Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

Background Endocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility. Methods A panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies. Results Here, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001. Conclusions Our data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER

Src Is a Potential Therapeutic Target in Endocrine-Resistant Breast Cancer Exhibiting Low Estrogen Receptor-Mediated Transactivation.

Despite the effectiveness of endocrine therapies in estrogen receptor positive (ER+) breast cancer, approximately 40% of patients relapse. Previously, we identified the Focal-adhesion kinase canonical pathway as a major contributor of resistance to estrogen deprivation and cellular-sarcoma kinase (c-src) as a dominant gene in this pathway. Dasatinib, a pan-src inhibitor, has recently been used in clinical trials to treat ER+ patients but has shown mixed success. In the following study, using isogenic cell line models, we provide a potential explanation for these findings and suggest a sub-group that may benefit. A panel of isogenic cell lines modelling resistance to aromatase inhibitors (LTED) and tamoxifen (TAMR) were assessed for response to dasatinib ± endocrine therapy. Dasatinib caused a dose-dependent decrease in proliferation in MCF7-TAMR cells and resensitized them to tamoxifen and fulvestrant but not in HCC1428-TAMR. In contrast, in estrogen-deprived conditions, dasatinib increased the proliferation rate of parental-MCF7 cells and had no effect on MCF7-LTED or HCC1428-LTED. Treatment with dasatinib caused a decrease in src-phosphorylation and inhibition of downstream pathways, including AKT and ERK1/2 in all cell lines tested, but only the MCF7-TAMR showed a concomitant decrease in markers of cell cycle progression. Inhibition of src also caused a significant decrease in cell migration in both MCF7-LTED and MCF7-TAMR cells. Finally, we showed that, in MCF7-TAMR cells, in contrast to tamoxifen sensitive cell lines, ER is expressed throughout the cell rather than being restricted to the nucleus and that treatment with dasatinib resulted in nuclear shuttling of ER, which was associated with an increase in ER-mediated transcription. These data suggest that src has differential effects in endocrine-resistant cell lines, particularly in tamoxifen resistant models, with low ER genomic activity, providing further evidence of the importance of patient selection for clinical trials testing dasatinib utility in ER+ breast cancer

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance

Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer.

BACKGROUND:Endocrine therapies are still the main strategy for the treatment of oestrogen receptor-positive (ER+) breast cancers (BC), but resistance remains problematic. Cross-talk between ER and PI3K/AKT/mTORC has been associated with ligand-independent transcription of ER. We have previously reported the anti-proliferative effects of the combination of everolimus (an mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape via AKT signalling can lead to resistance; therefore, the use of dual mTORC1/2 inhibitors has met with significant interest. METHODS:To address this, we tested the effect of vistusertib, a dual mTORC1 and mTORC2 inhibitor, in a panel of endocrine-resistant and endocrine-sensitive ER+ BC cell lines, with varying PTEN, PIK3CA and ESR1 mutation status. End-points included proliferation, cell signalling, cell cycle and effect on ER-mediated transcription. Two patient-derived xenografts (PDX) modelling endocrine resistance were used to assess the efficacy of vistusertib, fulvestrant or the combination on tumour progression, and biomarker studies were conducted using immunohistochemistry and RNA-seq technologies. RESULTS:Vistusertib caused a dose-dependent decrease in proliferation of all the cell lines tested and reduced abundance of mTORC1, mTORC2 and cell cycle markers, but caused an increase in abundance of EGFR, IGF1R and ERBB3 in a context-dependent manner. ER-mediated transcription showed minimal effect of vistusertib. Combined therapy of vistusertib with fulvestrant showed synergy in two ER+ PDX models of resistance to endocrine therapy and delayed tumour progression after cessation of therapy. CONCLUSIONS:These data support the notion that models of acquired endocrine resistance may have a different sensitivity to mTOR inhibitor/endocrine therapy combinations

Src Is a Potential Therapeutic Target in Endocrine-Resistant Breast Cancer Exhibiting Low Estrogen Receptor-Mediated Transactivation.

Despite the effectiveness of endocrine therapies in estrogen receptor positive (ER+) breast cancer, approximately 40% of patients relapse. Previously, we identified the Focal-adhesion kinase canonical pathway as a major contributor of resistance to estrogen deprivation and cellular-sarcoma kinase (c-src) as a dominant gene in this pathway. Dasatinib, a pan-src inhibitor, has recently been used in clinical trials to treat ER+ patients but has shown mixed success. In the following study, using isogenic cell line models, we provide a potential explanation for these findings and suggest a sub-group that may benefit. A panel of isogenic cell lines modelling resistance to aromatase inhibitors (LTED) and tamoxifen (TAMR) were assessed for response to dasatinib ± endocrine therapy. Dasatinib caused a dose-dependent decrease in proliferation in MCF7-TAMR cells and resensitized them to tamoxifen and fulvestrant but not in HCC1428-TAMR. In contrast, in estrogen-deprived conditions, dasatinib increased the proliferation rate of parental-MCF7 cells and had no effect on MCF7-LTED or HCC1428-LTED. Treatment with dasatinib caused a decrease in src-phosphorylation and inhibition of downstream pathways, including AKT and ERK1/2 in all cell lines tested, but only the MCF7-TAMR showed a concomitant decrease in markers of cell cycle progression. Inhibition of src also caused a significant decrease in cell migration in both MCF7-LTED and MCF7-TAMR cells. Finally, we showed that, in MCF7-TAMR cells, in contrast to tamoxifen sensitive cell lines, ER is expressed throughout the cell rather than being restricted to the nucleus and that treatment with dasatinib resulted in nuclear shuttling of ER, which was associated with an increase in ER-mediated transcription. These data suggest that src has differential effects in endocrine-resistant cell lines, particularly in tamoxifen resistant models, with low ER genomic activity, providing further evidence of the importance of patient selection for clinical trials testing dasatinib utility in ER+ breast cancer

Abstract P4-04-09: New oral SERD elacestrant (RAD1901) shows efficacy in breast cancer models harbouring ESR1 mutations and enhances the antiproliferative activity of mTORC1 and CDK4/6 inhibitors

Abstract Background: Targeting estrogen receptor (ER) signalling is the main therapeutic option for ER+ breast cancer (BC). However, over 30% of patients relapse with endocrine resistance emphasising the need for improved therapeutic strategies. The prevalence of ESR1 mutations in relapsed tumours highlights the sustained reliance on ER signalling, rationalising continued targeting of ER. Unlike other endocrine therapies such as aromatase inhibitors (AI) and tamoxifen, selective ER degraders (SERDs) are competitive ER antagonists, that induce a conformational change in ER resulting in ubiquitination and degradation via the proteasomal pathway. Fulvestrant has shown clinical utility in advanced BC but is limited by its pharmaceutical properties highlighting the need for SERDs with enhanced bioavailability and pharmacokinetic properties. Here, we show that elacestrant (RAD1901) an orally bioavailable SERD, has antitumor activity in endocrine sensitive and resistant models of ER+ BC. Furthermore, elacestrant enhances the efficacy of mTORC1 inhibitor, everolimus and CDK4/6 inhibition, in model systems. Methods: Several ER+ BC lines adapted to long-term E deprivation (LTED) and harbouring wild-type or a naturally occurring ESR1 mutation, were treated with elacestrant or fulvestrant +/- estradiol (E2). Effects on cell proliferation, cell signalling, cell cycle, transcription, ER protein stability and ER genomic binding were assessed. Efficacy in combination with everolimus, palbociclib and abemaciclib was also evaluated. Results: Cell proliferation assays in 2D and spheroids in the presence of 0.01nM E2 showed a concentration-dependent decrease in proliferation in response to elacestrant and fulvestrant. GI50 values for elacestrant in general were 10-fold higher than fulvestrant but equated to doses that are clinically achievable for the drug. Most importantly, elacestrant suppressed proliferation of two LTED models harbouring ESR1 mutations, MCF7 LTEDY537C (GI50 5nM) and SUM44-LTEDY537S (GI50 100nM). GI50 values of elacestrant and fulvestrant showed similar reduction of ER, progesterone receptor (PGR) and cyclinD1 together with decreased phosphorylation of retinoblastoma (RB), concordant with cell cycle arrest. Chromatin immunoprecipitation (ChIP) for ER in response to elacestrant or fulvestrant showed a reduction in recruitment of ER to TFF1, GREB1 and PGR promoters and concomitant reduction in mRNA expression of these genes in the presence of E2. Elacestrant-mediated ER depletion was dependent on the 26S proteasome, as addition of the proteasome inhibitor MG132, fully blocked elacestrant depletion of ER, similar to the effect of MG132 treatment in preventing fulvestrant-mediated ER turnover. The combination of elacestrant with CDK4/6 inhibitors, palbociclib or abemaciclib, demonstrated additivity compared with monotherapy. In addition, elacestrant inhibited growth of palbociclib-resistant MCF7 LTED cells. Conclusion: These findings highlight the potential utility of elacestrant as a 1st or 2nd line drug in the treatment of ER+ BC. The results support the testing of elacestrant versus fulvestrant after relapse on an AI, either alone or in combination with a CDK4/6 inhibitor. Citation Format: Martin L-A, Pancholi S, Simigdala N, Nikitorowicz-Buniak J, Ribas R, Garner F, Bihani T, Dowsett M. New oral SERD elacestrant (RAD1901) shows efficacy in breast cancer models harbouring ESR1 mutations and enhances the antiproliferative activity of mTORC1 and CDK4/6 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-09.</jats:p

Abstract P3-03-09: Resistance to palbociclib depends on multiple targetable mechanisms highlighting the potential of drug holidays and drug switching to improve therapeutic outcome

Abstract Background: Combination of CDK4/6 inhibitors and endocrine therapy has been shown to improve clinical outcome in advanced estrogen receptor-positive (ER+) breast cancer (BC) patients. However, most patients will progress with acquired resistance, making the identification of new therapeutic strategies and potential biomarkers of great importance. Here, we show for the first time that certain resistance mechanisms are amenable to drug holidays and drug switching while others cause irreversible resistance to further perturbation of G1/S and highlight the potential of targeting these tumours by blockade of G2/M with inhibitors of Wee1 or CDK7. Methods: To identify adaptive mechanisms associated with resistance to palbociclib, MCF7 and T47D cells adapted to long term oestrogen deprivation (LTED), which either retain or lose expression of ER respectively, were grown in the presence of palbociclib until they became resistant (LTED991R). Cell lines were subjected to exome sequencing, global gene expression analysis and siRNA knockdown of 709 cellular kinases to identify candidates associated with resistance. Candidate drug targets were evaluated in proliferation assays. Data was validated in vivo using MCF7-LTED xenografts. Results: Exome data showed few genetic changes were associated with resistance to palbociclib. MCF7-LTED991R had a copy number (CN) gain of CCNE2 and ESR1 compared to MCF7-LTED while T47D-LTED991R showed loss of RB. Global gene expression analysis revealed increased expression of CDK4, CDK2, CDK7 and CCNE1 in MCF7-LTED991R and CCNE2 and CDK2 in T47D-LTED991R. In order to assess if these changes were drug specific, cell lines were treated with another CDK4/6 inhibitor, abemaciclib. Strikingly, MCF7-LTED991R cells showed sensitivity, possibly as a result of increased CDK4 expression, for which abemaciclib has increased potency. We next assessed the plasticity of the 991R phenotype by giving the cells a drug holiday followed by re-challenge. T47D-LTED991R cells remained resistant whilst the MCF7-LTED991R cells were re-sensitised, an observation we confirmed in a MCF7-LTED xenograft after long term treatment with palbociclib. Finally, siRNA kinome knockdown highlighted CDK4, CDK7 and Wee1 as associated with the resistant phenotypes suggesting targeting of G2/M in both RB+ and RB- 991R tumours may provide benefit; this possibility was confirmed by increased sensitivity to THZ1 (CDK7 inhibitor) or MK1775 (Wee1 inhibitor). Conclusion: Few genetic changes are associated with resistance to palbociclib in ER+ BC in vitro but kinase re-wiring provides resistance. RB loss of function appears an irreversible mechanism of resistance while gain of cyclin E, and overexpression of CDK2 and CDK4 are amenable to a drug holiday, leading to re-sensitisation to palbociclib in vitro and in vivo. Of note, the palbociclib-resistant cells that retained RB expression were sensitive to abemaciclib possibly as a result of increased expression of CDK4 acting as compensatory mechanism. Finally, LTED cell lines retaining ER that were resistant to palbociclib also showed a gain in ESR1 CN, highlighting the potential for combination therapy with an alternate endocrine agent, such as fulvestrant. Citation Format: Martin L-A, Pancholi S, Ribas R, Gao Q, Simigdala N, Nikitorowicz-Buniak J, Johnston SR, Dowsett M. Resistance to palbociclib depends on multiple targetable mechanisms highlighting the potential of drug holidays and drug switching to improve therapeutic outcome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-09.</jats:p

Abstract P1-09-03: Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors

Abstract Background Estrogen-receptor positive (ER+) breast cancer (BC) accounts for over 75% of diagnosed cases. Despite treatment, a large proportion relapse with de novo or acquired endocrine resistant disease, making it one of the greatest challenges for BC research. Using a kinome siRNA library screen, we identified MPS1 that is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to endocrine therapy and palbociclib. Until now, the target population for MPS1 inhibitors has focused on triple negative BC. Our unexpected finding shows, potential efficacy of MPS1 inhibitors in ER+ BC resistant to endocrine therapies and palbociclib, a prominent contemporary first-line combination for advanced disease. Methods ER+ BC cell lines (MCF7, SUM44, ZR75.1, HCC1428 and T47D) adapted to estrogen independent growth (LTED) and sequential resistance to palbociclib (991R) were subjected to a siRNA screen targeting 709 kinases. Z-scores were used to identify the most robust candidates. Cell viability upon MPS1 inhibition with CCT289346 (MPS1i) was assessed 2D and 3D. The class effect was confirmed with other compounds targeting MPS1. Impact of MPS1i on ER co-localisation and ER-transactivation was assessed using confocal microscopy and reporter assays, respectively. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. BrdU incorporation and cell cycle were assessed by FACS. PARP cleavage was used to measure apoptosis. Global gene expression analysis of MPS1 was carried out in two independent neoadjuvant studies of aromatase inhibitor (AI) treated patients. Results Kinome knockdown identified targets associated with the G2/M checkpoint as strongly implicated in the LTED phenotype. In particular, MPS1 was the top common hit in LTED and 991R cell lines. Increase in MPS1 was evident in MCF7-LTED at both the transcript and protein level. Notably, the MPS1 inhibitor CCT289346 caused a significant reduction in viability of the majority of LTED and 991R cell lines tested. Table. Sensitivity of ER+ BC cell lines to CCT289346Cell LineModelIC50 nMMCF7Wt90 LTEDESR1wt80 LTEDESR1Y537C50 TAMR55 ICIR50 LTEDICIR60 991R40 LTED991R45 LTED991R-ICIR25T47DWt110 LTED110 991R50 LTED991R60SUM44LTED40 Upon inhibition of MPS1, cells demonstrated shorter time in mitosis, aberration of cell cycle and amplified mitotic errors, resulting in increased apoptosis. To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoadjuvant AI therapy. In the anastrozole cohort, on-treatment gene expression of MPS1 (p&amp;lt;0.0001) was significantly associated with poor response to anastrozole based on a 2-week residual Ki67 score &amp;lt;10%. In the letrozole cohort, increased on-treatment expression of MPS1 (p=0.0118) was associated with poor response based of tumor shrinkage ≥50%. Conclusion This novel finding shows MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC models resistant to endocrine therapy and palbociclib providing a new therapeutic strategy. Citation Format: Nikitorowicz-Buniak J, Pancholi S, Simigdala N, Ribas R, Linardopoulos S, Dowsett M, Johnston SR, Martin L-A. Global knockdown of cellular kinases identifies MPS1 as a novel modulator of endocrine and palbociclib resistance highlighting a new role for MPS1 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-03.</jats:p