Biology subjects

Miller, T. W.

Publications and source records attributed to Miller, T. W..

9 recordsLinked to original sources

bioRxiv2025

Engineering SIRPα conformational plasticity to reveal a cryptic pocket suitable for structure-based drug design

The protein-protein interaction between Signal Regulatory Protein alpha (SIRP) and CD47 is a critical immune checkpoint that enables tumor immune escape, making it a key target for cancer immunotherapy. While antibody-based therapies exist, the development of small-molecule inhibitors has been hindered by the flat, featureless binding interface. Here, we report the discovery of a novel, druggable cryptic pocket within the SIRP D1 domain (the WYF pocket), revealed through a structure-based fragment screening campaign using x-ray crystallography. This pocket, defined by residues Trp38, Tyr50, and Phe74, is only accessible in a conformation that is incompatible with CD47 binding, making it a candidate for structure-based drug design and immune checkpoint inhibitor development. Through a combination of NMR spectroscopy, molecular dynamics simulations, and biophysical assays, we demonstrate that access to this cryptic site is dynamically controlled by a single "gatekeeper" residue, Gln52. The rotameric state of Gln52 dictates a conformational equilibrium between a "closed," state and a ligand-accessible "open" state. We validated this mechanism by engineering SIRP mutants to bias this equilibrium. A Q52F mutation locked the pocket in a closed state, abolishing both CD47 and fragment binding, while Q52A and Q52R mutations biased the protein toward an open state. These "open-biased" mutants not only exhibited decreased affinity for CD47 but also significantly improved binding to small-molecule fragments that inhibit the SIRP-CD47 interaction. This work reveals the intrinsic conformational plasticity of SIRP and establishes a validated structure-based roadmap for a new class of allosteric inhibitors. This flexibility-for-inhibition strategy functions by trapping a non-binding conformation and represents a broadly applicable framework for targeting this and other challenging immune checkpoints.

biochemistry↗

bioRxiv2025

Epithelial-Mesenchymal Transition is Associated with Altered Immune Composition and Cytotoxic Function in Triple-Negative Breast Cancer

Epithelial-mesenchymal transition (EMT) is a dynamic process that contributes to breast cancer progression, metastasis, and therapy resistance. EMT also influences the tumor immune microenvironment, shaping immune cell infiltration and function. Although the immunological features of fully epithelial or fully mesenchymal tumor states have been characterized, the immune landscape associated with intermediate or partial EMT states remains poorly understood. To address this gap, we established five single-cell-derived clonal populations from the triple-negative 4T1 mouse mammary tumor cell line, representing a spectrum of EMT phenotypes, and analyzed tumors derived from these clones using single-cell RNA sequencing. We show that tumors derived from these clones retained their relative EMT states in vivo, and that EMT progression is associated with a graded reduction in tumor immunogenicity, accompanied by alterations in immune cell composition and function. Along the EMT spectrum, tumor cells exhibited progressive downregulation of major histocompatibility complex (MHC) class I and II gene expression, along with decreased infiltration of cytotoxic CD8+ T cells, and reduced expression of key effector and trafficking genes (Gzmb, Ccr5, Cxcr6). B cell composition also shifted, with decreased frequencies of IgG1-producing plasma cells and an enrichment of regulatory-like B cells. Natural killer (NK) cells similarly demonstrated progressive functional suppression, marked by reduced expression of cytotoxic molecules and the downregulation of key effector pathways. These findings reveal that EMT is associated with immune cell recruitment and function in a graded manner, providing a rationale for integrating EMT phenotyping into therapeutic strategies to overcome immune resistance in breast cancer.

cancer biology↗

bioRxiv2025

Endocrine therapy induces oxidative stress in ER+ breast cancer that sensitizes persister cells to ferroptosis

Despite endocrine therapy, recurrence and progression of estrogen receptor alpha (ER)-positive breast cancer remain significant clinical problems. We therefore sought to identify mechanisms underlying endocrine-tolerant persistence. Endocrine-tolerant persister ER+ breast cancer cells were oxidatively stressed during endocrine therapy. Proteomic analysis revealed upregulation of antioxidant-driving enzymes including glutathione peroxidase 4 (GPX4) in persisters. Relief of oxidative stress enhanced persister fitness. The increased oxidative state of persisters enabled lipid peroxidation and ferroptosis. Persisters had an altered lipidome with increased levels of polyunsaturated fatty acids prone to peroxidation, which was attributable in part to increased lysophosphatidylcholine acyltyransferase 3 (LPCAT3, MBOAT5) expression via loss of ER-mediated repression during endocrine therapy. Treatment with the GPX4 inhibitor RSL3 enhanced the anti-persister effects of endocrine-based therapies in xenograft-bearing mice. These findings supporting the development of therapeutic strategies to leverage the oxidative stress induced by endocrine-based therapies and drive ferroptosis as a treatment for ER+ breast cancer. Statement of SignificanceEndocrine therapy increases oxidative stress and sensitizes endocrine-tolerant persister ER+ breast cancer cells to ferroptosis, indicating that therapies targeting this metabolic dependency could help prevent disease recurrence and progression.

cancer biology↗

bioRxiv2024

Endocrine persistence in ER+ breast cancer is accompanied by metabolic vulnerability in oxidative phosphorylation

Despite adjuvant treatment with endocrine therapies, estrogen receptor-positive (ER+) breast cancers recur in a significant proportion of patients. Recurrences are attributable to clinically undetectable endocrine-tolerant persister cancer cells that retain tumor-forming potential. Therefore, strategies targeting such persister cells may prevent recurrent disease. Using CRISPR-Cas9 genome-wide knockout screening in ER+ breast cancer cells, we identified a survival mechanism involving metabolic reprogramming with reliance upon mitochondrial respiration in endocrine-tolerant persister cells. Quantitative proteomic profiling showed reduced levels of glycolytic proteins in persisters. Metabolic tracing of glucose revealed an energy-depleted state in persisters where oxidative phosphorylation was required to generate ATP. A phase II clinical trial was conducted to evaluate changes in mitochondrial markers in primary ER+/HER2-breast tumors induced by neoadjuvant endocrine therapy (NCT04568616). In an analysis of tumor specimens from 32 patients, tumors exhibiting residual cell proliferation after aromatase inhibitor-induced estrogen deprivation with letrozole showed increased mitochondrial content. Genetic profiling and barcode lineage tracing showed that endocrine-tolerant persistence occurred stochastically without genetic predisposition. Mice bearing cell line- and patient-derived xenografts were used to measure the anti-tumor effects of mitochondrial complex I inhibition in the context of endocrine therapy. Pharmacological inhibition of complex I suppressed the tumor-forming potential of persisters and synergized with the anti-estrogen fulvestrant to induce regression of patient-derived xenografts. These findings indicate that mitochondrial metabolism is essential in endocrine-tolerant persister ER+ breast cancer cells and warrant the development of treatment strategies to leverage this vulnerability in the context of endocrine-sensitive disease. Statement of SignificanceEndocrine-tolerant persister cancer cells that survive endocrine therapy can cause recurrent disease. Persister cells exhibit increased energetic dependence upon mitochondria for survival and tumor re-growth potential.

cancer biology↗

bioRxiv2023

Alteration of DNMT1/DNMT3A by eribulin elicits global DNA methylationchanges with potential therapeutic implications for triple-negative breastcancer

Triple-negative breast cancer (TNBC) is an aggressive disease subtype with limited treatment options. Eribulin is a chemotherapeutic approved for the treatment of advanced breast cancer that has been shown to elicit epigenetic changes. We investigated the effect of eribulin treatment on genome-scale DNA methylation patterns in TNBC cells. Following repeated treatment, The results showed that eribulin-induced changes in DNA methylation patterns evident in persister cells. Eribulin also affected the binding of transcription factors to genomic ZEB1 binding sites and regulated several cellular pathways, including ERBB and VEGF signaling and cell adhesion. Eribulin also altered the expression of epigenetic modifiers including DNMT1, TET1, and DNMT3A/B in persister cells. Data from primary human TNBC tumors supported these findings: DNMT1 and DNMT3A levels were altered by eribulin treatment in human primary TNBC tumors. Our results suggest that eribulin modulates DNA methylation patterns in TNBC cells by altering the expression of epigenetic modifiers. These findings have clinical implications for using eribulin as a therapeutic agent.

cancer biology↗

bioRxiv2023

Pharmacological Induction of mesenchymal-epithelial transition chemosensitizes breast cancer cells and prevents metastatic progression

The epithelial-mesenchymal transition (EMT) is a developmental program co-opted by tumor cells that aids the initiation of the metastatic cascade. Tumor cells that undergo EMT are relatively chemoresistant, and there are currently no therapeutic avenues specifically targeting cells that have acquired mesenchymal traits. We show that treatment of mesenchymal-like triple-negative breast cancer (TNBC) cells with the microtubule-destabilizing chemotherapeutic eribulin, which is FDA-approved for the treatment of advanced breast cancer, leads to a mesenchymal-epithelial transition (MET). This MET is accompanied by loss of metastatic propensity and sensitization to subsequent treatment with other FDA-approved chemotherapeutics. We uncover a novel epigenetic mechanism of action that supports eribulin pretreatment as a path to MET induction that curtails metastatic progression and the evolution of therapy resistance. SummaryWhile the advent of targeted therapy has led to vast improvements in outcomes for certain types of breast cancer, a mainstay for triple-negative breast cancer (TNBC) remains cytotoxic chemotherapy. A major clinical hurdle in successfully managing this disease is the eventual development of therapeutic resistance and disease relapse in more aggressive forms. Our data reveal that epigenetic modulation of EMT state using the FDA-approved therapeutic eribulin curtails metastatic propensity of breast tumors and, when administered in the treatment-naive setting, sensitizes to subsequent treatment with other chemotherapeutics.

cancer biology↗

bioRxiv2023

Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer

PurposeClinical evidence indicates that treatment with estrogens elicits anti-cancer effects in [~]30% of patients with advanced endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains under-utilized. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. Experimental DesignWe performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived (LTED) ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17{beta}-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDXs), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. ResultsCells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2-mutant and BRCA1/2-wild-type cell line and PDX models. ConclusionsE2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

cancer biology↗

bioRxiv2020

AMPK activation by metformin promotes survival of dormant ER+ breast cancer cells

PurposeDespite adjuvant anti-estrogen therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental DesignMimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant anti-estrogen therapy. ResultsDormant breast cancer cells exhibited upregulated 5 adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the anti-diabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. ConclusionsAMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer. Statement of Translational RelevanceDormant cancer cells that survive adjuvant therapy can ultimately give rise to recurrent/advanced tumors that frequently develop resistance to all approved therapies. Patients with early-stage estrogen receptor alpha (ER)-positive breast cancer are typically treated with surgical resection followed by [≥]5 years of adjuvant anti-estrogen therapy that neutralizes ER and suppresses, but often does not eliminate, tumor-initiating cells. Estrogen withdrawal, which mimics aromatase inhibitor therapy, induced activation of the metabolic sensor 5 adenosine monophosphate-activated protein kinase (AMPK) and upregulated fatty acid oxidation (FAO) in preclinical models. Treatment with the anti-diabetes AMPK-activating drug metformin or high dietary fat intake promoted survival of dormant ER+ breast cancer cells, while anti-anginal drugs that inhibit FAO induced clearance of dormant tumor cells. These findings caution against using AMPK modulators with anti-estrogens in patients with ER+ breast cancer, and warrant testing of FAO inhibitors as anti-cancer agents in combination with anti-estrogens.

cancer biology↗

bioRxiv2019

A Homogeneous SIRPα-CD47 Cell-Based, Ligand-Binding Assay: Utility for Small Molecule Drug Development in Immuno-oncology.

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRP. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRP receptors, that block the SIRP-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRP and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRP variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. The LSC assay also confirmed published studies, such as the inhibition of amino-terminal pyroglutamate formation in CD47 disrupts SIRP binding. SIRP-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRP-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.

immunology↗