Biology subjects

McDonnell, D. P.

Publications and source records attributed to McDonnell, D. P..

10 recordsLinked to original sources

bioRxiv2025

Mitoxantrone inhibits and downregulates ERα through binding at the DBD-LBD interface

Targeting the estrogen receptor (ER or ER) through competitive antagonists, receptor downregulators, or estrogen synthesis inhibition remains the primary therapeutic strategy for luminal breast cancer. We have identified a novel mechanism of ER inhibition by targeting the critical interface between its DNA-binding domain (DBD) and ligand-binding domain (LBD). We demonstrate that mitoxantrone (MTO), a topoisomerase II inhibitor, binds at this previously unexplored DBD-LBD interface. Using comprehensive computational, biophysical, biochemical, and cellular analyses, we show that independent of its DNA damage response activity, MTO binding induces distinct conformational changes in ER, leading to its cytoplasmic redistribution and subsequent proteasomal degradation. Notably, MTO effectively inhibits clinically relevant ER mutations (Y537S and D538G) that confer resistance to current endocrine therapies, outperforming fulvestrant in both in vitro and in vivo assays. Our findings establish domain-domain interaction targeting as a viable therapeutic strategy for ER, with translational implications for other nuclear receptors.

cancer biology↗

bioRxiv2024

Estrogens increase cancer cell efferocytosis to establish an immunosuppressive tumor microenvironment

Phagocytic clearance of apoptotic cancer cells (efferocytosis) by tumor-associated macrophages (TAMs) contributes in a substantial manner to the establishment of an immunosuppressive tumor microenvironment. This puts in context our observation that the female steroid hormone 17{beta}- estradiol (E2) facilitates tumor immune resistance through cancer cell extrinsic Estrogen Receptor (ER) signaling in TAMs. Notable was the finding that E2 induces the expression of CX3CR1 in TAMs to enable efferocytosis of apoptotic cancer cells which results in the suppression of type I interferon (IFN) signaling. Mechanistically, E2 facilitates calcium-dependent activation of the transcription factor NFATC1, which in turn induces CX3CR1 expression. This drives macrophage polarization towards an immune-suppressive state, increasing the ability of TAMs to engulf pro- inflammatory apoptotic cancer cells. Genetic or pharmacological inhibition of the E2/ER/CX3CR1 axis reversed the efferocytic activity of TAMs, rescued E2-dependent suppression of type I IFN signaling, and potentiated intratumoral adaptive immune cell function. Efferocytosis following radiation-induced cancer cell apoptosis limits the efficacy of radiation therapy. Importantly, we determined that preconditioning with either ER-directed endocrine therapies or CX3CR1 inhibition enhanced the antitumor efficacy of radiation therapy by reversing macrophage suppression and reviving intratumoral T cell activation. Our work defines the mechanisms by which E2 increases the efferocytotic activity of TAMs to establish an immunosuppressive tumor microenvironment and demonstrates how this process can be reversed with endocrine therapies which target ER.

cancer biology↗

bioRxiv2024

Estrogen receptor-related receptor (Esrra) induces ribosomal protein Rplp1-mediated adaptive hepatic translation during prolonged starvation

Protein translation is an energy-intensive ribosome-driven process that is reduced during nutrient scarcity to conserve cellular resources. During prolonged starvation, cells selectively translate specific proteins to enhance their survival (adaptive translation); however, this process is poorly understood. Accordingly, we analyzed protein translation and mRNA transcription by multiple methods in vitro and in vivo to investigate adaptive hepatic translation during starvation. While acute starvation suppressed protein translation in general, proteomic analysis showed that prolonged starvation selectively induced translation of lysosome and autolysosome proteins. Significantly, the expression of the orphan nuclear receptor, estrogen-related receptor alpha (Esrra) increased during prolonged starvation and served as a master regulator of this adaptive translation by transcriptionally stimulating 60S acidic ribosomal protein P1 (Rplp1) gene expression. Overexpression or siRNA knockdown of Esrra expression in vitro or in vivo led to parallel changes in Rplp1 gene expression, lysosome/autophagy protein translation, and autophagy. Remarkably, we have found that Esrra had dual functions by not only regulating transcription but also controling adaptive translation via the Esrra/Rplp1/lysosome/autophagy pathway during prolonged starvation.

molecular biology↗

bioRxiv2023

Breast cancer cells can recognize and respond to different levels of progestins to achieve different phenotypic outputs

The steroid hormone progesterone, acting through its nuclear progesterone receptor (PR), has complex physiologic activities with different levels of hormones manifesting distinct and sometimes opposing phenotypic responses in target tissues. However, most of what is currently known about the transcriptional activity of PR comes from studies performed using progestins at levels that are in the high physiologic range ([≥]10nM), relevant only in the luteal phase of the reproductive cycle and pregnancy in humans. These studies do not consider the non-linearity of responses to progestins that exist in physiology and are not informative as to the mechanisms by which low levels of progestins, as occurs during menopause, exert their biological activities. Thus, we undertook to define the mechanisms which enable cells to recognize and respond to different levels of progestins. Using a PR expressing cell model of luminal breast cancer (T47D cells) we demonstrated that low concentration progestins (0.1-0.3nM) drive proliferation while high dose progestins ([≥]10nM) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 and SGK1, which are both required to initiate a signaling cascade that leads to increased phospho-Rb and E2F1 transcriptional activity. CCND1 cooperates with CDK4/6 to phosphorylate Rb, while SGK1 phosphorylates p21, thereby excluding it from the nucleus and inhibiting its anti-proliferative function. Expression of CCND1 and SGK1 mRNAs are primary responses to low dose progestin treatment. However, these responses occur at very low levels of receptor occupancy and in the absence of receptor phosphorylation events that have been shown to be required for nuclear translocation and transcriptional activity. These findings challenge the assumption of linearity in response to progestin dose. Further, they suggest that concentrations of progestins found in post-menopausal women (0.1-0.3nM) have the potential to exert proliferative responses in PR expressing cancers.

cancer biology↗

bioRxiv2023

Ca2+/Calmodulin Dependent Protein Kinase Kinase-2 (CaMKK2) promotes Protein Kinase G (PKG)-dependent actin cytoskeletal assembly to increase tumor metastasis

Triple-negative breast cancers (TNBCs) tend to become highly invasive early during cancer development. Despite some successes in the initial treatment of patients diagnosed with early-stage localized TNBC, the rate of metastatic recurrence remains high with poor long-term survival outcomes. Here we show that elevated expression of the serine/threonine-kinase, Calcium/Calmodulin (CaM)-dependent protein kinase kinase-2 (CaMKK2), is highly correlated with tumor invasiveness. We determined that genetic disruption of CaMKK2 expression, or inhibition of its activity, disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor-prognosis ovarian cancer subtype, shares many genetic features with TNBC, and importantly, CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Probing the mechanistic links between CaMKK2 and metastasis we defined the elements of a new signaling pathway that impacts actin cytoskeletal dynamics in a manner which increases cell migration/invasion and metastasis. Notably, CaMKK2 increases the expression of the phosphodiesterase PDE1A which decreases the cGMP-dependent activity of protein kinase G1 (PKG1). This inhibition of PKG1 results in decreased phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate contraction/cell movement. Together, these data establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis. Further, it credentials CaMKK2 as a therapeutic target that can be exploited in the discovery of agents for use in the neoadjuvant/adjuvant setting to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

cancer biology↗

bioRxiv2022

Effects of breast fibroepithelial tumor associated retinoic acid receptor alpha ligand binding domain mutations on receptor function and retinoid signaling

Point mutations in the ligand binding domain of retinoic acid receptor alpha (RAR) have been implicated in breast fibroepithelial tumors development. However, their role in the tumorigenesis of solid tumors is currently unknown. In this study, using a combination of biochemical and cellular assays, we evaluated the functional consequences of known tumor associated RAR mutations on retinoic acid signaling. All of the clinically associated mutants tested showed diminished transcriptional activities compared to wild type RAR. These mutants also exhibited a dominant negative effect, an activity which has previously been linked to developmental defects and tumor formation in mice. X-ray crystallography showed that mutants remain relatively intact structurally and the loss of transcriptional activity is due to altered co-activator recruitment. In agreement with our biochemical analyses, transcriptomics and cell growth analysis showed that the mutant RAR proteins confer resistance to growth inhibition in the presence of its ligand in phyllodes tumor cells. Although the mutations impair the receptor responses to retinoic acid, certain mutant RAR are partially reactivatable with alternative synthetic agonists. Our data provide insights into the mechanisms by which RAR mutations impact tumorigenesis.

cancer biology↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

Increased CaMKK2 expression is an adaptive response that maintains the fitness of tumor-infiltrating natural killer cells

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of energy homeostasis in several cell types. Expression of this enzyme in tumor cells promotes proliferation and migration, and expression in tumor-associated immune cells facilitates M2 macrophage polarization and the development of myeloid-derived suppressor cells. Thus, there has been considerable interest in developing CaMKK2 inhibitors as potential cancer therapeutics. However, the roles of CaMKK2 in other cellular compartments within the tumor immune environment remain to be established, an impediment to the clinical development of these agents. We report that CaMKK2 is expressed at low basal levels in natural killer (NK) cells but is significantly upregulated in tumor-infiltrating NK cells where it suppresses apoptosis and promotes proliferation. It was further demonstrated that NK cell-intrinsic deletion of CaMKK2 increased metastatic progression across several murine models, establishing a critical role for this enzyme in NK cell tumor immunity. Interestingly, ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK cell survival. These results indicate an important scaffold function for CaMKK2 in NK cells and suggest that competitive CaMKK2 inhibitors and ligand-directed degraders (LDDs) are likely to have distinct therapeutic utilities. Finally, we determined that intracellular lactic acid is a key driver of CaMKK2 expression, suggesting that upregulated expression of this enzyme is an adaptive mechanism by which tumor-infiltrating NK cells mitigate the deleterious effects of a lactate-rich tumor environment. The findings of this study should inform strategies to manipulate the CaMKK2 signaling axis as a therapeutic approach in cancer.

cancer biology↗

bioRxiv2022

A new chemotype of chemically tractable nonsteroidal estrogens based on a thienopyrimidine core.

Despite continued interest in development of nonsteroidal estrogens and antiestrogens, there are only a few chemotypes of estrogen receptor ligands. Using targeted screening in a ligand sensing assay we identified a phenolic thieno[2,3-d]pyrimidine with affinity for estrogen receptor . An efficient three-step synthesis of the heterocyclic core and structure-guided optimization of the substituents resulted in a series of potent nonsteroidal estrogens. The chemical tractability of the thieno[2,3-d]pyrimidine chemotype will support the design of new estrogen receptor ligands as therapeutic hormones and antihormones. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=48 SRC="FIGDIR/small/488344v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@4a0c18org.highwire.dtl.DTLVardef@16001f5org.highwire.dtl.DTLVardef@20a58forg.highwire.dtl.DTLVardef@1558bdf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Estrogen-related receptor alpha and Rplp1 ribosome protein-dependent translation coordinately regulate starvation response and decrease NASH progression

BackgroundCurrently, little is known about the mechanism(s) regulating global and specific protein translation during non-alcoholic steatohepatitis (NASH). MethodsWe used puromycin-labelling, polysome profiling, ChIPseq and ChIP-qPCR, and gene manipulation in vitro and in dietary mouse models of NASH in this study. ResultsUsing unbiased label-free quantitative proteome, puromycin-labelling and polysome profiling, we observed a global decrease in protein translation during lipotoxicity in human primary hepatocytes, mouse hepatic AML12 cells, and livers from a dietary mouse model of NASH. Interestingly, proteomic analysis showed that Rplp1, which regulates ribosome and translation pathways, was one of the most downregulated proteins. Moreover, decreased Esrra expression and binding to the Rplp1 promoter, diminished Rplp1 gene expression during lipotoxicity. This, in turn, reduced global protein translation and Esrra/Rplp1-dependent translation of lysosome (Lamp2, Ctsd) and autophagy (sqstm1, Map1lc3b) proteins. Of note, Esrra did not increase its binding to these gene promoters or their gene transcription, confirming its regulation of their translation during lipotoxicity. Notably, hepatic Esrra-Rplp1-dependent translation of lysosomal and autophagy proteins also was impaired in NASH patients and liver-specific Esrra knockout mice. Remarkably, alternate day fasting induced Essra-Rplp1-dependent expression of lysosomal proteins, restored autophagy, and reduced lipotoxicity, inflammation, and fibrosis in hepatic cell culture and in vivo models of NASH. ConclusionEsrra regulation of Rplp1-mediated translation of lysosome / autolysosome proteins was downregulated during NASH. Alternate day fasting activated this novel pathway and improved NASH, suggesting that Esrra and Rplp1 may serve as therapeutic targets for NASH. Our findings also provided the first example of a nuclear hormone receptor, Esrra, to not only regulate transcription but also protein translation, via induction of Rplp1.

molecular biology↗