Background:We had previously reported a novel small molecule, ERX-11, that directly interacts with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. To enhance the clinical translation of ERX-11, we sought to pursue both lead optimization and evaluate combinations of ERX-11 with other approved agents in breast cancer. Methods: We designed, synthesized and tested 500 derivatives of ERX-11 in multiple models of ER+ breast cancer. We also tested combinations of ERX-11 with multiple agents, including other ER targeting agents, chemotherapies and CDK4/6 inhibitors. We tested the effect of combination therapy using breast cancer cells with acquired resistance (Tamoxifen, Letrozole, Ribociclib resistant) and engineered models that express ER mutations. In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays and RNA-seq analysis. Xenograft, patient derived xenograft (PDX), patient derived explant (PDE) and xenograft derived explant (XDE) models were used for preclinical evaluation and toxicity. Result: Evaluation of 500 analogs of ERX-11 identified a number of leads with differential activity against ER+ and ER- breast cancer cells, identified several analogs including ERX-144, 208, 296, 315 with nanomolar potency against ER+ and therapy-resistant ER+ breast cancers. Validation of the mechanism of action of these analogs is ongoing. The combination of ERX-11 and palbociclib significantly blocked ER-mediated and ER-coregulators mediated oncogenic signaling and showed potent anti-proliferative activity against both endocrine therapy-sensitive and resistant breast cancer cells. In addition, ERX-11 inhibited ribociclib-resistant ER+ cell proliferation in a dose dependent manner. Mechanistic studies using IP-Mass spectrometry demonstrated that ERX-11 and palbociclib blocks the interaction between larger subset of coregulators with ER in therapy resistant breast cancer models. ERX-11 and palbociclib both exhibited potent anti-proliferative activity against therapy-sensitive and therapy-resistant ER+ve breast cancer cells, in xenograft models and in PDEs. Importantly, combination therapy of ERX-11 and palbociclib synergistically reduced the growth of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. Mass spec based DIA analyses and RNA-seq studies revealed that combinational treatment uniquely activated p53, unfolded response mediated apoptotic pathways, altered DNA damage response and suppressed E2F and Myc target genes. Biochemical studies confirmed combination therapy significantly altered E2F1, ER and DNA damage response pathways. Conclusion: We have successfully pursued two avenues to improving ERX-11 for clinical translation. We have developed ERX-11 analogs with higher potency against ER+ breast cancer. We have shown that combinational treatment with ERX-11 and palbociclib may overcome endocrine therapy resistance and CDK4/6 inhibitor (ribociclib) resistance. Citation Format: Viswanadhapalli S, Ma S, Lee T-K, Sareddy GR, Liu X, Ekoue D, Alluri A, Luo Y, Kassees K, Arteaga C, Alluri P, Weintraub SE, Tekmal RR, Ahn J-M, Raj GV, Vadlamudi RK. Enhancing the activity of a novel estrogen receptor coregulator binding modulator (ERX-11) against ER-positive therapy resistant 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 P5-04-23.
BACKGROUND: Recently,CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) is approved for the treatment of ER+ advanced breastcancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. Emerging studies indicate many therapy-resistant tumors retainER signaling, via interaction with critical oncogenic coregulatorproteins. Considering complex signaling interplay of ER and CDK4/6 axis, combination therapy of CDK inhibitor with other potent ER-targeted agents that block ER coregulatory signaling may extend the efficacy and may prevent the development of resistance to the CDK4/6 inhibitors. We recently developed a small organic molecule, ER coregulator binding modulator ERX-11 (EtiraRx-11). The objective of this study is to test the utility of novel combination therapy of ERX-11 with CDK4/6 inhibitor palbociclib in treating therapy resistant advanced BCa. METHODS: We have utilized multiple therapy sensitive and therapy-resistant BCa models with various genetic backgrounds. We tested efficacy using both acquired resistance and engineered models that express ER mutations or oncogenes. Efficacy of combination therapy was tested using established in vitro assays including, MTT, colony formation, apoptosis, and cell cycle progression. Mechanistic studies were conducted using reporter gene assays, gene expression, RNA-seq analysis and signaling alterations. Patient-derived BCa explant and Xenograft studies were used to determine the in vivo efficacy of the combination therapy. RESULTS: ERX-11 effectively blocked ER-mediated and ER-coregulator mediated oncogenic signaling and has potent anti-proliferative activity against both endocrine therapy-sensitive and therapy-resistant BCa cells. Mechanistic studies using IP-Mass spectrometry showed that ERX-11 blocks the interaction between a subset of coregulators with ER in resistant BCa models. ERX-11 exhibited potent anti-proliferative activity against therapy-sensitive and therapy-resistant ER-driven BCa cells in vitro, in xenograft models in vivo and in patient-derived breast tumor explants ex vivo. Co-treatment of ERX-11 with palbociclib synergistically reduced cell viability and induced apoptosis of therapy sensitive and resistant BCa model cells. Importantly, combination therapy of ERX-11 and the palbociclib synergistically reduced the growth and induced apoptosis of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. RNA-seq studies revealed that combinational treatment with ERX-11 and palbociclib uniquely activated p53 and unfolded response mediated apoptotic pathways and suppressed E2F and Myc target genes. Biochemical studies confirmed combination therapy significantly altered E2F1 and ER signaling pathways and promoted apoptosis. CONCLUSIONS: Our data support a critical role of blocking ER coregulator signaling in treating therapy resistance in advanced ER+ BCa. Combinational treatment with ERX-11 and palbociclib may overcome/delay endocrine therapy resistance. Citation Format: Viswanadhapalli S, Sareddy GR, Zhou M, Ali E, Li X, Ma S-H, Lee T-K, Tekmal RR, Ahn J-M, Raj GV, Vadlamudi RK. Blocking ER coregulator signaling enhances CDK4/6 inhibitor palbociclib therapy in ER-positive advanced breast cancer [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 P1-09-06.
Proline, Glutamic-acid and Leucine-rich Protein 1 (PELP1) is a proto-oncogene that modulates ER signaling by functioning as an ER-coregulator. Emerging studies demonstrated that in a subset of breast tumors, PELP1 is predominantly localized in the cytoplasm and that PELP1 participates in extranuclear signaling by facilitating ER interactions with Src, PI3K, and AKT. PELP1 expression is upregulated in breast cancer, its deregulation contributes to therapy resistance, and PELP1 is a prognostic marker of poor survival. However, the mechanism by which PELP1 extranuclear actions contributes to cancer progression and therapy resistance remains unknown. We have recently discovered that PELP1 has the potential to interact with mammalian target of rapamycin (mTOR), a serine/threonine kinase that forms two distinct complexes called mTORC1 (containing Raptor and PRAS40) and mTORC2 (containing Rictor and Protor). The objective of this application is to test whether crosstalk occurs between mTOR and PELP1 signaling axis and to test whether mTOR targeting drugs can be used to target PELP1 oncogenic functions. We have used breast cancer cells with PELP1 overexpression (MCF7-PELP1, ZR75-PELP1, T47D-PELP1) or PELP1 down regulation (MCF7-PELP1shRNA, ZR75-PELP1shRNA) along with controls to study the role of PELP1 in the regulation of mTOR axis. PELP1 knockdown significantly reduced downstream mTOR signaling components as analyzed by Western analysis using phospho-S6K, -4EBP1, -mTOR and -Akt, antibodies. Overexpression of PELP1 activated mTOR signaling components. Using immunoprecipitation, we have demonstrated that PELP1 interacts with mTOR. Further immunopreciptation analysis using Rictor and Raptor specific antibodies revealed that PELP1 associates with both TORC1 and TORC2 complexes. Using PELP1WT and PELP1cyto (that predominantly localizes in the cytoplasm), we have demonstrated the differential activation of mTOR signaling components: PELP1WT activated both TORC1 and TORC2 pathways, while PELP1cyto uniquely activated TORC2. mTOR targeting drugs (Rapamycin or AZD8055) showed a significant effect on the in vitro proliferation of PELP1 model cells. AZD8055 is more potent in reducing PELP1 driven tumor growth in vivo compared to rapamycin. Immunohistochemical studies on xenografts derived from MCF7, MCF7-PELP1WT and MCF7-PELP1cyto models demonstrated that PELP1 signaling modulates mTOR signaling in vivo and inhibition of mTOR signaling rendered PELP1 driven tumors to be highly sensitive to therapeutic inhibition. Further, mTOR inhibitors sensitized tamoxifen therapy resistant PELP1cyto model cells to hormonal therapy. IHC analysis of mammary glands and mammary tumors from PELP1Tg mice revealed deregulation of mTOR signaling components with excessive activation of S6K and 4EBP1. Using breast tumor tissue arrays (n = 100), we found significant correlation of PELP1 cytosolic localization with mTOR signaling. Collectively, the experimental results from these studies identified PELP1-mTOR axis as a novel component of PELP1 oncogenic functions and suggests, mTOR inhibitor(s) will be effective chemotherapeutic agents for down regulating PELP1 oncogenic functions and for blocking PELP1-mediated therapy resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-07.
INTRODUCTION: Fatty acid synthase (FASN) is a key enzyme in tumor cell biology controlling endogenous lipid biosynthesis. It is overexpressed in a biologically aggressive subset of tumors, including breast carcinoma. We previously reported prolonged stabilization of disease with TVB-2640 in patients with advanced metastatic breast cancer, including some endocrine resistant ER+ tumors. Using in vitro and in vivo models, we assessed the role of FASN inhibition by TVB-3166 (preclinical version of TVB-2640) for treatment of endocrine resistant breast cancer. METHODS: Breast tumor cells were incubated with TVB-3166 (200nM), imaged and analyzed by automated Live-Cell analysis system (IncuCyte). For tumor growth inhibition, cells (2X106)were subcutaneously injected into SCID mice implanted with estrogen pellets. Once tumors were measurable, mice were divided into treatment groups: tamoxifen (4mg/kg), TVB-3166 (60mg/kg) and the combination. Patient tumor explants were incubated for 72h on gelatin sponges in culture medium in the absence or presence of 200nM TVB-3166. Tissue were fixed in 10% formalin and processed into paraffin blocks. Sections were stained with H&E, ERα and Ki67. RESULTS: The effectiveness of FASN inhibition on the growth of tumor cells has been confirmed in a number of breast cancer cell lines such as MCF7, ZR75, MDA-MB-231 and others. TVB-3166 leads to a marked inhibition of growth in tamoxifen resistant (TamR) cells, which 15% greater than in the parent line. IHC and Western blot showed FASN inhibition leads to significantly reduction of ERα levels. Immunofluorescent confocal microscopy showed inhibition of FASN by TVB-3166 alters subcellular localization of ERα. TVB-3166 was able to significantly inhibit tamoxifen resistant breast tumor growth in mice (p<0.05). Additionally, TVB-3166 treatment of primary tumor explants decreased their proliferation (Ki67) compared to untreated controls (21% vs 38%, p<0.01). CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treating of endocrine resistant breast cancer. RNA sequencing of tumor explants is being performed to evaluate FASN inhibition impact on canonical and non-canonical ERα signaling pathways. Citation Format: Gruslova A, Sareddy GR, Vadlamudi RK, Viswanadhapalli S, Brenner A. FASN inhibition as a potential treatment for therapy of endocrine resistant 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 P2-02-03.
BACKGROUND: Estrogen contribute to the progression of breast cancer via estrogen receptor 1 (ESR1) and current therapies involve either antiestrogens or aromatase inhibitors. However, most patients develop resistance to these drugs. Critically, therapy-resistant tumors retain ESR1-signaling. Mechanisms of therapy resistance involve the activation of ESR1 in the absence of ligand or mutations in ESR1 that allow interaction between the ESR1 and coregulators leading to sustained ESR1 signaling and proliferation. For patients with therapy-resistant breast cancers, there is a critical unmet need for novel agents to disrupt ESR1 signaling by blocking ESR1 interactions with its coregulators. METHODS: Using rational design, we synthesized and evaluated a small organic molecule (ESR1 coregulator binding inhibitor, ECBI) that mimics the ESR1 coregulator nuclear receptor box motif. Using in vitro cell proliferation and apoptosis assays, we tested the effect of ECBI on several breast cancer and therapy-resistant model cells. Mechanistic studies were conducted using established biochemical assays, reporter gene assays, RT-qPCR and RNA-Seq analysis. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA). ESR1 positive (MCF7 and ZR75) xenografts were used for preclinical evaluation and toxicity. The efficacy of ECBI was tested using ex vivo cultures of freshly extirpated primary human breast tissues. RESULTS: In estrogen induced proliferation assays using several ESR1 positive model cells, ECBI significantly inhibited growth and promoted apoptosis. Importantly, ECBI showed little or no activity on ESR1 negative cells. Further, ECBI also reduced the proliferation of several ESR1 positive hormonal therapy resistant cells. Mechanistic studies showed that ECBI interacts with ESR1, efficiently blocks ESR1 interactions with coregulators and reduces the ESR1 driven ERE reporter gene activity. Further, ECBI directly interacted with mutant-ESR1 with high affinity and significantly inhibited mutant-ESR1 driven oncogenic activity. RNA sequencing analysis revealed that ECBI blocks multiple ESR1 driven pathways, likely representing the ability of a single ECBI compound to block multiple ESR1-coregulator interactions. Treatment of ESR1-positive xenograft tumors with ECBI (10 mg/kg/day/oral) significantly reduced the tumor volume compared to control. Further, ECBI also significantly reduced the tumor growth of coregulator-overexpressed breast cancer cells in xenograft model. Using human primary breast tissue ex vivo cultures, we have provided evidence that ECBI has potential to dramatically reduce proliferation of human breast tumors. CONCLUSIONS: The ECBI is a novel agent that targets ESR1 with a unique mechanism of action. ECBI has distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. Remarkably, ECBI block both native and mutant forms of ESR1 and have activity against therapy resistant breast cancer cell proliferation both in vitro and in vivo and against primary human tumor tissues ex vivo. Thus development of ECBI represents a quantum leap in therapies to target ESR1. Citation Format: Vadlamudi RK, Sareddy GR, Viswanadhapalli S, Lee T-K, Ma S-H, Lee WR, Mann M, Krishnan SR, Gonugunta V, Strand DW, Tekmal RR, Ahn J-M, Raj GV. ESR1 coregulator binding inhibitor (ECBI) as a novel therapeutic to target hormone therapy resistant metastatic 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 S3-04.
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