ER Target Research Articles

SPTLC1 Mutations Associated with Early Onset Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by selective degeneration of lower and upper motor neurons leading to progressive muscle weakness, swallowing difficulties, and respiratory insufficiency that ultimately causes death usually within 2–5 years of diagnosis. The majority of ALS cases occur sporadically, but a significant number (>10%) display Mendelian inheritance. Very recently, four unique de novo or dominantly inherited SPTLC1 mutations associated with early childhood‐onset ALS without clinical sensory involvement have been identified in eight independent families. SPTLC1 encodes a subunit of serine palmitoyltransferase (SPT), the committed and rate‐limiting enzyme of sphingolipid synthesis. The ALS mutations flank the first membrane spanning domain of SPTLC1, which is not required for ER targeting, association with the SPTLC2 and ssSPT subunits, or enzymatic activity. Rather, this domain is critical for binding of the ORMDL proteins that negatively regulate SPT. This suggested that the SPTLC1 mutations might interfere with homeostatic regulation of SPT and that elevated de novo sphingolipid synthesis could underlie the ALS disease pathology. Our studies reveal that these highly penetrant SPTLC1 mutations do indeed abrogate negative regulation of SPT by the ORMDL proteins and result in elevated levels of sphingolipids. Using cultured cells, patient fibroblasts, iPSC‐induced MNs, and mouse models, we are investigating the importance of ORMDL regulation in the maintenance of sphingolipid homeostasis and the mechanisms responsible for sphingolipid mediated motor neuron death. Significantly, the ALS SPTLC1 mutations confer disease pathology by a distinctly different mechanism than the previously characterized SPTLC1 and SPTLC2 mutations associated with hereditary sensory and autonomic neuropathy type 1 (HSAN1). Whereas the ALS mutations result in elevated levels of canonical sphingolipids, the HSAN1 mutations, which compromise amino acid substrate selectivity of SPT, cause accumulation of atypical deoxy‐sphingoid bases that are implicated in neuronal cell death.

Abstract P3-06-08: Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer

Abstract Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for the treatment of estrogen receptor positive (ER+) metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: p21-activated kinase 1 (PAK1) is a serine/threonine kinase amplified in several human cancer types. Specifically, the survey of TCGA for copy number alterations and/or expression showed PAK1 amplification and/or overexpression in ~23% of ER+ breast cancer. To test the hypothesis that PAK1-amplification is related to CDK4/6 inhibitors resistance, we generated two ER+ breast cancer cell lines T47D (harbouring PAK1 amplification) and MCF7 (non PAK1-amplified) resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor), namely T47DFAR and MCF7FAR. T47DFAR and MCF7FAR were obtained, exposing the parental cells to increasing doses of drugs. Interestingly, only the T47DFAR but not the MCF7FAR showed higher level of PAK1 expression and activation compared to the parental cell line. PAK1 pathway activation was evaluated measuring by immunoblot the levels of phosphorylation of PAK1, MEK, ERK and Beta-Catenin. Moreover, the genomic knockdown of PAK1 through RNAi causes a decrease in cell proliferation of T47DFAR, but not of MCF7FAR and parental cells, suggesting the role of PAK1 as a possible mediator of drug resistance. Conversely, overexpression of PAK1 in MCF7 and T47D parental cells was able to generate resistance to the drug combination. PAK-1 has shown to have a different role in the cytosol as pathways mediator and in the nucleus as transcriptional factor. Our data obtained by subcellular fractionation showed that after treatment with fulvestrant-abemaciclib, PAK1 protein accumulates into the cytosol in T47D-FAR compared to parental T47D, probably mediating an enhancement in cytoplasmatic cell signalling and pathway activation. To univocally demonstrate that PAK1 mediates tumor resistance to fulvestrant-abemaciclib combination, we are generating data from RNA-Seq and whole exome sequencing from T47DFAR and MCF7FAR and the relative parental cell lines. Also, we will assess the efficacy of PAK pharmacological inhibition in overcoming such resistance. Conclusions: In conclusion, our data suggest the role of PAK1 as a novel therapeutic target in ER+/PAK1-amplified breast cancer model resistant to endocrine therapy and CDK4/6 inhibitors. Citation Format: Stefania Belli, Alberto Servetto, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Fabiana Napolitano, Antonio Santaniello, Pietro De Placido, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Roberto Bianco, Luigi Formisano. Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-08.

Abstract P6-04-04: Functional characterization of ESR1 fusions in breast cancer

Abstract Background: We recently reported two in-frame estrogen receptor alpha gene (ESR1) fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, in endocrine therapy (ET)-refractory, metastatic estrogen receptor positive (ER+) breast cancer patients. Both fusions were causal in driving ET resistance and metastasis in experimental models. CDK4/6 inhibition suppressed tumor growth driven by ESR1 fusions, but did not induce tumor regression (Lei et al., Cell Rep., 2018). Additional ESR1 fusions with diverse partner genes have been recently reported (ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9) (Hartmaier et al., Ann Oncol., 2018), or communicated to us (ESR1-ARNT2, ESR1-PCMT1 and ESR1-ARID1B) (Dr. Dan R. Robinson, University of Michigan). All were identified in ET-refractory tumors. We therefore undertook detailed functional characterization of these new ESR1 fusions to examine their role as disease drivers. We also used mass spectrometry-based kinomics to identify therapeutic targets induced by individual ESR1 fusions. Methods: Unreported ESR1 fusions were identified by RNA sequencing followed by informatics designed to identify fusion gene reads. Stable ER+ breast cancer cell lines (T47D and MCF7) expressing in-frame ESR1 fusion proteins were generated by lentiviral transduction. ET-resistant cell proliferation was assessed in low estradiol medium and/or in the presence of anti-estrogen. Expression of estrogen responsive and epithelial-to-mesenchymal transition (EMT) genes were measured by mRNA-qPCR. Cell motility driven by these fusions was examined using scratch wound assay. Kinase expression patterns were examined using a Kinase Inhibitor Pulldown (KIP) mass spectrometry-based assay. Results: All the fusions we studied contain the first 6 exons (ESR1-e6) of ESR1 that provides the first 4 coding exons. In-frame C-terminal sequences were provided by different partner genes. In addition to previously described ESR1-YAP1 and ESR1-PCDH11X fusions, ESR1-SOX9 and ESR1-ARNT2 conferred estrogen-independent and fulvestrant-resistant growth in stable ER+ breast cancer cells (T47D and MCF7). In T47D cells, these ESR1 fusions upregulated not only classical ER targets, including GREB1, PGR, and TFF1, but also EMT-associated genes, SNAI1 (Snail) and VCAN in an estradiol-independent, fulvestrant-resistant manner. In addition, transcriptionally active ESR1 fusions promoted cell motility. KIP profiling of T47D cells expressing transcriptionally active ESR1 fusions and a patient-derived xenograft naturally harboring the ESR1-YAP1 fusion revealed an increase in abundance of multiple receptor tyrosine kinases including RET and insulin like growth factor 1 receptor (IGF1R). Responses to targeting these kinases have been observed in vitro. Other ESR1-e6 fusions produced stable proteins (ESR1-DAB2, ESR1-GYG1, ESR1-PCMT1, ESR1-ARID1B) but had much less clear transcriptional phenotypes and fewer upregulated kinases identified by KIP. Conclusion: A classification of ESR1 fusions in ET-refractory breast cancer is emerging from this study. About half of ESR1-e6 fusions studied are transcriptionally active, presumably because the 3’ partner in these examples is encoded by transcription factor gene (TF) (SOX9 or ARNT2) or co-activator gene (CoA) (YAP1). These can be classified as ESR1-e6 TF/CoA fusions. The phenotype of the remaining fusions remains under investigation because of the functionally diverse nature of the 3’ partners and the low activity in functional assays. While some of these may be passengers, the example of PCDH11X suggests a non-TF/CoA partner ESR1-e6 fusion can still be active and further studies on additional fusions will be necessary. Downstream targeting of fusion-dependent kinases may be effective. Citation Format: Xuxu Gou, Jonathan T Lei, Sinem Seker, Beom-Jun Kim, Saif Rehman, Adrian V Lee, Dan R Robinson, Matthew J Ellis. Functional characterization of ESR1 fusions in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-04.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

The LncRNA HOTAIRM1 Promotes Tamoxifen Resistance by Mediating HOXA1 Expression in ER+ Breast Cancer Cells.

Breast cancer is one of the most commonly diagnosed cancers in women worldwide. Approximately 40% of patients with breast cancer acquire endocrine resistance following therapy with tamoxifen. Many explanations for the development of endocrine resistance have been put forward, one of them being the dysregulation of long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1, known to be involved in myelopoiesis as well as transcriptional regulation of the HOXA genes in embryonic stem cells, is also expressed in breast cancer cells. This study explored the molecular mechanisms of HOTAIRM1 involved in acquired tamoxifen resistance. We showed that HOTAIRM1 and HOXA1 are concurrently up-regulated in tamoxifen-resistant MCF7 (TAMR) cells. Knockdown of HOTAIRM1 down-regulated HOXA1 expression and restored sensitivity to tamoxifen. In addition, the knockdown of HOXA1 showed similar effects, suggesting that the HOTAIRM1/HOXA1 axis regulates tamoxifen resistance. Furthermore, we showed that HOTAIRM1 directly interacts with EZH2 and prevents the PRC2 complex from binding and depositing H3K27me3 on the putative promoter of HOXA1. Together, our findings suggest that HOXA1 and its neighboring lncRNA, HOTAIRM1, might serve as potential therapeutic targets for ER+ breast cancer patients who have acquired tamoxifen resistance.

J-Domain Proteins Promote Client Relay from Hsp70 During Tail-Anchored Membrane Protein Targeting

J-domain proteins (JDPs) play essential roles in assisting Hsp70 function in diverse aspects of protein biogenesis. Previous work showed that JDPs act by recruiting Hsp70 to client proteins, assisting Hsp70 in client trapping, and regulating the ATPase cycle of Hsp70. Here we report that JDPs can further enhance the conformational quality of Hsp70-bound client proteins during tail-anchored membrane protein (TA) targeting. In the guided-entry-of-tail-anchored protein (GET) pathway in yeast, newly synthesized TAs are captured and solubilized by the cytosolic Hsp70 Ssa1, which further initiates sequential TA handovers to downstream chaperones, Sgt2 and Get3, to enable their delivery to the endoplasmic reticulum. We found that two J-domain proteins (JDPs), Ydj1 and Sis1, function in parallel to support the ER targeting of TAs in vivo . Biochemical analyses showed that, while Ydj1 and Sis1 differ in their ability to assist Ssa1 in TA trapping, both JDPs enhance the transfer of Ssa1-bound TA substrate to Sgt2. The ability of the JDPs to remodel the conformation and ATPase activity of Ssa1, rather than their client binding activity, is essential for enhancing the transfer competence of Ssa1-bound TAs and for supporting TA insertion in vivo. Our results define the Hsp70 cochaperone requirement for the GET pathway and demonstrate a role of JDPs in improving the conformational quality of Hsp70-bound membrane protein clients.

Cystatin SN Affects Cell Proliferation by Regulating the ERα/PI3K/AKT/ERα Loopback Pathway in Breast Cancer.

BackgroundCystatin SN (CST1) has been reported to act as an oncogene in cancers, but its underlying mechanism remains unclear.MethodsWe performed Western blotting analyses to observe protein expression and conducted transwell invasion, wound healing, and colony formation assays to assess cell invasion, migration, and proliferation, respectively. We also performed cell cycle analyses by flow cytometry to determine the role of CST1 in the cell cycle. In vivo experiments used subcutaneous tumor models in BALB/c-nu athymic female mice to evaluate the effect of CST1 on tumor growth.ResultsWestern blotting analyses showed that CST1 was upregulated in ER+ breast cancer cells such as MCF7, T47D, and BT474. CST1 knockdown led to slower cell growth and inhibited the G1 to S phase transition in ER+ breast cancer cells. In vivo experiments showed that CST1 deletion inhibited tumor growth, and led to decreased expression of estrogen receptor α (ERα) and p-AKT. In vitro experiments showed that the over-expression of CST1 led to the upregulation of ERα, and inhibition of CST1 inhibited the expression of ERα. Western blotting analyses showed that CST1 regulated the activity of the PI3K/AKT signaling pathway in breast cancer cells. We confirmed that CST1 acted as an oncogene in ER+ breast cancer by regulating the ERα/PI3K/AKT/ERα loopback pathway.ConclusionCST1 acts as an oncogene in ER+ breast cancer, and CST1 contributes to cancer development by regulating the ERα/PI3K/AKT/ERα loopback pathway in ER+ breast cancer. Our findings indicate that CST1 could be a significant therapeutic target for ER+ breast cancer patients. Our discovery should inspire further studies on the role of CST1 in cancers.

340P - Dose-escalation study of G1T48, an oral selective estrogen receptor degrader (SERD), in postmenopausal women with ER+/HER2- locally advanced or metastatic breast cancer (ABC)

BackgroundThe SERD fulvestrant is a well-established therapy for ER+/HER2- ABC. However, its intramuscular route of administration may be painful and limits systemic exposure. G1T48 is a highly potent and efficacious oral SERD in both mutant and WT ESR1 preclinical models with the potential to achieve higher exposure and provide clinical benefit in patients with ER+ ABC. MethodsPostmenopausal women with ER+/HER2- ABC following progression on endocrine therapy are eligible for this 3+3 dose escalation and expansion study. Patients receive G1T48 QD until disease progression or unacceptable toxicity. The objectives are to evaluate DLTs, safety, tolerability, PK, ER target engagement via [18F] FES-PET, and anti-tumor efficacy, and to determine the recommended phase 2 dose. ResultsCurrently, 11 patients (mean age 64 years) have received G1T48 doses ranging from 200-800mg QD for up to 7 months. Prior lines (median of 3) included chemotherapy in 45%, fulvestrant in 91%, and CDK4/6i in 54% of patients. G1T48 is well tolerated: no DLTs, G1T48-related SAEs, or withdrawals due to an AE have occurred. The most common Gr 1/2 G1T48-related TEAEs are diarrhea, headache, hot flush, and nausea (27% each) with a single Gr 3 TEAE (fatigue) and no Gr 4 TEAEs. G1T48 exposure increased with dose (200 to 800mg), and minimum to moderate accumulation of G1T48 was observed following repeated doses. Median maximum standard tumor [18F] FES-PET uptake values decreased (ranging from 70% (200mg) to 88% (600mg)) after 4 weeks of treatment. Of 6 response evaluable patients (RECIST v1.1), 1 patient had a PR (600mg, prior palbociclib/fulvestrant) and 1 patient had SD≥24 weeks (200mg, prior abemaciclib/fulvestrant). ConclusionsThe oral SERD G1T48 is well tolerated with no DLTs reported to date in patients with ER+/HER2- ABC. Early efficacy, safety, PK, and FES-PET data are encouraging, and support continued dose escalation followed by expansion. Updated safety, anti-tumor activity, and cfDNA data will be presented (NCT#03455270). Clinical trial identificationNCT03455270. Legal entity responsible for the studyG1 Therapeutics, Inc. FundingG1 Therapeutics. DisclosureE.C. Dees: Research grant / Funding (institution): G1 Therapeutics; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Merck; Research grant / Funding (institution): H3Biosciences; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Cerulean; Research grant / Funding (institution): Meryx; Advisory / Consultancy, Research grant / Funding (institution): Novartis. P.G. Aftimos: Advisory / Consultancy: Boehringer Ingleheim; Advisory / Consultancy: Macrogenics; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Amcure; Honoraria (self), Research grant / Funding (institution): Synthon; Honoraria (self), Travel / Accommodation / Expenses: Amgen; Research grant / Funding (institution), Travel / Accommodation / Expenses: MSD; Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Radius; Research grant / Funding (institution): Servier. C.W. Menke-van der Houven van Oordt: Advisory / Consultancy: G1 Therapeutics; Advisory / Consultancy: Novartis. E.G..E. De Vries: Research grant / Funding (institution): G1 Therapeutics; Advisory / Consultancy: NSABP; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Daiichi Sankyo; Advisory / Consultancy: Pfizer; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Roche; Research grant / Funding (institution): Chugai; Research grant / Funding (institution): CytomX Therapeutics; Research grant / Funding (institution): Nordic Nanovector; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Synthon. M.D. Pegram: Advisory / Consultancy: G1 Therapeutics. J. Xiao: Full / Part-time employment: G1 Therapeutics. C. Sipes: Full / Part-time employment: G1 Therapeutics. C. Li: Full / Part-time employment: G1 Therapeutics. J.A. Sorrentino: Full / Part-time employment: G1 Therapeutics. R. Malik: Full / Part-time employment: G1 Therapeutics. A.P. Beelen: Full / Part-time employment: G1 Therapeutics. All other authors have declared no conflicts of interest.

Bone Marrow Stromal Cells Transcriptionally Repress ESR1 but Cannot Overcome Constitutive ESR1 Mutant Activity.

Estrogen receptor α (ER) is the target of endocrine therapies in ER-positive breast cancer (BC), but their therapeutic effectiveness diminishes with disease progression. Most metastatic BCs retain an ER-positive status, but ER expression levels are reduced. We asked how the bone tumor microenvironment (TME) regulates ER expression. We observed ESR1 mRNA and ER protein downregulation in BC cells treated with conditioned media (CM) from patient-derived, cancer-activated bone marrow stromal cells (BMSCs) and the BMSC cell line HS5. Decreases in ESR1 mRNA were attributed to decreases in nascent transcripts as well as decreased RNA polymerase II occupancy and H3K27Ac levels on the ESR1 promoter and/or distal enhancer (ENH1). Repression extended to neighboring genes of ESR1, including ARMT1 and SYNE1. Although ERK/MAPK signaling pathway can repress ER expression by other TME cell types, MAPK inhibition did not reverse decreases in ER expression by BMSC-CM. ESR1 mRNA and ER protein half-lives in MCF7 cells were unchanged by BMSC-CM treatment. Whereas ER phosphorylation was induced, ER activity was repressed by BMSC-CM as neither ER occupancy at known binding sites nor estrogen response element-luciferase activity was detected. BMSC-CM also repressed expression of ER target genes. In cells expressing the Y537S and D538G ESR1 mutations, BMSC-CM reduced ESR1, but expression of target genes PGR and TFF1 remained significantly elevated compared with that of control wild-type cells. These studies demonstrate that BMSCs can transcriptionally corepress ESR1 with neighboring genes and inhibit receptor activity, but the functional consequences of the BMSC TME can be limited by metastasis-associated ESR1 mutations.

Abstract 3009: FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling

Abstract Palbociclib is the first CDK4/6 inhibitor approved by the FDA for treating ER+/HER2- breast cancer. Palbociclib resistance is emerging as a clinical challenge. Identification of the factors that determine the sensitivity to Palbociclib is of pivotal significance. Fibroblast Growth Factor Receptor (FGFR) overexpression is frequently detected in breast cancer, which has been associated with poor prognosis. To investigate the impact of FGFR overexpression on Palbociclib resistance, we first established stable sublines of MCF7 and T47D cells that overexpress FGFR1, and examined their responses to Palbociclib. Data from MTT and clonogenic assays demonstrated that overexpression of FGFR1 rendered ER+ MCF7 and T47D cells resistant to Palbociclib significantly. Cell cycle analysis showed that the percentage of cells in S phase in Palbociclib treated MCF7/FGFR1 and T47D/FGFR1 cells was significantly higher than that of the control. FGFR overexpression also significantly attenuated Palbociclib-associated inhibition of cancer cell invasion. We further found that Palbociclib-induced inhibition of cancer cell stemness, as indicated by colony formation in soft agar and mammosphere assays, was significantly reduced in MCF7/FGFR1 and T47D/FGFR1 cells, as compared to control. The data indicate that FGFR1 overexpression in ER+ breast cancer cells are resistant to Palbociclib. We next examined the signaling of the key pathways in the paired cell lines treated with Palbociclib. We found that Palbociclib-induced inhibition of phospho-Rb, E2F, cyclin D3, cyclin B1, cdc-2, c-myc, and cdc-25 was significantly attenuated in MCF-7/FGFR1 and T47D/FGFR1 cells. The protein levels of p-ERK, p-AKT, and p-p38 in MCF-7/FGFR1 and T47D/FGFR1 cells were significantly higher than the control cells with/without Palbociclib treatment. The results also showed that ER alpha phosphorylation, estrogen response element (ERE)-luciferase activity and mRNA levels of ER target genes, including PS2, c-myc, E2F, FGF2 and FGFR2, in MCF-7/FGFR1 and T47D/FGFR1 cells were maintained at higher levels post Palbociclib, as compared to control. We then treated control and FGFR1 overexpressing MCF-7 and T47D cells with Palbociclib in combination with FGFR inhibitor, AZD 4547. The combined drug treatment resulted in remarkably synergistic effect on MCF-7/FGFR1 and T47D/FGFR1 cells, as indicated by MTT and clonogenic assays. Key markers in cell cycle regulation, RTK signaling and ER pathway were significantly inhibited in the cells treated with both Palbociclib and AZD 4547. Taken together, our results demonstrated that FGFR1 overexpression is a critical factor affecting Palbociclib sensitivity. The underlying mechanisms involve FGFR1 overexpression-associated RTK and ER signaling. Combination of Palbociclib with FGFR targeting agents may overcome FGFR-associated Palbociclib resistance. Citation Format: Yujie Shi, Zhikun Ma, Yudan Wu, Amanda B. Parris, Qiong Cheng, Lingfei Kong, Xiaohe Yang. FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3009.

Phase I dose escalation of H3B-6545, a first-in-class highly Selective ERα Covalent Antagonist (SERCA), in women with ER-positive, HER2-negative breast cancer (HR+ BC).

1059 Background: H3B-6545 inactivates both wild-type and mutant ERα by targeting cysteine 530 and enforcing a unique antagonist conformation. Methods: Women with locally advanced or metastatic HR+ BC are treated (tx) with H3B-6545 administered once daily orally over a 28 day cycle after progression on at least one hormonal therapy and at least one additional therapy/regimen. Dose escalation uses a 3+3 design with the option to backfill previously cleared doses and allows for intrapatient dose escalation. This phase 1 explores the safety, pharmacokinetics and pharmacodynamics of H3B-6545 in women with HR+ BC to identify the recommended Phase 2 dose. Results: As of 10-Dec-2018, 32 pts have been tx with H3B-6545 at doses of 100 to 450 mg/day; 97% had prior tx with a CDK4/6 inhibitor and 56% had received ≥3 lines of prior anti-cancer therapy. No dose-limiting toxicities and only one Grade 3 treatment related adverse event (TRAE) have been observed (lymphocyte count decrease). The most common (≥10%) TRAEs include asymptomatic sinus bradycardia, diarrhea, nausea, fatigue, anemia, decreased appetite, and hot flush. H3B-6545 was rapidly absorbed with a tmax of 2-4 h. Plasma concentration increased with dose from 100 to 450 mg, and was similar on C1D1 and C1D15. Consistent with the H3B-6545 mechanism of action and preclinical data, H3B-6545 inhibits ER target gene expression and shows a 50% decrease in Ki67 levels across all dose levels post-tx. ESR1 (60%) and PIK3CA (34%) mutations were detected in plasma at baseline and changes in mutant allele frequencies show correlation in response to tx. Stable disease was observed in 15 pts (47%) and 34% of pts completed at least 6 months of tx. Partial responses (PRs) were observed in 3 pts: 1 pt (mutant) received 2 prior lines of therapy and 2 pts (1 mutant and 1 wild-type) received >5 prior lines of therapy including fulvestrant and capecitabine; all 3 pts received a prior CDK4/6 inhibitor. Conclusions: H3B-6545 has been well-tolerated up to the 450 mg dose level with early signs of single-agent anti-tumor activity in a post CDK4/6 setting. Dose escalation continues in pts with advanced HR+ BC. Clinical trial information: NCT03250676.

Widespread PERK-dependent repression of ER targets in response to ER stress

The UPR (Unfolded Protein Response) is a well-orchestrated response to ER protein folding and processing overload, integrating both transcriptional and translational outputs. Its three arms in mammalian cells, the PERK translational response arm, together with the ATF6 and IRE1-XBP1-mediated transcriptional arms, have been thoroughly investigated. Using ribosome footprint profiling, we performed a deep characterization of gene expression programs involved in the early and late ER stress responses, within WT or PERK −/− Mouse Embryonic Fibroblasts (MEFs). We found that both repression and activation gene expression programs, affecting hundreds of genes, are significantly hampered in the absence of PERK. Specifically, PERK −/− cells do not show global translational inhibition, nor do they specifically activate early gene expression programs upon short exposure to ER stress. Furthermore, while PERK −/− cells do activate/repress late ER-stress response genes, the response is substantially weaker. Importantly, we highlight a widespread PERK-dependent repression program, consisting of ER targeted proteins, including transmembrane proteins, glycoproteins, and proteins with disulfide bonds. This phenomenon occurs in various different cell types, and has a major translational regulatory component. Moreover, we revealed a novel interplay between PERK and the XBP1-ATF6 arms of the UPR, whereby PERK attenuates the expression of a specific subset of XBP1-ATF6 targets, further illuminating the complexity of the integrated ER stress response.

Abstract PD7-10: Neoadjuvant trial with letrozole identifies PRR11 in 17q21-23 amplicon as a resistance mechanism to endocrine therapy in ER-positive breast cancer

Abstract Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy relapse with metastatic disease. Previously, we identified 3 amplicons (11q11.3, 8p11.23, and 17q21-23) associated with endocrine-resistance (Giltnane et al. Sci Transl Med 2017). The 17q21-23 amplicon has been associated with highly proliferative luminal B tumors and cancers with high genomic instability. A causal role of this region in endocrine resistance is unclear. We performed whole transcriptome analysis on RNA extracted from 58 ER+ breast cancers of patients treated with letrozole for 5.4-9.2 months (median 7.2 months). PRR11 (Proline rich 11), located in 17q21-23, was significantly upregulated in non-responding tumors as defined by cancer relapse after a median follow up of 5 years and/or a preoperative endocrine prognostic index (PEPI) ≥4. Differential gene expression analysis between tumors expressing low vs high PRR11 mRNA showed that BC signatures associated with proliferation, cell cycle, IGF-1 and PI3K signaling were enriched in tumors with high PRR11 expression. In the Metastatic Breast Cancer Project and TCGA, PRR11 amplification was higher in metastatic vs. primary BCs (16.5% and 8.5%, respectively; Fisher's p=0.0088). Gene Set Enrichment Analysis of mRNA expression in METABRIC and TCGA revealed significant enrichment of hallmark gene sets associated with proliferation in PRR11 amplified ER+ BCs. Genome-scale RNAi screening in Project Achilles showed that among all genes in the 17q21-23 amplicon, PRR11 knockdown results in the 4th strongest anti-proliferative effect in MCF7 cells. PRR11 knockdown with siRNA inhibited proliferation, cell cycle progression, and RB phosphorylation in HCC1428 LTED (long-term estrogen deprived), MCF7 LTED, and fulvestrant-resistant MCF7 cells. Using a PCR array with 84-cell cycle genes, we identified SKP2, CDKN1A, CCNB2, CCNA2, CKS2 and CCNB1 as genes downregulated by PRR11 knockdown. Except for CDKN1A, expression of all those genes was elevated ER+ BCs with PRR11 gain or amplification in TCGA. PRR11 associates with the p85 regulatory subunit of PI3K via its SH3 domain. We speculated this association would suppress p85 homodimers, thus permitting binding of PI3Kα (p110α)-p85 dimers to IRS1 and, hence, activating PI3K/AKT. To test this, we co-transfected HEK293T cells with HA-p85 and FLAG-p85. Transfection of PRR11 into these HEK293T cells reduced HA-p85 and FLAG-p85 homodimers as shown by HA and FLAG pulldowns followed by FLAG and HA immunoblots, respectively. Finally, PRR11 knockdown resulted in a reduction of p110a and S473 P-AKT levels and inhibition of IGF-1/2 stimulated P-AKT. Not inconsistent with these data, PRR11 amplification and PIK3CA mutations in METABRIC and TCGA are exclusive of each other, suggesting these alterations are functionally linked with the same signaling pathway. These data support a role of PRR11 in PI3K/AKT activation that may be causal to resistance to estrogen deprivation. We propose PRR11, located in the 17q21-23 amplicon, is a potential mediator of resistance to antiestrogen therapy by amplifying PI3K/AKT signaling, suggesting PI3K may be a therapeutic target in ER+ BCs harboring PRR11 amplification. Citation Format: Lee K-M, Guerrero-Zotano A, Formisano L, Jansen V, Gonzalez Ericsson P, Arteaga C. Neoadjuvant trial with letrozole identifies PRR11 in 17q21-23 amplicon as a resistance mechanism to endocrine therapy in ER-positive breast cancer [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 PD7-10.

Abstract P3-01-23: Targeting of endocrine therapy-induced estrogen-receptor/HER2-cross-talk in circulating tumor cells from metastatic ER+/HER2- breast cancer: Implications for treatment of ER+/HER2- breast cancer

Abstract Background: Transient induction of HER2 expression upon inhibition of the estrogen-receptor (ERα) might be an underestimated rescue mechanism that enables ER+/HER2- breast cancer cells to survive and to grow in the presence of endocrine therapies. Besides protecting ER+/HER2- BC cells from endocrine treatment, transient HER2 expression is also considered to save circulating, disseminated and dormant ER+/HER2- BC cells from apoptotic cell death in hostile environments and might therefore be of importance for ER+ BC progression, metastasis formation and recurrence. Thus therapeutic strategies that efficiently target both ERα and HER2 simultaneously might improve treatment of patient with ER+/HER2- BCs. Methods: Circulating tumor cells (CTCs) were originally isolated from a patient with bilateral ER+/HER2- metastatic BC. These cells have been treated with either the proteasome inhibitor (PI) carfilzomib or fulvestrant alone or with both drugs in combination. The consequences of these drugs upon ERα and HER2 expression were monitored by western blotting or qPCR. Induction of cell death upon treatment was measured by PARP and caspase cleavage or by quantification of SubG1 cells using propidium iodide staining or by the use of colony forming assays. Results: Fulvestrant treatment of CTCs decreases the amount of ERα but immediately increases HER2 expression whereas carfilzomib markedly inhibits the expression of both ERα and HER2 simultaneously. Combined treatment of CTCs with carfilzomib + fulvestrant cause reduced expression of HER2 and lead to a much stronger decrease of ERα than carfilzomib or fulvestrant alone. Fulvestrant causes no significant reduction of proliferation and no induction of cell death. Conversely the combination of carfilzomib and fulvestrant causes a significant induction of apoptotic cell death and a massive reduction of colonies in colony forming assays. Conclusion: These findings suggest that rapid and transient up-regulation of HER2 expression following endocrine treatment might be an important so far underestimated adaptive mechanism which enables ER+/HER2- BC cells to sustain proliferation in the presence of ERα-inhibitory drugs and to stay alive during the metastatic process. Furthermore these data also lead to the assumption that PIs such as carfilzomib in combination with ERα degraders could be a potential therapeutic strategy for efficient targeting of metastatic ER+/HER2- BC cells. Citation Format: Thaler S, Roßwag S, Pantel K, Sleeman JP, Schmidt M, Cotarelo CL. Targeting of endocrine therapy-induced estrogen-receptor/HER2-cross-talk in circulating tumor cells from metastatic ER+/HER2- breast cancer: Implications for treatment of ER+/HER2- breast cancer [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 P3-01-23.

Abstract P5-11-01: Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD)

Abstract Background: Modulation of estrogen activity and/or synthesis is the mainstay therapeutic strategy in the treatment of ER positive breast cancer. However, despite the effectiveness of available endocrine therapies, many patients ultimately relapse or develop resistance to these agents via estrogen-dependent and estrogen-independent mechanisms, including mutations in ESR1 affecting the ER ligand binding domain that drive ER-dependent transcription and proliferation in the absence of estrogen. Based on preclinical and clinical data, SERDs are expected be effective in patients harboring ESR1 mutations. Biomarker analysis was performed on plasma and tumor samples from the Phase I study of GDC-0927 in metastatic breast cancer (Dickler et al, SABCS 2017) with the goal of evaluating activity in both ESR1 mutant and wildtype tumors, and to assess ER pathway modulation. Methods: Hotspot mutations in ESR1, PIK3CA, and AKT1 were analyzed in baseline, on-treatment and end of treatment plasma derived circulating tumor DNA (ctDNA) using the BEAMing assay in patients treated at multiple dose levels of GDC-0927. A subset of samples was analyzed with Foundation Medicine's next generation sequencing ctDNA assay (FACT), which covers genomic alterations in 62 commonly altered genes. Paired pre- and on-treatment biopsies were collected to assess ER pathway modulation. ER, PR, and Ki67 protein levels were analyzed by immunohistochemistry. Gene expression analysis was performed using Illumina's RNA Access library preparation kit followed by paired-end (2x50b, 50M reads) sequencing on the HiSeq. Results: Baseline and on-treatment plasma samples were available for 40 patients. ESR1 and PIK3CA mutations were observed in 52% and 33% of patient baseline samples, respectively (BEAMing method). Mutant allele frequencies (MAF) generally declined in the first on-treatment samples collected for both ESR1 (16 out of 21 samples) and PIK3CA (7 out of 12 samples). The majority of the reductions were greater than 95% relative to baseline. Increases in ESR1 MAFs were observed in later time-points and were not associated with any particular ESR1 mutation. There were six instances for which an ESR1 mutation was detected in an on-treatment sample that was not detected in the baseline sample, three at L536P and one each at D538G, L536H, and S463P, and four out of six with MAFs close to the limit of detection. The FACT assay also detected alterations in CDH1, NF1, PTEN, and TP53 in baseline samples. The relationship between MAF changes and clinical benefit to GDC-0927 will be presented. A predefined, experimentally-derived set of ER target genes were evaluated in pre- and on-treatment tumor biopsy pairs from six patients. Four of the six patients showed evidence of suppression in ER pathway activity, one patient treated at the 1000 mg dose level and three at the 1400 mg dose. The degree of pathway suppression was associated with pre-treatment pathway levels and decreases of ER and Ki67 protein levels. Conclusions: We report here evidence of consistent reduction of ESR1 and PIK3CA ctDNA in patients treated with GDC-0927. ER pathway suppression was observed at both the transcript and protein level confirming pharmacodynamic activity of the SERD. Citation Format: Spoerke JM, Daemen A, Chang C-W, Giltnane J, Metcalfe C, Dickler MN, Bardia A, Perez Fidalgo JA, Mayer IA, Boni V, Winer EP, Hamilton EP, Bellet M, Urruticoechea A, Gonzalez Martin A, Cortes J, Martin M, Gates M, Cheeti S, Fredrickson J, Wang X, Friedman LS, Liu L, Li R, Chan IT, Mueller L, Milan S, Lauchle J, Humke EW, Lackner MR. Phamacodynamic and circulating tumor DNA evaluation in a phase I study of GDC-0927, a selective estrogen receptor antagonist/ degrader (SERD) [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 P5-11-01.

Abstract P5-05-13: Quantitative combinatory indexed ChIP-seq reveals distinct transcriptional complexes containing estrogen receptor and GREB1 at chromatin in breast cancer cells

Abstract GREB1 is an estrogen-activated gene in estrogen receptor a (ER)-positive breast cancer cells. GREB1 is also required for estrogen-stimulated breast cancer cell growth and its level is highly correlated to ER level in breast cancer cells and tumor samples. In endocrine resistant diseases, GREB1 is often dysregulated. GREB1 has been shown to interact with ER and bind to the same ER binding sites throughout the genome. There is no identified functional domain in GREB1 and it is not completely known how GREB1 exerts its function to regulate the transcription of ER target genes. In order to demonstrate whether GREB1 is present in the ER-containing transcriptional complex at chromatin, we have adapted and developed a quantitative combinatory indexed ChIP-seq assay suitable for dissecting components in a transcriptional cofactor complex in a genome-wide scale. In ER-positive MCF-7 breast cancer cells, we found that almost all GREB1 binding sites are the same sites bound by ER or its bona fide coactivator SRC-3. We further found that GREB1 and SRC-3 are both present in the same ER-containing complex at chromatin. Thus, both GREB1 and SRC-3 are integral members of the ER transcriptional complex at chromatin. Moreover, we discovered that only a portion of GREB1 at chromatin is present in the ER complex while the other portion of GREB1 is present in a different complex lacking ER or SRC-3 at the same genomic loci. Thus, two distinct GREB1-containing complexes are identified in equilibrium at chromatin: one contains ER/SRC-3 and the other one lacks ER/SRC-3. Our results suggest a non-traditional role of GREB1 in transcriptional regulation of ER target genes. The method used in our study can be widely applied for probing components of transcriptional complexes at chromatin. Citation Format: Sun J, Lippman ME. Quantitative combinatory indexed ChIP-seq reveals distinct transcriptional complexes containing estrogen receptor and GREB1 at chromatin in breast cancer cells [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 P5-05-13.

Variable Consequences of Membrane Targeting Motifs for Genetically Encoded Voltage Indicators

Golgi and ER export signals have widely been used to improve membrane trafficking of optical bio-sensors. Previously, we reported a linker modified version of a genetically encoded voltage indicator engineered to image action potential spikes of neurons. Optimization of membrane expression was expected to improve the signal to noise ratio by minimizing unwanted none voltage dependent fluorescence. Accordingly, either Golgi or ER trafficking motif, or both were introduced into Bongwoori-R3 and affected its voltage sensing properties such as the kinetics, responsive voltage range and ΔF/F signal size. The ER motif improved the fluorescence response but it also caused increased time constants and a shifted V1/2 toward hyperpolarization. Introducing spacers between the fluorescent protein and the ER targeting motif recovered the response speed while maintaining the improved fluorescence signal size. This research was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under the award number U01NS099691.

Extended Adjuvant Therapy with Neratinib Plus Fulvestrant Blocks ER/HER2 Crosstalk and Maintains Complete Responses of ER+/HER2+ Breast Cancers: Implications to the ExteNET Trial.

The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses. Mice receiving "extended adjuvant" therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2- MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells. These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.

Monetarne faze i okviri emergentnih zemalja Evropske unije

The paper reviews the monetary stages and frameworks towards the eurozone (EZ) for emerging EU members. At the first pre-EU stage, three monetary frameworks were identified, whereas at the second pre-EZ stage, the emerging EU members combined ER targeting with the rigid/fixed exchange rate regimes (ERRs), on the one hand, and inflation targeting (IT) with the ERM II, on the other. The last stage of monetary convergence assumes monetary nonautonomy within the monetary union as a rigid ERR, where the ER and monetary policies are sacrificed as countercyclical instruments. Countries with rigid ERRs are more prone to macroeconomic overheating, performing worse under a crisis impact compared to the floaters. The difficulties to maintain stability under a rigid ER environment, such as the monetary union, as well as the inherent vulnerabilities of the EZ, are crucial for the delayed entry of some EU members into the ERM II.

Production and separation of 163Ho for nuclear physics experiments.

This paper describes the production and chemical separation of the 163Ho isotope that will be used in several nuclear physics experiments aiming at measuring the neutrino mass as well as the neutron cross section of the 163Ho isotope. For this purpose, several batches of enriched 162Er have been irradiated at the Institut Laue-Langevin high flux reactor to finally produce 6 mg or 100 MBq of the desired 163Ho isotope. A portion of the Er/Ho mixture is then subjected to a sophisticated chemical separation involving ion exchange chromatography to isolate the Ho product from the Er target material. Before irradiation, a thorough analysis of the impurity content was performed and its implication on the produced nuclide inventory will be discussed.