Biology subjects

Toker, A.

Publications and source records attributed to Toker, A..

13 recordsLinked to original sources

bioRxiv2026

MAPK signaling links BRD2 chromatin occupancy to PI3K/AKT inhibitor sensitivity

Bromodomain and extra-terminal (BET) proteins, BRD2, BRD3, BRD4, and BRDT, couple histone acetylation to transcription by recruiting elongation and transcription factor complexes to chromatin. Although BET proteins are promising therapeutic targets, the functions of individual family members remain incompletely understood. We identify BRD2 as a co-targetable vulnerability with PI3K/AKT inhibition in breast cancer. Pan-BET inhibition and BRD2 knockout synergized with PI3K pathway inhibitors in breast cancer cell lines, patient-derived organoids, and in vivo models. BRD2 knockout impaired proliferation of triple-negative breast cancer cells and upregulated signaling and stress-response pathways, including the DNA damage response. Mechanistically, BRD2 is phosphorylated at Ser37 by the mitogen- and stress-activated kinases MSK and RSK, and Ser37 phosphorylation is required for chromatin binding and reader function. Finally, mechanistic digital twin modeling identified BETi-PI3Ki regimens that maintained efficacy while reducing drug exposure. Together, these findings identify BRD2 as a phosphorylation-dependent, co-targetable vulnerability in PI3K-inhibited breast cancer.

cancer biology↗

bioRxiv2025

The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation

The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls key cellular processes such as survival, proliferation, metabolism and growth. Protein glycosylation is essential for proper protein folding and is also often deregulated in cancer. Cancer cells depend on increased protein folding to sustain oncogene-driven proliferation rates. The N-glycosyltransferase asparagine-linked glycosylation 3 homolog (ALG3), a rate-limiting enzyme during glycan biosynthesis, catalyzes the addition of the first mannose to glycans in an alpha-1,3 linkage. Here we show that ALG3 is phosphorylated downstream of the PI3K/AKT pathway in both growth factor-stimulated cells and PI3K/AKT hyperactive cancer cells. AKT directly phosphorylates ALG3 in the amino terminal region at Ser11/Ser13. CRISPR/Cas9-mediated depletion of ALG3 leads to improper glycan formation and induction of endoplasmic reticulum stress, the unfolded protein response, and impaired cell proliferation. Phosphorylation of ALG3 at Ser11/Ser13 is required for glycosylation of cell surface receptors EGFR, HER3 and E-cadherin. These findings provide a direct link between PI3K/AKT signaling and protein glycosylation in cancer cells.

cancer biology↗

bioRxiv2024

Structural and systems characterization of phosphorylation on metabolic enzymes identifies sex-specific metabolic reprogramming in obesity

Coordination of adaptive metabolism through cellular signaling networks and metabolic response is essential for balanced flow of energy and homeostasis. Post-translational modifications such as phosphorylation offer a rapid, efficient, and dynamic mechanism to regulate metabolic networks. Although numerous phosphorylation sites have been identified on metabolic enzymes, much remains unknown about their contribution to enzyme function and systemic metabolism. In this study, we stratify phosphorylation sites on metabolic enzymes based on their location with respect to functional and dimerization domains. Our analysis reveals that the majority of published phosphosites are on oxidoreductases, with particular enrichment of phosphotyrosine (pY) sites in proximity to binding domains for substrates, cofactors, active sites, or dimer interfaces. We identify phosphosites altered in obesity using a high fat diet (HFD) induced obesity model coupled to multiomics, and interrogate the functional impact of pY on hepatic metabolism. HFD induced dysregulation of redox homeostasis and reductive metabolism at the phosphoproteome and metabolome level in a sex-specific manner, which was reversed by supplementing with the antioxidant butylated hydroxyanisole (BHA). Partial least squares regression (PLSR) analysis identified pY sites that predict HFD or BHA induced changes of redox metabolites. We characterize predictive pY sites on glutathione S-transferase pi 1 (GSTP1), isocitrate dehydrogenase 1 (IDH1), and uridine monophosphate synthase (UMPS) using CRISPRi-rescue and stable isotope tracing. Our analysis revealed that sites on GSTP1 and UMPS inhibit enzyme activity while the pY site on IDH1 induces activity to promote reductive carboxylation. Overall, our approach provides insight into the convergence points where cellular signaling fine-tunes metabolism. Summary StatementBy employing a multi-disciplinary approach we stratify structural features of phosphorylation sites on metabolic enzymes, map the systems level changes induced by obesity, identify key pathways with sex specific phosphoproteomic responses, and validate the functional role of phosphorylation sites for select enzymes.

biochemistry↗

bioRxiv2024

Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved post-translational modification that facilitates rapid and reversible modulation of enzyme activity, localization or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in non-tumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the epidermal growth factor receptor (EGFR) inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (PKM, PGAM1) and two novel (GSTP1, GLUD1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference (CRISPRi) system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous re-expression of wild-type, phosphomutant or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

biochemistry↗

bioRxiv2024

Cholesterol biosynthesis inhibition synergizes with AKT inhibitors in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer deaths due to its molecular heterogeneity, high recurrence rate and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. We performed a genome-wide CRISPR/Cas9 screen with PI3K and AKT inhibitors to find targetable synthetic lethalities in TNBC. We identified cholesterol homeostasis as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived, estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single agent or combination treatment with AKT inhibitor and pitavastatin. This was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. Our work motivates combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC.

cancer biology↗

bioRxiv2022

Progressive loss of conserved spike protein neutralizing antibody sites in Omicron sublineages is balanced by preserved T-cell recognition epitopes

The continued evolution of the SARS-CoV-2 Omicron variant has led to the emergence of numerous sublineages with different patterns of evasion from neutralizing antibodies. We investigated neutralizing activity in immune sera from individuals vaccinated with SARS-CoV-2 wild-type spike (S) glycoprotein-based COVID-19 mRNA vaccines after subsequent breakthrough infection with Omicron BA.1, BA.2, or BA.4/BA.5 to study antibody responses against sublineages of high relevance. We report that exposure of vaccinated individuals to infections with Omicron sublineages, and especially with BA.4/BA.5, results in a boost of Omicron BA.4.6, BF.7, BQ.1.1, and BA.2.75 neutralization, but does not efficiently boost neutralization of sublineages BA.2.75.2 and XBB. Accordingly, we found in in silico analyses that with occurrence of the Omicron lineage a large portion of neutralizing B-cell epitopes were lost, and that in Omicron BA.2.75.2 and XBB less than 12% of the wild-type strain epitopes are conserved. In contrast, HLA class I and class II presented T-cell epitopes in the S glycoprotein were highly conserved across the entire evolution of SARS-CoV-2 including Alpha, Beta, and Delta and Omicron sublineages, suggesting that CD8+ and CD4+ T-cell recognition of Omicron BQ.1.1, BA.2.75.2, and XBB may be largely intact. Our study suggests that while some Omicron sublineages effectively evade B-cell immunity by altering neutralizing antibody epitopes, S protein-specific T-cell immunity, due to the very nature of the polymorphic cell-mediated immune, response is likely to remain unimpacted and may continue to contribute to prevention or limitation of severe COVID-19 manifestation.

immunology↗

bioRxiv2022

Multi-omic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation

Over 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, there remains scope to improve upon the therapeutic window of the current generation of AKT inhibitors. Here we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-driven phenotypes in breast cancer cell lines. A systematic growth inhibition screen across 288 cancer cell lines confirmed a substantially higher potency for INY-05-040 (median GI50adj = 1.1 {micro}M) compared to our first-generation AKT degrader (INY-03-041; median GI50adj = 3.1 {micro}M), with both compounds outperforming catalytic AKT inhibition with GDC-0068 (median GI50adj > 10 {micro}M). Using multi-omic profiling and causal network integration in breast cancer cells, we demonstrate that the enhanced efficacy of INY-05-040 is associated with sustained suppression of AKT signaling, followed by a potent induction of the stress mitogen activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low baseline JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Collectively, our study presents a systematic framework for mapping the network-wide signaling effects of therapeutically relevant compounds, and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

cancer biology↗

bioRxiv2022

Exposure to BA.4/BA.5 Spike glycoprotein drives pan-Omicron neutralization in vaccine-experienced humans and mice

The SARS-CoV-2 Omicron variant and its sublineages show pronounced viral escape from neutralizing antibodies elicited by vaccination or prior SARS-CoV-2 variant infection owing to over 30 amino acid alterations within the spike (S) glycoprotein. We and others have recently reported that breakthrough infection of vaccinated individuals with Omicron sublineages BA.1 and BA.2 are associated with distinct patterns of cross-neutralizing activity against SARS-CoV-2 variants of concern (VOCs). BA.2 breakthrough infection mediated overall stronger cross-neutralization of BA.2 and its descendants (BA.2.12.1, BA.4, and BA.5) compared to BA.1 breakthrough infection. Here we characterized the effect of Omicron BA.4/BA.5 S glycoprotein exposure on the magnitude and breadth of the neutralizing antibody response upon breakthrough infection in vaccinated individuals and in mice upon booster vaccination. We show that immune sera from triple mRNA-vaccinated individuals with subsequent Omicron BA.4/BA.5 breakthrough infection display broad and robust neutralizing activity against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5. Administration of a prototypic BA.4/BA.5-adapted mRNA booster vaccine to mice following SARS-CoV-2 wild-type strain-based primary immunization is associated with similarly broad neutralizing activity. Immunization of naive mice with a bivalent mRNA vaccine (wild-type + Omicron BA.4/BA.5) induces strong and broad neutralizing activity against Omicron VOCs and previous variants. These findings suggest that when administered as boosters, mono- and bivalent Omicron BA.4/BA.5-adapted vaccines may enhance neutralization breadth, and in a bivalent format may also have the potential to confer protection to individuals with no pre-existing immunity against SARS-CoV-2.

immunology↗

bioRxiv2022

Omicron BA.2 breakthrough infection enhances cross-neutralization of BA.2.12.1 and BA.4/BA.5

Recently, we reported that BNT162b2-vaccinated individuals after Omicron BA.1 breakthrough infection have strong serum neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 variants of concern (VOCs), yet less against the highly contagious Omicron sublineages BA.4 and BA.5 that have displaced previous variants. As the latter sublineages are derived from Omicron BA.2, we characterized serum neutralizing activity of COVID-19 mRNA vaccine triple-immunized individuals who experienced BA.2 breakthrough infection. We demonstrate that sera of these individuals have broadly neutralizing activity against previous VOCs as well as all tested Omicron sublineages, including BA.2 derived variants BA.2.12.1, BA.4/BA.5. Furthermore, applying antibody depletion we showed that neutralization of BA.2 and BA.4/BA.5 sublineages by BA.2 convalescent sera is driven to a significant extent by antibodies targeting the N-terminal domain (NTD) of the spike glycoprotein, whereas their neutralization by Omicron BA.1 convalescent sera depends exclusively on antibodies targeting the receptor binding domain (RBD). These findings suggest that exposure to Omicron BA.2, in contrast to BA.1 spike glycoprotein, triggers significant NTD specific recall responses in vaccinated individuals and thereby enhances the neutralization of BA.4/BA.5 sublineages. Given the current epidemiology with a predominance of BA.2 derived sublineages like BA.4/BA.5 and rapidly ongoing evolution, these findings are of high relevance for the development of Omicron adapted vaccines.

immunology↗

bioRxiv2021

FGFR inhibitor mediated dismissal of SWI/SNF complexes from YAP-dependent enhancers induces adaptive therapeutic resistance

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodeling leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Collectively, these findings reveal a novel feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance.

cancer biology↗

bioRxiv2020

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia

The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial cells that comprise the vasculature are poorly connected causing vessels to be leaky and exposing the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are generally inefficient and result in acquired resistance to increased hypoxia due to elimination of the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of endothelial cells in response to hypoxia has been relatively unexplored. Here, we measured polar metabolite levels in microvascular endothelial cells exposed to short- and long-term hypoxia with the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor vasculature and improve drug delivery. Many amino acid-related metabolites were altered by hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-methionine metabolism. Additionally, there were significant changes in de novo pyrimidine synthesis as well as glutathione and taurine metabolism. These results provide key insights into the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a foundation for future studies to develop therapies that lead to vessel normalization and more efficient drug delivery.

cancer biology↗

bioRxiv2020

Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy

Treatment of triple-negative breast cancer (TNBC) is limited by a lack of effective molecular targeted therapies. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. We found that exposure of TNBC cells to cytotoxic chemotherapy drugs leads to alterations in arginine and polyamine metabolites due to a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme ornithine decarboxylase (ODC). The reduction in ODC was mediated by its negative regulator, antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor DFMO sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines showed greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. Alterations in polyamine metabolism in response to chemotherapy, as well as preferential sensitization of TNBC cells to chemotherapy by DFMO, suggest that ODC may be a targetable metabolic vulnerability in TNBC.

cancer biology↗

bioRxiv2019

Discovery of an AKT Degrader with Prolonged Inhibition ofDownstream Signaling

The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 hours, even after compound washout. Our findings indicate that AKT degradation may confer prolonged pharmacological effects compared to inhibition, and highlight the potential advantages of AKT-targeted degradation.

cancer biology↗