H3B-5942 suppresses co-activator recruitment and shows potency in endocrine sensitive and resistant lines

H3B-5942 in combination with palbocyclib shows synergy in vitro and in vivo

ESR1 mutants are resistant to endocrine therapies in vitro and in vivo and H3B-5942 can suppress ER pathway activity.

Supplementary table showing the relative expression of 43 target genes in a panel of EphA2-positive cell lines.

Supplementary methods and materials, and Supplemental Figures

H3B-5942 exhibits dose-dependent inhibition of ER target genes and shows significant efficacy ER Wt and mutant in vivo models

Crystallography and amino acid coordinates

C530 residue is critical for H3B-5942 potency

Supplementary Data from Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

Patient-Derived Xenografts (PDX) represent a versatile tool for preclinical drug development because they recapitulate many key features of the parent tumors, including molecular and histopathological profiles, tumor microenvironment, and tumor heterogeneity. The clinical relevance of PDX thus offer several advantages over other commonly used tools such as cell lines and genetically modified mice. Many large collections of PDX have been developed across a wide range of tumor types that profile the genetic diversity of each disease and the ability to bank PDX material allows repeated generation of mice for in vivo drug screens. As a result, PDX are used in several parts of the drug development pipeline, including early cancer biology studies, biomarker development, evaluation of therapeutic efficacy, and assessing/overcoming drug resistance. However, there remain drawbacks to this approach, most notably the large number of mice required for comprehensive drug screens and the associated time and costs. One approach to streamline PDX model selection and in vivo study execution would be to predict in vivo drug responses by assessing responses to the same drugs in an ex vivo platform utilizing PDX-derived dissociated tumor cells. KIYATEC’s KIYA-PREDICT™ PDX assay is a 3D spheroid-based ex vivo platform that has been used to screen a wide range of drugs and tumor types to predict drug responses in primary patient-derived tumors and PDX-derived tumors, including several PDX from XenoSTART’s extensive library of XPDX models. Here, we dissociated XPDX-derived tumors from a panel of 20 breast, ovarian, and lung cancer models provided by XenoSTART and evaluated their ex vivo responses to a panel of chemotherapy agents in our 3D KIYA-PREDICT™ assay. After exposure to drugs for 3-7 days, viability was assessed and both IC50s and percent survival values were calculated. The percent survival was then compared to in vivo drug response data provided by XenoSTART, and correlations between in vivo and ex vivo data were assessed. Drug responses were highly correlative between ex vivo and in vivo models, including the ability to recapitulate palbociclib resistance and platinum and taxane response. These results indicate that the KIYA-PREDICT™ PDX assay is a valuable tool to incorporate into drug development pipelines to accelerate the screening of new drug compounds on a wide range of clinically relevant samples and to guide selection of PDX models for in vivo studies. Citation Format: Aaron L. Carlson, Ashley K. Elrod, Natalie A. Williams, Alyssa D. Moriarty, Michael J. Wick, Teresa M. DesRochers. Ex vivo 3D drug response profiling of XPDX-derived tumor cells for acceleration of preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 183.

Abstract Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy–resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). Summary: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Abstract Ras protein plays a critical role in cell growth and proliferation by transmitting signal from cell surface receptor tyrosine kinase to downstream cellular proteins such as RAF and MEK. KRas is one of the most frequently mutated oncogenes, with mutations at residues G12, G13, and Q61, found in lung, colon, pancreatic and other solid malignancies. We have developed an orally bioavailable, covalent inhibitor of KRas G12C, D-1553, with potent in vitro biochemical, cellular activity and in vivo efficacy. Here we report the efficacy of D-1553 in a large panel of patient-derived xenograft (PDX) tumor models with KRas G12C mutation. In the lung cancer PDX models, D-1553 exhibited tumor growth inhibition (TGI) from 43.6% to 124.3%, with 4 out of 8 models showing tumor regression. In the colorectal cancer PDX models, the range of TGI was from 60.9% to 105.7%, with 3 out of 9 models showing regression. Combination treatment of D-1553 with chemotherapy and targeted agents demonstrated enhanced anti-tumor activity resulting in more tumor regression compared to single agent treatment. These observations from PDX models support the combination treatment as a key to increased overall response rate to KRas G12C inhibitor in clinical trial. D-1553 is currently in a Phase 1/2 clinical trial for patients with advanced solid tumors harboring KRas G12C mutation (NCT04585035). Citation Format: Zhe Shi, Jifang Weng, Xiaochong Fan, Qingqing Zhu, Emily Robb, Alyssa Moriarty, Michael Wick, Yueheng Jiang, Ling Zhang, Xing Dai, Yaolin Wang. Potent in vivo anti-tumor activity of D-1553 as a single agent and in combination with targeted therapeutics in a broad spectrum of patient-derived xenograft tumor models with KRas G12C mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1056.

Abstract Background: Palbociclib is one of three CDK 4/6 inhibitors approved in combination with an aromatase inhibitor for treatment of hormone receptor-positive breast cancer. Although this combination therapy has been found effective in some patients, resistance often develops. To aid in developing new therapies for CDK4/6-resistant breast cancer and better understand resistance mechanisms, we established a PDX model, designated ST1799, from a patient with luminal A breast cancer which was responsive to palbociclib in vivo and then conditioned the model with chronic drug administration until resistant. The resistant model, designated ST1799/PBR, and the parent were characterized for receptor expression, genomic alterations and in vivo drug sensitivity. Methods: ST1799 was established from an axillary lymph node FNA taken from a 66-year-old woman pretreated with various chemo and hormone therapies. The resulting model was passaged and a cohort challenged with chronic palbociclib treatment to produce drug resistance. The parent ST1799 and resistant ST1799/PBR were subjected to various comparative analyses including receptor expression, NGS and RNAseq and response to various relevant therapies. Results: Both models retained high ER staining (3+) expression and demonstrated comparable histopathology. DNA-based analysis of the parent model confirmed variants concordant with clinical mutation analysis. Comparison of the parent and resistant clone using RNAseq identified several alterations in variants and expression. In vivo palbociclib reported significant (p<0.0001) activity towards ST1799 (T/C=11%) while ST1799/PBR was resistant towards the therapy (T/C=80%). Tamoxifen or fulvestrant treatment of ST1799 resulted in partial tumor regressions while activity in ST1799/PBR was less pronounced. Activity of abemaciclib or ribociclib was greater in ST1799/PBR compared with palbociclib treatment. Conclusion: We have established and characterized paired palbociclib-sensitive and resistant breast PDX models which can be utilized as valuable tools in better understanding CDK4/6i resistance and in developing new therapies for CDK4/6 inhibitor resistant patients. Citation Format: Lizette Gamez, Alyssa Moriarty, Tomoyuki Mashimo, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Michael J Wick. Establishment and characterization of paired palbociclib-sensitive and resistant luminal A breast PDX models [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 P1-03-10.

Abstract Background: Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for treatment of HER2 positive (HER2+), trastuzumab-resistant breast cancers. While this agent is initially effective, resistance often develops. To aid in developing new therapies for HER2+, T-DM1-resistant breast cancer and better understanding resistance mechanisms, we established PDX models designated ST340, ST1339, and ST2167 from three patients with trastuzumab-resistant HER2+ breast cancer and sensitive to T-DM1 treatment in vivo. A cohort of each model was conditioned in vivo with chronic T-DM1 treatment to generate drug resistance. These models, designated ST340/TDR, ST1339/TDR, and ST2167/TDR were subjected to characterization and efficacy studies, and the results compared with parent models. Methods: ST340, ST1339, and ST2167 were established from patients who responded then progressed on trastuzumab and other therapies. The models were passaged, and cohorts were challenged with chronic T-DM1 treatment to produce drug resistance. The parent and resistant models were subjected to various comparative analyses including receptor expression, RNAseq, and response to various relevant therapies. Results: T-DM1-resistant models retained high HER2 expression and demonstrated receptor staining and histopathology comparable to respective parent lines. Comparison of parent and resistant clones using RNAseq identified several alterations in variants and expression. In vivo T-DM1 reported significant activity towards ST340: %T/C=3%, ST1339: %T/C=-40% and ST2167: %T/C=13% versus control, including tumor regressions in ST1339. T-DM1-resistant models reported the following values following treatment: ST340/TDR: %T/C=65%, ST1339/TDR: %T/C=58% and ST2167/TDR: T/C=91%. Conclusion: We have established and characterized three paired T-DM1-sensitive and resistant breast PDX models, which can be utilized as valuable tools in developing new therapies for T-DM1-resistant patients. Citation Format: Alyssa Moriarty, Lizette Gamez, Tomoyuki Mashimo, Kyriakos Papadopoulos, Amita Patnaik, Drew Rasco, Amy Lang, Ronald Drengler, Michael J Wick. Development and characterization of HER2+ T-DM1-resistant breast PDX models in athymic nude mice [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A017. doi:10.1158/1535-7163.TARG-19-A017

Background: Overall, 10-30% of patients with metastatic breast cancer (BC) will develop brain metastases. Depending on the subtype of the primary breast cancer the incidence of brain metastases varies ranging from 14% (hormone receptor positive BC) to 46% (triple-negative BC) and approximately about 34% for patients with HER2-positive BC. The major impact of the blood-brain-barrier (BBB), BBB efflux pumps and the local microenvironment in the brain represent a challenge for treatment of BC brain metastasis and treatment options remain limited. The aim of this study was to develop a model system of breast cancer brain metastases for evaluation of drugs directed at BC brain metastases. Methods: Models of BC brain metastasis were established by intracranial stereotactic injection of enzymatically digested PDX tumors (ST340, ST941, ST1339, ST1616B, ST1360B, and ST3338) or by intracardiac or intracarotid injection of cell suspensions in nude mice. Contrast-enhanced T1- and T2-weighted magnetic resonance imaging (MRI) or bioluminescence imaging was used to determine tumor take and growth. Drug sensitivity to single agent trastuzumab emtansine (T-DM1) administered intravenously were performed at confirmed tumor take in a subset of the models. Mice were treated with either saline or T-DM1, 10 mg/kg/week x4 or 2 mg/kg/week x4. PET imaging with 18F-FES was applied in estrogen receptor (ER) positive models as a PD biomarker of anti-estrogen receptor endocrine therapy. Results: T-DM1 treatment in the intracranial ST1339 or ST3338 HER2 positive (3+) PDX models inhibited tumor growth and prolonged survival compared to non-treated animals. However, T-DM1 treatment only caused a modest growth delay in the ST3338 model established intracranially compared to a complete response in the model established subcutaneously. Delivery of T-DM1 across the BBB to the intracranial tumors was visualized by 64Cu-trastuzumab PET/CT and high tumor uptake was associated with a response to T-DM1 therapy in the ST1339 model. Models of BC brain metastasis were successfully established by the intracardiac or intracarotid methods for the triple negative BC cell line MDA-MB-231, the HER2 positive cell line BT474, and an ER positive cell line (ST941C). In vivo imaging with 18F-FES PET/CT was able to visualize intracranial ER positive BC tumors, and animals treated with fulvestrant exhibited reduced 18F-FES tumor uptake. Conclusion: We have established model systems of BC brain metastasis and drug efficacy evaluation. Treatment response to T-DM1 was observed in intracranial HER2 positive models and delivery of T-DM1 to the tumors was visualized by 64Cu-trastuzumab PET/CT imaging. Together, the established breast cancer brain metastases models in combination with advanced non-invasive imaging can be used as a relevant translational platform for evaluation of new drugs. Citation Format: Carsten H. Nielsen, Michael J. Wick, Lotte K. Kristensen, Henriette S. Joergensen, Maria Z. Alfsen, Mark U. Juul, Alyssa Moriarty, Kyriakos P. Papadopoulos, Andreas Kjaer. Preclinical models of breast cancer brain metastasis for drug efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A011. doi:10.1158/1535-7163.TARG-19-A011

Background: FDA-approved CDK 4/6 inhibitors (CDK4/6i) including palbociclib, abemaciclib, and ribociclib have demonstrated clinical benefit in treating hormone receptor-positive (HR+) breast cancer. However, whether there is differential sensitivity to each therapy in vivo is unknown. In addition, whether these inhibitors demonstrate activity towards other tumor types is unclear. To better understand potential differences in CDK4/6i activity, we compared activity of each in a panel of 100 HR+/- breast cancer PDX models. To identify additional tumor types sensitive to CDK4/6i, we screened single agent palbociclib in an additional panel of 350 PDX models representing solid and hematologic malignancies. FFPE samples were collected from control and treatment groups in all studies at endpoint, and %T/C values in each model were calculated and in breast studies compared. Methods: START PDX models were established and validated as previously described and further characterized using IHC, WES, RNAseq, and drug sensitivity studies. For each CDK4/6i study, models were implanted SC in immune-deficient mice and studies initiated at 200-300 mm3 (n=1-3/grp). CDK4/6i agents were administered orally once daily at 50-75 mg/kg for a minimum of twenty-eight days or until study completion. Models tested with palbociclib included breast, lung, ovary, pancreas, head/neck, renal, gastric, uterine, colorectal, melanoma, and various hematologic malignancies. Study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a %T/C of ≤ 20% versus control was considered sensitive. Partial tumor regression (PR) (%PR=1-99%) and complete tumor regression (CR) (%CR=100%) values versus Day 0 tumor volume of treated groups were also reported. Results: HR+/- breast models demonstrated variable sensitivity to the three CDK4/6i with HR+ models established from chemo-naive patients reporting greatest tumor growth inhibition (TGI). Two of four HR+ ESR1 mutant models reported modest sensitivity to CDK4/6i (ST941Y537S and ST2535D538G), while ST2177Y537S was sensitive and ST2056Y537S refractory to the three therapies. Evaluation of palbociclib in additional indications reported notable TGI versus control in several indications with the following percentage of tested models reporting sensitivity: Lung: 10%, Ovary: 15%, Pancreas: 30%, Head/Neck: 35%, Renal: 45%, Gastric: 60%, Uterine: 20%, Colorectal: 45%, Melanoma: 40% and Hematologic: 15%. In addition, several indications reported tumor regressions including one CR (lung: ST1748) and partial responses in ovary, pancreas, head/neck, renal, colorectal, and melanoma. Conclusion: We evaluated and compared three FDA-approved CDK4/6i therapies in a panel of HR+/- breast PDX models and identified models with differential responses. In addition, we screened single agent palbociclib in a panel of 350 PDX models representing solid and hematologic malignancies and identified several sensitive models in multiple indications, including some with partial or complete tumor regressions. Citation Format: Lizette Gamez, Tomoyuki Mashimo, Alyssa Moriarty, Kyriakos Papadopoulos, Drew Rasco, Amita Patnaik, Amy Lang, Ronald Drengler, Lon Smith, Michael J Wick. PDX-based screen to evaluate and compare approved CDK4/6 inhibitors in breast cancer and determine palbociclib efficacy in additional tumor types [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A008. doi:10.1158/1535-7163.TARG-19-A008

Metastatic breast cancer patients with coexistent HER2 mutation and amplification respond to neratinib.

Abstract Background: Overexpression of the human epidermal growth factor receptor 2 (HER2) is an independent risk factor for development of brain metastases. Between 37-55% of patients with HER2-positive metastatic breast cancer develop brain metastases and the incidence is increasing. A reason for the increase of brain metastases is the improved control of systemic disease by novel therapeutics such as ado-trastuzumab emtansine (T-DM1). However, the major impact of the blood-brain-barrier (BBB), BBB efflux pumps and the local microenvironment in the brain represent a challenge for treatment of brain metastases. The aim of this study was to develop a model system of HER2 positive breast cancer brain metastases for evaluation of drugs directed at brain metastases. Methods: Subcutaneous tumors from a panel of HER2-positive PDX breast cancer models designated ST340, ST1339, ST1616B, ST1360B and ST3338 were enzymatically digested and used for intracranial stereotactic injection or intra carotid injection in nude mice. Contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) were used to determine tumor take and growth. Drug sensitivity studies to single agent T-DM1 administered intravenously were performed at confirmed tumor take in a subset of the models. Mice were treated with either saline or T-DM1, 10 mg/kg/week x4 or 2 mg/kg/week x4. Delivery of T-DM1 to the tumor site was evaluated by PET/CT imaging with 64Cu-trastuzumab. Results: Intracranial tumor growth was detected by MR imaging in all models. T-DM1 treatment of animals with intracranial ST1339 or ST3338 tumors inhibited tumor growth and prolonged survival compared to non-treated animals. A variable response within the treatment groups was observed. Delivery of T-DM1 across the BBB to the intracranial tumors was visualized by 64Cu-trastuzumab PET/CT and high tumor uptake was associated with a response to T-DM1 therapy in the ST1339 model. Conclusion: We have established a model system for evaluating drug sensitivity in HER2 positive breast cancer brain metastases. Treatment response to T-DM1 was observed in the models. 64Cu-trastuzumab PET imaging confirmed delivery of trastuzumab to the tumors, and high uptake was associated with an increased response to T-DM1 therapy. Together, the established PDX models of breast cancer and brain metastases can be used as a relevant translational platform in combination with advanced non-invasive imaging for evaluation of new drugs. Citation Format: Carsten H. Nielsen, Michael J. Wick, Mette M. Jensen, Lotte K. Kristensen, Alyssa Moriarty, Melissa Rundle, Kyriakos P. Papadopoulos, Andreas Kjaer. Intracranial PDX models of breast cancer brain metastases and PET imaging for drug efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2174.

Abstract Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs. Significance: Nearly 30% of endocrine therapy–resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176–93. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1047