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Biology subjects

Fanning, S. W.

Publications and source records attributed to Fanning, S. W..

8 recordsLinked to original sources

A non-enzymatic role for METTL3 as an Androgen Receptor co-regulator that promotes prostate cancer proliferation.

Metastatic prostate cancer (PCa) continues to be a major cause of death in males, despite advances in treatment. Most treatment focuses on targeting the Androgen Receptor (AR), the main oncogene responsible for driving most prostate tumors. Despite these therapies targeting AR, the majority of patients still succumb to AR-driven disease. Therefore, there is a critical need for understanding how AR functions to promote prostate cancer growth and identify alternative therapeutic targets in AR-driven PCa. One avenue garnering attention is targeting epigenetic regulators that promote AR-activity; however, the importance of epitranscriptomic regulators, like those that modify mRNAs, is not well understood. Here, we identify a new role for the key catalytic subunit of the RNA N6-methyladenosine (m6A) transferase complex, METTL3, as an AR-coregulator. METTL3 is overexpressed in prostate tumors compared to normal tissue, and METTL3 protein is elevated in AR-expressing cell lines. Depletion of METTL3 significantly reduces proliferation of cancer cells and has no effect on the growth of non-transformed prostate epithelial cells, despite decreasing global m6A levels on mRNA. The catalytic activity of METTL3 is dispensable for the growth of both non-transformed and PCa cell lines, as pharmacologic inhibition of METTL3 does not inhibit proliferation, despite the reduction of global m6A on mRNA. Overexpression of both wild-type and catalytically inactive METTL3 mutants enhances cell viability and rescues cells in which METTL3 is knocked down. Finally, we report on direct interaction between AR and METTL3, their co-localization on chromatin, and reduced AR-cistromic occupancy within cells with METTL3 knockdown. Together, these findings identify a non-enzymatic role for METTL3 in supporting AR-driven transcriptional programs and PCa proliferation.

cancer biology↗

SOX2 utilizes FOXA1 as a heteromeric transcriptional partner to drive proliferation in therapy-resistant prostate cancer

Treatment options and diagnostic outlook for men with advanced, therapy resistant prostate cancer (PCa) are extremely poor; this is primarily due to the common lack of durable response to androgen receptor (AR) targeted therapies and phenotypic transdifferentiation into a particularly lethal subtype known as neuroendocrine prostate cancer (NEPC). In this study, we mechanistically determine that SOX2 (a transcription factor originally repressed by AR) physically binds and acts in a concerted manner with FOXA1 (a key AR pioneering cofactor) to regulate a subset of genes which promote cell cycle progression, and lineage plasticity in AR-refractory prostate cancers. Our findings assert the SOX2/FOXA1 interaction as an important mediator of resistance to AR-targeted therapy and a driver of NEPC and lineage plasticity; their coordinated action and downstream signaling offers a potential novel therapeutic opportunity in late-stage PCa.

cancer biology↗

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ER) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ER ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ER breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29s activities in breast cancer cells harboring Y537S ER. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

cancer biology↗

Structural determinants of pure antiestrogenicity

Inhibitory activities of antiestrogens on estrogen receptor alpha (ER) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ER turnover (selective ER degraders; SERDs). Here, we show using a panel of SERMs, SERDs and a PROTAC that efficient induction of ER SUMOylation better accounts for complete transcriptional repression than increased suppression of coactivator recruitment or accelerated ER degradation. Antiestrogen-induced ER SUMOylation depends on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix 12 (H12). L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished SUMOylation with all antiestrogens. Structures of the L536S human ER LBD bound to fulvestrant analogs and molecular dynamics simulations predict dynamic side chain interactions with wild-type ER H12 in the coactivator-binding groove and model the impact of L536 mutations. Together, our results show that induced SUMOylation contributes to the complete suppression of ER activity and provide insights into its structural determinants.

molecular biology↗

Re-education of myeloid immune cells to reduce regulatory T cell expansion and impede breast cancer progression

Immune checkpoint blockade (ICB) has revolutionized cancer therapy but has had limited utility in several solid tumors such as breast cancer, a major cause of cancer-related mortality in women. Therefore, there is considerable interest in alternate strategies to promote an anti-cancer immune response. We demonstrate that NR0B2, a protein involved in cholesterol homeostasis, functions within myeloid immune cells to modulate the NLRP3 inflammasome and reduce the expansion of immune-suppressive regulatory T cells (Treg). Loss of NR0B2 increased mammary tumor growth and metastasis. Small molecule agonists, including one developed here, reduced Treg expansion, reduced metastatic growth and improved the efficacy of ICB. This work identifies NR0B2 as a target to re-educate myeloid immune cells providing proof-of-principle that this cholesterol-homeostasis axis may have utility in enhancing ICB. Brief SummaryImmune therapy has been disappointing for breast cancer. NR0B2 within myeloid immune cells reduces the expansion of Tregs, a highly immune suppressive subtype historically challenging to target. NR0B2 within myeloid immune cells represses the inflammasome, leading to reduced Treg expansion and subsequent tumor growth/metastasis. Activation of NR0B2 with small molecule agonists, including one developed herein, attenuates tumor growth and metastasis in murine models of mammary cancer.

cancer biology↗

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↗

Stereo-specific Lasofoxifene Derivatives Reveal the Interplay between Estrogen Receptor Alpha Stability and Antagonistic Activity in ESR1 Mutant Breast Cancer Cells

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend ER cellular lifetime, accumulation, and are antagonists in the breast and agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders (SERDs) reduce ER cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ER cellular lifetime and accumulation influences antagonistic activity, we synthesized a series of methylpyrollidine lasofoxifene derivatives that maintained the drugs antagonistic activities while uniquely tuning ER cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ER cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ER ligand binding domain in complex with the methylated lasofoxifene derivatives, SERMs, and SERDs show that molecules that favor a highly buried helix 12 conformation achieve the greatest transcriptional suppression activities. Together these results show that chemical reduction of ER cellular lifetime does not necessarily correlate with transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our approach shows how minor chemical additions modulate receptor cellular lifetime while maintaining other activities to achieve desired SERM or SERD profiles. SIGNIFICANCEThis study shows that antiestrogens that enforce a wild-type-like antagonist conformation demonstrate improved therapeutic activities in hormone-resistant breast cancer cells harboring Y537S and D538G ESR1.

cancer biology↗

The SERM/SERD Bazedoxifene Disrupts ESR1 Helix 12 to Overcome Acquired Hormone Resistance in Breast Cancer Cells

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ER) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER+ breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We find BZA possesses improved inhibitory potency against the Y537S and D538G ER mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show that BZAs selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ER mutations.\n\nSignificanceBazedoxifene (BZA) is a potent orally available antiestrogen that is clinically approved for use in hormone replacement therapy (DUAVEE). We explore the efficacy of BZA to inhibit activating somatic mutants of ER that can arise in metastatic breast cancers after prolonged exposure to aromatase inhibitors or tamoxifen therapy. Breast cancer cell line, biophysical, and structural data show that BZA disrupts helix 12 of the ER ligand binding domain to achieve improved potency against Y537S and D538G somatic mutants compared to 4-hydroxytamoxifen.

cancer biology↗