Metastatic estrogen receptor α (ERα)–positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14 to 21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did not undergo complete regression and regrew remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression.

Approximately 30% of metastatic breast cancers harbor estrogen receptor α (ERα) mutations associated with resistance to endocrine therapy and reduced survival. Consistent with their constitutive proliferation, T47D and MCF7 cells in which wild-type ERα is replaced by the most common mutations, ERαY537S and ERαD538G, exhibit partially estrogen-independent gene expression. A novel invasion/dissociation/rebinding assay demonstrated that the mutant cells have a higher tendency to dissociate from invasion sites and rebind to a second site. Compared to ERαD538G breast tumors, ERαY537S tumors exhibited a dramatic increase in lung metastasis. Transcriptome analysis showed that the ERαY537S and ERαD538G mutations each elicit a unique gene expression profile. Gene set enrichment analysis showed Myc target pathways are highly induced in mutant cells. Moreover, chromatin immunoprecipitation showed constitutive, fulvestrant-resistant, recruitment of ERα mutants to the Myc enhancer region, resulting in estrogen-independent Myc overexpression in mutant cells and tumors. Knockdown and virus transduction showed Myc is necessary and sufficient for ligand-independent proliferation of the mutant cells but had no effect on metastasis-related phenotypes. Thus, Myc plays a key role in aggressive proliferation-related phenotypes exhibited by breast cancer cells expressing ERα mutations.

Metastatic estrogen receptor α (ERα) positive breast cancer is presently incurable and most patients die within 7 years. From a medicinal chemistry program, we identified a novel small molecule that acts through ERα to kill breast cancer cells and often induces complete regression without recurrence of large, therapy-resistant primary breast tumors and of lung, bone, and liver metastases. To target metastatic ERα positive breast cancer, we exploited our finding that estrogen-ERα activates an extranuclear tumor-protective, signaling pathway, the anticipatory unfolded protein response (UPR). We repurposed this tumor protective pathway by targeting it with the small molecule, ErSO. ErSO kills cancer cells by acting non-competitively through ERα to induce lethal hyperactivation of the anticipatory UPR, triggering rapid necrotic cell death. Using luciferase to image primary tumors and metastases containing lethal ERαD538G and ERαY537S mutations seen in metastatic breast cancer, oral and injected ErSO exhibited unprecedented antitumor activity. In mouse xenografts bearing large breast tumors, oral and injected ErSO induced complete regression (>115,000 fold mean regression) in about 45% of mice (18/39). Although durable response for 4-6 months without additional treatment was common, tumors that did recur remained fully sensitive to ErSO re-treatment. Consistent with the essential nature of the UPR pathway targeted by ErSO, in more than 100 tumor-bearing mice, we have never seen an ErSO-resistant tumor. In just 7 days, oral ErSO induced complete regression of most lung, bone, and liver metastases. ErSO is well-tolerated in mice and blood-brain-barrier penetrant. Injected ErSO induced profound regression of challenging brain tumors. On average, ErSO-treated tumors were >180-fold smaller than vehicle-treated tumors. These xenograft studies used human cancer cells in mice that lack a functional immune system and therefore did not exploit the known ability of inducers of necrotic cell death to activate immune cells and induce immunogenic cell death. Notably, medium from breast cancer cells killed by ErSO contained high levels of immune cell activators, robustly activated mouse and human macrophages and increased macrophage migration. Moreover, use of ErSO is not limited to breast cancer. ErSO rapidly kills ERα positive ovarian and endometrial cancer cells that do not require estrogen for growth. ErSO’s potent activity against advanced primary and metastatic ERα-positive breast cancers represents a paradigm shift in leveraging ERα for anticancer efficacy.

Treatment of advanced ERα-positive breast cancer remains a major clinical challenge, as the standard therapeutics are mostly cytostatic agents, ultimately leading to drug resistance. Resistant ERα-positive tumors typically retain ERα expression, suggesting an opportunity to develop new therapeutics that move beyond inhibition of this nuclear receptor. Through a medicinal chemistry campaign, we have discovered an estrogen receptor selective activator, the compound ErSO, that – in stark contrast to current ERα modulators – potently kills ERα-positive breast cancer cells. ErSO is effective even against breast cancer cell lines with known mutations in ERα that confer drug resistance. ErSO hyperactivates the anticipatory unfolded protein response, leading to quantitative cell death. ErSO is well-tolerated in mice, orally bioavailable, blood-brain-barrier penetrant. Using sensitive tumor imaging, ErSO often leads to quantitative tumor eradication (without recurrence) in multiple mouse models of ERα-positive breast cancer, including those utilizing breast cancer cell lines harboring mutant ERα. This potent activity against advanced primary and metastatic ERα-positive breast cancers represents a paradigm shift in leveraging of ERα for anticancer efficacy. Citation Format: Matthew W Boudreau, Paul J Hergenrother, Darjan Duraki, Lawrence Wang, Chengjian Mao, Ben H Park, Tim M Fan, Eric R Nelson, David J Shapiro, Ramon Moreno, Elizabeth Bruckheimer, Jeffrey Kiefer, Theodore M Tarasow. Quantitative regression of estrogen receptor alpha positive 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 P5-05-16.

Metastatic estrogen receptor α (ERα) positive breast cancer is presently incurable and most patients die within 7 years. From a medicinal chemistry program, we identified a novel small molecule that acts through ERα to kill breast cancer cells and often induces complete regression without recurrence of large, therapy-resistant primary breast tumors and of lung, bone, and liver metastases. We exploited our finding that estrogen-ERα activates an extranuclear tumor-protective, signaling pathway, the anticipatory unfolded protein response (UPR). We repurposed this tumor protective pathway by targeting it with the small molecule, ErSO. ErSO kills cancer cells by acting non-competitively through ERα to induce lethal hyperactivation of the anticipatory UPR (a-UPR), triggering rapid necrotic cell death. Using luciferase to image primary tumors and metastases containing lethal ERαD538G and ERαY537S mutations seen in metastatic breast cancer, oral and injected ErSO exhibited unprecedented antitumor activity. In mouse xenografts bearing large breast tumors, oral and injected ErSO induced complete regression (>115,000 fold mean regression) in about 45% of mice (18/39). Although durable response without treatment for 4-6 months was common, tumors that did recur remained fully sensitive to ErSO re-treatment. Consistent with the essential nature of the a-UPR pathway targeted by ErSO, in more than 100 tumor-bearing mice, we have never seen an ErSO-resistant tumor. In just 7 days, oral ErSO induced complete regression of most lung, bone, and liver metastases. ErSO is well-tolerated in mice and blood-brain-barrier penetrant. Injected ErSO induced profound regression of challenging brain tumors. On average, ErSO-treated tumors were >180-fold smaller than vehicle-treated tumors. Moreover, use of ErSO is not limited to breast cancer. With its unique mechanism of action through the a-UPR, ErSO eradicated orthotoptic ERα positive ovarian tumors that do not require estrogen for growth. These xenograft studies used human cancer cells in immune compromised mice and therefore did not exploit the known ability of inducers of necrotic cell death to activate immune cells and induce immunogenic cell death. Notably, medium from breast cancer cells killed by ErSO contained high levels of the established immune cell activators, HMGB1 and ATP, robustly activated mouse and human macrophages and increased macrophage migration. ErSO’s potent activity against advanced primary and metastatic ERα-positive breast cancers represents a paradigm shift in leveraging ERα for anticancer efficacy.

Immunotherapy has dramatically impacted cancer therapy, but it has been challenging to apply immunotherapy to estrogen receptor (ER) positive breast cancer and many other solid tumors that do not display neoantigens. One way to target these tumors is to induce necrosis, which robustly activates immune cells, inducing immunogenic cell death. However, anticancer therapy-induced necrosis was primarily characterized by morphological changes, and the molecular drivers of necrosis were largely obscure. To probe necrosis, we used our necrosis inducing anticancer agents, the small molecules BHPI and second-generation ErSO, which kill cancer cells by hyperactivating the anticipatory unfolded protein response (a-UPR). In orthotopic mouse xenografts, ErSO induces complete regression without recurrence of large, therapy-resistant primary ER positive breast tumors, of most lung, bone, and liver metastases, near complete regression of challenging breast cancer brain metastases and robust responses in PDX and patient derived organoids (PDOs) models. ErSO also induces complete or near complete regression in mouse xenograft models of ER positive ovarian and endometrial cancer. Using genome wide CRISPR-Cas9 screens with negative selection against our necrosis-inducing a-UPR hyperactivators, BHPI and ErSO, we identified the calcium-activated, ATP-inhibited, plasma membrane sodium channel, Transient Receptor Potential Melastatin Member 4 (TRPM4) as critical for anticancer therapy induced necrosis. TRPM4 knockout in multiple models abolished ErSO-induced ATP depletion, sustained UPR activation, cell swelling, necrotic cell death and increased migration of immune cells. Notably, knockout of TRPM4 completely abolished the ability of ErSO to induce regression of ER positive breast tumors in mice. Supporting a broad role for the TRPM4 pathway in anticancer therapy induced necrosis, rapid cancer cell death induced by four necrosis-inducing cancer therapies unrelated to ErSO, that range from FDA-approved to preclinical, is strongly reversed by TRPM4 knockout. ErSO treatment induces migration of macrophage into regressing tumors. Medium from cancer cells killed by necrosis-inducing ErSO, but not by an apoptosis inducer, dramatically increases macrophage migration and activation, as shown by induction of pro-inflammatory cytokines. This work identifies a protein that plays a pivotal role in the action of diverse anticancer therapies inducing immunogenic necrosis. Since increasing levels of TRPM4 increase sensitivity of breast cancer cells to killing by ErSO, TRPM4 is a novel biomarker whose levels can be used to identify patients most likely to benefit from ErSO and other necrosis-inducing cancer therapies. Citation Format: Santanu Ghosh, Rachel Yang, Darjan Duraki, Ji Eun Kim, Junyao Zhu, Mara Livezey, Matthew Boudreau, Ben H. Park, TImothy Fan, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-01.

In orthotopic xenografts and a PDX, our anticancer drug ErSO eradicates primary and metastatic therapy-resistant estrogen receptor alpha (ERα) positive breast cancer, and induces near-complete regression of ovarian cancer. The mechanism by which ErSO induces necrosis and kills ER positive cancer cells was unknown. From genome-wide CRISPR-Cas9 screens in MCF-7 and T47D cells with negative selection against first-generation BHPI and second-generation ErSO, and follow-on experiments, we identified the Ca2+ activated, plasma membrane Na+ channel TRPM4 as the executioner protein that BHPI and ErSO use to induce necrosis. Notably, in 6 ERα+ breast and ovarian cancer cell lines, knockout of TRPM4 completely abolished the ability of ErSO to induce death of cancer cells. Moreover, TRPM4 mRNA and protein were dramatically down-regulated in breast cancer cells selected for resistance to BHPI and ErSO. Furthermore, in a mouse xenograft, while ErSO induced near complete regression of orthotopic MCF-7-ERY537S-luciferase tumors, ErSO had no effect on the TRPM4 knockout tumors, which continued their robust growth. Since necrosis, but not most other death pathways, activates immune cells, inducing immunogenic cell death, this provides a new avenue for enhancing cancer immunotherapy. Importantly, medium from ErSO-treated wild type MCF-7 cells, but not medium from TRPM4 knockout cells, robustly activates human THP-1 monocytes and greatly increases their migration. BHPI and ErSO-induced initial anticipatory unfolded protein response (a-UPR) activation results in elevated cytosolic Ca2+, opening the plasma membrane TRPM4 channel, eliciting a rapid influx of external Na+, accompanying Cl- to balance the charge, and water to maintain osmolality. This swells the cells, causing osmotic stress. Importantly, it is the osmotic stress that sustains UPR hyperactivation, leading to ATP depletion, which contributes to membrane rupture and rapid necrotic cell death and to near complete inhibition of protein synthesis that ultimately kills any surviving cancer cells . Suggesting a broad role of TRPM4 in the actions of necrosis inducing anticancer drugs, TRPM4 knockout also inhibited necrosis induced by unrelated anticancer therapies, the mitochondrial targeting oncolytic peptide, LTX-315 and the Ca2+ channel targeting agent, Englerin A. Since increasing expression TRPM4 by viral transduction results in progressively increased sensitivity of ER positive breast cancer cells to killing by ErSO, this enables identification of breast cancer patients whose elevated TRPM4 levels make them most likely to benefit from this novel therapy. The TRPM4 pathway is a new mechanism for sustained lethal activation of the UPR and for targeting ER positive breast and ovarian cancer. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 p.m.

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