrant is unique amongst currently approved ER ligand therapeutics due to its classification as a full ER antagonist, which is thought to be achieved through degradation of ERα protein. However, the full clinical potential of fulvestrant is believed to be limited by poor physiochemical properties and exposure limitations due to its administration by intramuscular injection. Strategies to generate orally bioavailable molecules that retain fulvestrant's full antagonist profile but with considerably improved drug-like properties are thus being widely employed to identify next generation ER therapeutics. However, we find that therapeutic candidates that have recently emerged from prospective optimization of ER degradation, including GDC-0810 and GDC-0927, are not mechanistically equivalent. GDC-0810, GDC-0927, and fulvestrant display unique profiles in terms of ER degradation, transcriptional phenotypes and anti-proliferative potential across a panel of ER+ breast cancer cell lines. In HCI-011 (ER.WT) and HCI-013 (ER.Y537S) ER+ patient-derived breast cancer xenograft (PDX) models, GDC-0927 achieves more robust transcriptional suppression of ER than GDC-0810, and also and greater efficacy. Although displaying a more desirable mechanistic profile than GDC-0810, GDC-0927 has more rapid clearance and poor oral bioavailability, leading to a high pill burden and potential exposure limitation. Here, we describe for the first time GDC-9545, in which the distinct liabilities of GDC-0810, GDC-0927 and fulvestrant are addressed. GDC-9545 is a non-steroidal ER ligand that is highly potent in competing with estradiol for binding and in driving an antagonist conformation within the ER ligand binding domain. Like fulvestrant, and displaying some improvements over GDC-0927, GDC-9545 consistently induces ER turnover and drives deep transcriptional suppression of ER, resulting in robust in vitro anti-proliferative activity. GDC-9545 exhibits reduced metabolism and increased oral bioavailability relative to GDC-0927, resulting in an overall improved oral exposure in multiple species. As a result of both its mechanistic pharmacology and improved oral exposure, GDC-9545 can achieve the same degree of anti-tumor activity as GDC-0927 but at 100-fold lower doses in the HCI-013 PDX model. The in vivo efficacy of GDC-9545 in this model is greater than GDC-0810 and fulvestrant at clinically relevant exposures. The highly potent in vivo efficacy of GDC-9545 likely arises due to the particular combination of high binding potency, full suppression of ER signaling, and an improved DMPK profile when compared to GDC-0927 and fulvestrant. GDC-9545 is currently being evaluated in Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT03332797). Citation Format: Metcalfe C, Ingalla E, Blake RA, Chang J, Daemen A, De Bruyn T, Giltnane JM, Guan J, Hafner M, Hartman S, Kategaya L, Kleinheinz T, Liang J, Mody V, Nannini M, Oeh J, Ubhayakar S, Wertz I, Young A, Zbieg J, Zhou W, Sampath D, Friedman LS, Wang X. GDC-9545: A novel ER antagonist and clinical candidate that combines desirable mechanistic and pre-clinical DMPK attributes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-07.

ER+ breast cancers can depend on ER signaling throughout disease progression, including after acquired resistance to existing endocrine agents, providing a rationale for further optimization and development of ER-targeting agents. Fulvestrant is unique amongst currently approved ER ligand therapeutics due to classification as a full ER antagonist, which is thought to be achieved through degradation of ER protein. However, the full clinical potential of fulvestrant is believed to be limited by poor bioavailability, spurring attempts to generate ligands capable of driving ER degradation but with improved drug-like properties. Here, we evaluate three ER ligand clinical candidates that recently emerged from prospective optimization of ER degradation – GDC-0810, AZD9496 and GDC-0927 - and show that they display distinct mechanistic features. GDC-0810 and AZD9496 are more limited in their ER degradation capacity relative to GDC-0927 and fulvestrant, display evidence of weak transcriptional activation of ER in breast cancer cells (i.e. partial agonist activity), and do not achieve the same degree of in vitro anti-proliferative activity as GDC-0927 and fulvestrant. In the HCI-013 (ER.Y537S) and HCI-011 (ER.WT) ER+ patient-derived xenograft models, GDC-0927 drives greater transcriptional suppression of ER, and greater anti-tumor activity relative to GDC-0810. We found that despite their full antagonist phenotype, GDC-0927 and fulvestrant promote association of ER with DNA, including at canonical ERE motifs, prior to ER degradation. Interestingly however, integration of ER ChIP-Seq and ATAC-Seq data revealed that ER complexed with fulvestrant or GDC-0927 fails to increase chromatin accessibility at DNA binding sites, in contrast to partial agonists which result in increased chromatin accessibility at ER binding sites. Thus, although ER contacts DNA when engaged with fulvestrant and GDC-0927, it is functionally inert. To further explore mechanistic features that might account for the differential activity of full antagonists and partial agonists that occurs prior to ER degradation, we used cell-based florescence recovery after photobleaching (FRAP) to measure the kinetics of ER diffusion within the nucleus. We demonstrate that while ER is generally highly mobile, including after engagement with GDC-0810 and AZD9496, GDC-0927 and fulvestrant immobilize intra-nuclear ER. A site saturating mutagenesis screen revealed a series of novel ER mutations that prevent ER immobilization by fulvestrant and GDC-0927. This class of “always mobile” ER variants promotes an antagonist-to-agonist transcriptional switch for fulvestrant and GDC-0927, and simultaneously prevents ER degradation by these molecules, implying that ER immobilization is a key functional determinant of robust transcriptional suppression. We thus propose that ER degradation is not a driver of full ER antagonism, but rather a downstream consequence of ER immobilization, occurring after a suppressive phenotype has been established at chromatin. We additionally argue that evaluating the transcriptional output of candidate ER therapeutics, both pre-clinically and clinically, will be critical for the identification of ER ligands with best-in-class potential. Citation Format: Metcalfe C, Zhou W, Guan J, Daemen A, Hafner M, Blake RA, Ingalla E, Young A, Oeh J, De Bruyn T, Ubhayakar S, Chen I, Giltnane JM, Li J, Wang X, Sampath D, Hager JH, Friedman LS. Prospective optimization of estrogen receptor degradation yields ER ligands with variable capacities for ER transcriptional suppression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS3-05.

Janus-kinase 2 (JAK2) is a receptor-coupled tyrosine kinase which transmits cytokine-mediated signals to the STAT pathway to drive signals of proliferation and differentiation. JAK2/STAT signaling has been shown to play a role in promoting breast cancer ‘stem-ness’ and driving the proliferation of CD44+/CD24- basal-like breast cancer cells. We performed targeted next-generation sequencing (tNGS) using HiSeq2000 at a CLIA-certified laboratory on 68 residual TNBCs after neoadjuvant chemotherapy (NAC), which are putatively enriched for drug-resistant cells. JAK2 amplifications were identified in 7/68 (10.2%) post-NAC tumors. Gene copy estimation in amplified cases ranged from 7-10 copies. All cases were confirmed by a novel JAK2-FISH assay that we developed using BAC-derived probes targeting the JAK2 locus on chromosome 9, using CEN9 as a normalization factor. Using JAK2-FISH, we identified only one amplified tumor in an independent cohort of 30 untreated TNBCs (3%). Rates of JAK2 amplification in post-NAC residual cancers in this study were also higher than those reported by TCGA (<1%), although their methodology differed. However, low level gains were more commonly identified in basal-like (46%) vs. luminal A/B tumors (4%) in the TCGA cohort, suggesting a subtype-specific copy number alteration pattern in breast cancer. Furthermore, drawing from a large database of breast tumors sequenced by identical methods, the frequency of JAK2 amplifications was 0.9%; the majority of these were reported as metastatic tumors, post-systemic therapy. These data suggest that JAK2 amplifications are enriched in frequency in drug-resistant and potentially metastatic TNBCs. JAK2 amplifications in the post-NAC residual cancer were associated with poor RFS (median: 7 mo. vs 17 mo; HR: 3.36; p = 0.006) and OS (median: 11.3 mo. vs 27.6 mo; HR: 4.16; p = 0.002), and were significantly associated with higher gene expression of IL6, CXCR1 and SNAI1, among others. The coexistence of these alterations suggests an association of amplified JAK2 with an epithelial-to-mesenchymal transition and cancer-stem cell programs. To explore the clonal evolution of JAK2 lesions in breast cancer, we sequenced sequential samples (diagnostic biopsy, post-NAC residual disease and metastatic recurrence) of two TNBCs where JAK2 amplifications were detected in the residual disease. In each of these, JAK2 gain was noted at levels below the amplification threshold at diagnosis, but was focally amplified in both the residual tumor at the time of definitive surgery as well as in the subsequent metastatic recurrence. To our knowledge, this is the first report of JAK2 gene amplification in breast cancer. These data suggest a role for JAK2 amplifications in driving chemotherapeutic resistance and disease progression. We hypothesize these lesions are therapeutically targetable with currently approved JAK2 or pan-JAK inhibitors. Additional data to be presented will include analysis of tumor-infiltrating lymphocytes in JAK2-amplified tumors and molecular studies of the role of JAK2 overexpression in breast cancer cell lines as well as their sensitivity to JAK2 inhibitors in clinical development. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-01.

Background: Estrogen receptor (ER)-positive breast cancers (BC) initially respond to antiestrogens but eventually become hormone-independent and recur. FGFR1 is amplified in ∼10% of ER+ BC and is associated with early recurrence on antiestrogen therapy. Notably, one third of FGFR1-amplified tumors have simultaneous amplification of CCND1, FGF3, FGF4 and FGF19 on chromosome 11q12-14. Herein, we investigated the mechanisms by which FGFR1 amplification confers resistance to antiestrogen therapy in ER+ BC cells. Results: We performed whole exome sequencing in tumor biopsies from 130 patients with an operable ER+/HER2- BC who had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log (ln) of post-letrozole Ki67 as sensitive (ln ≤1 or ≤2.7% Ki67+ cells; n=68) or resistant (ln ≥2 or ≥7.4%; n=18). We found amplifications in FGFR1 and/or 11q12-14 in 6/11 (55%) resistant tumors compared with 5/34 (15%) in sensitive tumors (p=0.006); all cases were confirmed by FGFR1-fluorescence in situ hydridization (FISH). Resistant tumors with FGFR1 and/or 11q12-14-amplification showed a marked increase in nuclear FGFR1 with letrozole. ER+/FGFR1-amplified CAMA1 and MDA134 cell lines also exhibited co-localization of ER and FGFR1 in the nucleus. Cell proliferation was partially reduced by estrogen deprivation, and FGFR1 siRNA further reduced cell growth in hormone-depleted medium. We generated CAMA1 and MDA134 cells resistant to long-term estrogen deprivation (LTED). These cells exhibited overexpression of FGF3/4/19 and ERα with a concomitant increase in ligand-independent ER transcriptional activity and growth. An ER-FGFR1 interaction was observed in the nucleus and cytosol of CAMA1 parental cells with enhanced interaction in CAMA1 LTED cells. Genetic (with siRNA) and pharmacologic (with lucitinib) inhibition of FGFR1 reduced a) nuclear localization of FGFR1; b) ER transcriptional activity; and c) cell proliferation. Nuclear localization and ER-FGFR1 interaction were disrupted by a kinase-deficient FGFR1. Conversely, addition of FGF3 ligand stimulated ER-FGFR1 interaction and ER transcriptional activity, suggesting FGFR activation can regulate ER function. Inhibition of FGF receptor-specific substrate (FRS2), a principal mediator of FGFR1 signal transduction to the MAPK and PI3K pathways, with siRNA or pharmacologic inhibition of PI3K with buparlisib or MEK with GSK1120212 did not reduce ER transcriptional activity suggesting that, in ER+/FGFR1-amplified cancer cells, ER function is not modulated by FGFR signal transducers. Finally, using chromatin immunoprecipitation (ChIP) we showed that FGFR1 binds directly to estrogen response elements (ERE). This association was reduced with lucitanib. We are currently investigating genes modulated by ER/FGFR1 in ER+ BC and the in vivo anti-tumor efficacy of dual inhibition of FGFR1 and ER in ER+/FGFR1-amplified patient-derived breast cancer xenografts. Conclusions: These data support a critical role of ER and FGFR1 interaction in endocrine resistance in ER+/FGFR1-amplified breast cancer. Targeting of FGFR1 in combination with antiestrogens may abrogate resistance to endocrine therapy in these tumors and is worthy of clinical investigation. Citation Format: Formisano L, Young CD, Bhola NE, Bulen B, Estrada VM, Wagle N, Van Allen E, Red Brewer ML, Jansen VM, Guerrero AL, Giltnane JM, Strcker T, Arteaga CL. Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-03.