Emch, Michael J.

Abstract Despite the availability of drugs that target ERα-positive breast cancer, resistance commonly occurs, resulting in relapse, metastasis, and death. Tamoxifen remains the most commonly-prescribed endocrine therapy worldwide, and “tamoxifen resistance” has been extensively studied. However, little consideration has been given to the role of endoxifen, the most abundant active tamoxifen metabolite detected in patients, in driving resistance mechanisms. Endoxifen functions differently from the parent drug and other primary metabolites, including 4-hydroxy-tamoxifen (4HT). Many studies have shown that patients who extensively metabolize tamoxifen into endoxifen have superior outcomes relative to patients who do not, supporting a primary role for endoxifen in driving tamoxifen responses. Therefore, “tamoxifen resistance” may be better modeled by “endoxifen resistance” for some patients. Here, we report the development of novel endoxifen-resistant breast cancer cell lines and have extensively compared these models to 4HT and fulvestrant (ICI)-resistant models. Endoxifen-resistant cells were phenotypically and molecularly distinct from 4HT-resistant cells and more closely resembled ICI-resistant cells overall. Specifically, endoxifen resistance was associated with ERα and PR loss, estrogen insensitivity, unique gene signatures, and striking resistance to most FDA-approved second- and third-line therapies. Given these findings, and the importance of endoxifen in the efficacy of tamoxifen therapy, our data indicate that endoxifen-resistant models may be more clinically relevant than existing models and suggest that a better understanding of endoxifen resistance could substantially improve patient care. Implications: Here we report on the development and characterization of the first endoxifen-resistant models and demonstrate that endoxifen resistance may better model tamoxifen resistance in a subset of patients.

Abstract Background: Epigenetic based therapies, such as histone deacetylase inhibitors, have proven successful in many cancer types including lymphomas and myelomas with ongoing phase III clinical trials in NSCLC. Epigenetic modulation in triple negative breast cancer (TNBC) has been increasingly explored in the past decade. A major issue preventing the uptake of these drugs in the clinic is the relatively high doses necessary to achieve therapeutic benefit. It is therefore of interest to identify combinatorial approaches that result in drug de-escalation and enhanced therapeutic benefit. We and others have demonstrated that estrogen receptor beta (ERβ) is expressed in approximately 20% of TNBC patients. ERβ targeted therapies, including estradiol and multiple ERβ selective agonists, result in suppression of cell proliferation, invasion, and migration in vitro and in vivo (Reese, PNAS 2018). For these reasons, we sought to determine if ERβ targeted therapies would synergize with epigenetic based therapies for the treatment of TNBC. Methods: Using multiple TNBC cell lines representative of ERβ+ and ERβ- disease, as well as a model of acquired resistance to ERβ targeted therapies, we performed an epigenetic drug screen assessing 145 compounds alone and in combination with estradiol treatment. The top hit from this screen was extensively profiled across multiple cell lines in the presence and absence of ERβ targeting agents and was compared to the efficacy observed in ERα+ models of breast cancer using cell proliferation assays, RT-PCR, and western blotting. Results: The most significant compound to elicit differential and therapeutically beneficial effects in ERβ+ TNBC was CUDC-101, a multitarget pan-HDACi, EGFRi, and HER2i that was developed based on the structures of Vorinostat, Erlotinib, and Lapatinib. CUDC-101 potently inhibits the growth of TNBC cells and synergizes with E2 stimulation in ERβ+ MDA-MB-231 TNBC cells, but not in ERβ- cells. CUDC-101 was also effective in ERβ-resistant MDA-MB-231 cells and restored the growth inhibitory effects of estrogen in this model. Treatment with Vorinostat (SAHA) alone elicited a similar, though far less potent, synergistic effect in the MDA-MB-231-ERβ+ cell line, suggesting that histone deacetylase inhibition is a critical, but not completely sufficient, component of the synergistic phenotype. CUDC-101, while effective at reducing the viability of MDA-MB-468-ERβ+ cell line, had no synergistic effect with E2 in this model suggesting differences in response between TNBC sub-types. Additionally, CUDC-101 was found to be ineffective in models of ERα+ disease (MCF7 and MCF7-ERβ) in both the presence and absence of E2 treatment. Conclusions: Therapeutic options remain extremely limited for TNBC patients and the development of novel treatment strategies for these patients continue to represent a major unmet clinical need. Here, we present evidence that CUDC-101 elicits potent anti-cancer effects in multiple models of TNBC and synergizes with ERβ-targeted therapies in some models. Our findings further strengthen the body of evidence suggesting ERβ as a valid therapeutic target and important biomarker in TNBC and further implicate HDACi/EGFRi as relevant therapeutic strategies within this patient population. Citation Format: Michael Justin Emch, David Benjamin, Elizabeth Bruinsma, Molly Nelson-Holte, Matthew Goetz, John Hawse. CUDC-101 enhances estrogen-mediated anticancer effects in ERβ+ TNBC [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 P6-05-10.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale . Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.