Biology subjects

Agarwal, S.

Publications and source records attributed to Agarwal, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

A genotoxic antibody drug conjugate targeting CD276/B7H3 demonstrates efficacy across multiple biomarker defined classes of treatment refractory metastatic prostate cancer

Antibody-drug conjugates (ADCs) are promising targeted cancer therapy; however, patient selection based solely on target antigen expression without consideration for cytotoxic payload vulnerabilities has plateaued clinical benefits. Biomarkers to capture patients who might benefit from specific ADCs have not been systematically determined for any cancer. We present a comprehensive therapeutic and biomarker analysis of a B7H3-ADC with pyrrolobenzodiazepine(PBD) payload in 26 treatment-resistant, metastatic prostate cancer(mPC) models. B7H3 is a tumor-specific surface protein widely expressed in mPC, and PBD is a DNA cross-linking agent. B7H3 expression was necessary but not sufficient for B7H3-PBD-ADC responsiveness. RB1 deficiency and/or replication stress, characteristics of poor prognosis, conferred sensitivity and were associated with complete tumor regression in both neuroendocrine (NEPC) and androgen receptor positive(ARPC) prostate cancer models, even with low B7H3 levels. Non-ARPC models, which are currently lacking efficacious treatment, demonstrated the highest replication stress and were most sensitive to treatment. In RB1 wild-type ARPC tumors, SLFN11 expression or select DNA repair mutations in SLFN11 non-expressors governed response. Significantly, wild-type TP53 predicted non-responsiveness (7/8 models). Overall, biomarker-focused selection of models led to high efficacy of in vivo treatment. These data enable a paradigm shift to biomarker-driven trial designs for maximizing clinical benefit of ADC therapies.

cancer biology↗

bioRxiv2022

Dietary αKG inhibits SARS CoV-2 infection and rescues inflamed lungs to restore normal O2 saturation in animals

Our recent works described the rescue effect of -ketoglutarate (KG, a metabolite of Krebs cycle) on thrombosis and inflammation in animals. KG augments activity of prolyl hydroxylase 2 (PHD2), which in turn degrades proline residues of substrates like phosphorylated Akt (pAkt) and hypoxia inducible factor (HIF). Here we describe the inhibitory effect of octyl KG on pAkt as well as on HIF1/HIF2, and in turn decreasing SARS CoV-2 replication in Vero E6 cells. KG failed to inhibit the viral replication and Akt phosphorylation in PHD2-knockdown U937 cells transiently expressing ACE2. Contrastingly, triciribine (TCN, an Akt-inhibitor) inhibited viral replication alongside a downmodulation of pAkt in PHD2-KD cells. Dietary KG significantly inhibited viral infection and rescued hamsters from thrombus formation and inflammation in lungs, the known causes of acute respiratory distress syndrome (ARDS) in COVID-19. KG supplementation also reduced the apoptotic death of lung tissues in infected animals, alongside a downmodulation of pAkt and HIF2. KG supplementation neither affected IgG levels against SARS CoV-2 RBD protein nor altered the neutralization antibody response against SARS CoV-2. It did not interfere with the percentage of interferon-{gamma} positive (IFN{gamma}+) CD4+ and IFN{gamma}+CD8+ T cells in infected animals. The extended work in balb/c mice transiently expressing ACE2 showed a similar effect of KG in reducing accumulation of inflammatory immune cells and cytokines, including IL6, IL1{beta} and TNF, in lungs as well as in circulation of infected animals. Pro-thrombotic markers like platelet microparticles and platelet-leukocyte aggregates were reduced significantly in infected mice after KG supplementation. Importantly, KG supplementation restored the O2 saturation (SpO2) in circulation of SARS CoV-2 infected hamsters and mice, suggesting a potential therapeutic role of this metabolite in COVID-19 treatment.

pathology↗

bioRxiv2022

The growth rate of DNA condensate droplets increases with the size of participating subunits

Liquid-liquid phase separation (LLPS) is a common phenomenon underlying the formation of dynamic membraneless organelles in biological cells, which are emerging as major players in controlling cellular functions and health. The bottom-up synthesis of biomolecular liquid systems with simple constituents, like nucleic acids and peptides, is useful to understand LLPS in nature as well as to develop programmable means to build new amorphous materials with properties matching or surpassing those observed in natural condensates. In particular, understanding which parameters determine condensate growth kinetics is essential for the synthesis of condensates with the capacity for active, dynamic behaviors. Here we use DNA nanotechnology to study artificial liquid condensates through programmable star-shaped subunits, focusing on the effects of changing subunit size. First, we show that LLPS is achieved in a six-fold range of subunit size. Second, we demonstrate that the rate of growth of condensate droplets scales with subunit size. Our investigation is supported by a general model that describes how coarsening and coalescence are expected to scale with subunit size under ideal assumptions. Beyond suggesting a route toward achieving control of LLPS kinetics via design of subunit size in synthetic liquids, our work suggests that particle size may be a key parameter in biological condensation processes.

bioengineering↗

bioRxiv2022

A cancer stem cell population underlies a multi-lineage phenotype and drug resistance in prostate cancer

PurposeTo resist lineage-dependent therapies such as androgen receptor inhibition in prostate cancer, cancer cells often adopt a stem-like state resulting in lineage-plasticity and phenotypic heterogeneity. We assessed the dynamics of lineage determination and cellular subpopulation expansion in treatment-resistant adenocarcinoma, amphicrine, and small cell neuroendocrine castrate resistant prostate cancers (CRPCs). Experimental DesignWe developed CRPC patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model harboring epigenetic driver mutations, ARID1A and ARID1B, and displaying a range of luminal and neuroendocrine phenotypes. We used single-cell RNA-seq, barcode lineage-tracing, single-cell ATAC-seq, and RNA-FISH to delineate the subpopulation structure of the heterogeneous organoids and define the lineage hierarchy, determine potential transcriptional regulators of amphicrine lineage-plasticity, and identify subpopulation-specific molecular targets for therapeutic intervention. ResultsTranscriptionally similar stem/progenitor cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent stem/progenitor cells that produced mainly quiescent differentiated amphicrine progeny. Amphicrine cells were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures. By contrast, adenocarcinoma CRPC had a less defined hierarchy, as progeny originated from stem/progenitor cells and self-renewing differentiated luminal cells. NEPC was composed almost exclusively of self-renewing stem/progenitor cells. Amphicrine stem cells demonstrated concurrent transcription factor activities associated with stem/progenitor, luminal epithelial and mesenchymal lineages. Finally, the amphicrine stem/progenitor subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. ConclusionsThese data illuminate distinct origins and dynamics of subtype-specific CRPC plasticity in addition to demonstrating a strategy for targeting differentiation-competent stem cells. Translational RelevanceFor advanced prostate cancer, therapeutic resistance to androgen signaling suppression increasingly involves the development of lineage plasticity. The cellular states of transition and subpopulation heterogeneity that underlie lineage-plasticity are not well understood, which is an ongoing challenge to the design of effective treatments. Using patient-derived organoid models of various CRPC lineage subtypes, we observed distinct patterns with respect to stem/progenitor activity and associated growth phenotypes. The simultaneous expression of AR-driven and neuroendocrine identities, so-called amphicrine tumors, are thought to be an early dedifferentiation stage in plasticity-mediated resistance. We observed in an epigenetically-driven, amphicrine model of CRPC that a rare but necessary bipotent stem/progenitor population is suppressed by AURKA inhibitors, leading to tumor regression, while ARPC demonstrates both self-renewing differentiated luminal cells and stem/progenitors. These data suggest that AURKA inhibition may block the amplification of a resistance dedifferentiation pathway and should be considered in combination with AR signaling inhibitors for ARPC with characteristics of lineage plasticity.

cancer biology↗

bioRxiv2022

Comparative analysis of MERFISH spatial transcriptomics with bulk and single-cell RNA sequencing

Spatial transcriptomics extends single cell RNA sequencing (scRNA-seq) by providing spatial context for cell type identification and analysis. Imaging-based spatial technologies such as Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) can achieve single-cell resolution, directly mapping single cell identities to spatial positions. MERFISH produces an intrinsically different data type than scRNA-seq and a technical comparison between the two modalities is necessary to ascertain how to best integrate them. We performed MERFISH on mouse liver and kidney and compared the resulting bulk and single-cell RNA statistics with those from the Tabula Muris Senis cell atlas as well as from two Visium datasets. MERFISH quantitatively reproduced the bulk RNA-seq and scRNA-seq results with improvements in overall dropout rates and sensitivity. Finally, we found that MERFISH independently resolved distinct cell types and spatial structure in both liver and kidney. Computational integration with the Tabula Muris Senis atlas did not enhance these results. We conclude that compared to scRNA-seq, MERFISH provides a quantitatively comparable method for measuring single-cell gene expression and can robustly identify cell types without the need for computational integration with scRNA-seq reference atlases.

bioinformatics↗

bioRxiv2021

Zebrafish xenografts to isolate unique human breast cancer metastatic cell populations

Cancer metastasis is a critical culprit frequently blamed for treatment failure, drug resistance, poor prognosis, and high mortality rate among all human cancers. Laboratory efforts to isolate metastatic cell populations have typically been confined to mouse models, which are time-consuming and expensive. Here, we present a model system based on xenografting zebrafish embryos to select for cells that are predisposed to progress through the early stages of metastasis. This model requires only 3-5 days to achieve distinct intravasation to the zebrafish circulatory system. The metastatic cells are easily tracked in real-time as they migrate, as well as isolated and propagated in vitro. Once expanded, molecular characterization of the serially derived invasive cell populations from the tails of the zebrafish accurately predicts genes, signaling pathways, protein-protein interactions, and differential splicing products that are important for an invasive phenotype. This zebrafish model therefore offers a high-throughput and robust method for identifying gene targets critical for cancer metastasis.

cancer biology↗

bioRxiv2021

Rapamycin microparticles induce autophagy, prevent senescence and are effective in treatment of Osteoarthritis

Trauma to the knee joint is associated with significant cartilage degeneration and erosion of subchondral bone, which eventually leads to osteoarthritis (OA), resulting in substantial morbidity and healthcare burden. With no disease-modifying drugs in clinics, the current standard of care focuses on symptomatic relief and viscosupplementation. Modulation of autophagy and targeting senescence pathways are emerging as potential treatment strategies. Rapamycin has shown promise in OA disease amelioration by autophagy upregulation, yet its clinical use is hindered by difficulties in achieving therapeutic concentrations, necessitating multiple weekly injections. Here, we have synthesized rapamycin - loaded poly (lactic-co-glycolic acid) microparticles (RMPs) that induced autophagy, prevented senescence and sustained sulphated glycosaminoglycans(sGAG) production in primary human articular chondrocytes from OA patients. RMPs were potent, nontoxic, and exhibited high retention time (up to 35 days) in mice joints. Intra-articular delivery of RMPs effectively mitigated cartilage damage and inflammation in surgery-induced OA when administered as a prophylactic or therapeutic regimen. Together, our studies demonstrate the feasibility of using RMPs as a potential clinically translatable therapy to prevent and treat post-traumatic osteoarthritis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/453073v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@29863org.highwire.dtl.DTLVardef@628da3org.highwire.dtl.DTLVardef@40ec6eorg.highwire.dtl.DTLVardef@626355_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Alpha-ketoglutarate augments prolyl hydroxylase-2 mediated inactivation of phosphorylated-Akt to inhibit induced-thrombosis and inflammation

Phosphorylation of Akt (pAkt) regulates multiple physiological and pathological processes including thrombosis and inflammation. In an approach to inhibit the pathological signalling of pAkt by prolyl-hydroxylase-2 (PHD2) we employed -ketoglutarate (KG), a cofactor of PHD2. Octyl-KG supplementation to platelets promoted PHD2 activity through elevated intracellular KG:succinate ratio and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline alongside enhanced binding of PHD2 to pAkt in KG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-KG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelet and leukocyte obtained from mice supplemented with dietary-KG, unaccompanied by alteration in their counts. Dietary-KG significantly reduced clot formation and leukocyte accumulation in various organs including lung of mice treated with thrombosis-inducing agent carrageenan. Importantly, we observed a significant rescue effect of dietary-KG on inflamed lung of SARS-CoV-2 infected hamsters. KG significantly reduced leukocyte accumulation, clot formation and viral load alongside downmodulation of pAkt in lung of the infected animals. Therefore, our study suggests a safe implementation of dietary-KG in prevention of Akt-driven anomalies including thrombosis and inflammation, highlighting a better pulmonary management in COVID-19.

pathology↗

bioRxiv2021

CanDIG: Secure Federated Genomic Queries and Analyses Across Jurisdictions

Rapid expansions of bioinformatics and computational biology have broadened the collection and use of -omics data including genomic, transcriptomic, methylomic and a myriad of other health data types, in the clinic and the laboratory. Both clinical and research uses of such data require co-analysis with large datasets, for which participant privacy and the need for data custodian controls must remain paramount. This is particularly challenging in multi-jurisdictional settings, such as Canada, where health privacy and security requirements are often heterogeneous. Data federation presents a solution to this, allowing for integration and analysis of large datasets from various sites while abiding by local policies. The Canadian Distributed Infrastructure for Genomics platform (CanDIG) enables federated querying and analysis of -omics and health data while keeping that data local and under local control. It builds upon existing infrastructures to connect five health and research institutions across Canada, relies heavily on standards and tooling brought together by the Global Alliance for Genomics and Health (GA4GH), implements a clear division of responsibilities among its participants and adheres to international data sharing standards. Participating researchers and clinicians can therefore contribute to and quickly access a critical mass of -omics data across a national network in a manner that takes into account the multi-jurisdictional nature of our privacy and security policies. Through this, CanDIG gives medical and research communities the tools needed to use and analyze the ever-growing amount of -omics data available to them in order to improve our understanding and treatment of various conditions and diseases. CanDIG is being used to make genomic and phenotypic data available for querying across Canada as part of data sharing for five leading pan-Canadian projects including the Terry Fox Comprehensive Cancer Care Centre Consortium Network (TF4CN) and Terry Fox PRecision Oncology For Young peopLE (PROFYLE), and making data from provincial projects such as POG (Personalized Onco- Genomics) more widely available.

genomics↗

bioRxiv2021

The clinical and functional effects of TERT variants in myelodysplastic syndrome

Germline pathogenic TERT variants are associated with short telomeres and an increased risk of developing myelodysplastic syndrome (MDS) among patients with a telomere biology disorder. We identified TERT rare variants in 41 of 1514 MDS patients (2.7%) without a clinical diagnosis of telomere biology disorder who underwent allogeneic transplantation. Patients with TERT rare variants had shorter telomere length (p<0.001) and younger age at MDS diagnosis (52 vs. 59 years, p=0.03) than patients without a TERT rare variant. In multivariable analyses, TERT rare variants were associated with inferior overall survival (p=0.034) driven by an increased incidence of non-relapse mortality (NRM) (p=0.015). Death from a non-infectious pulmonary cause was more frequent among patients with a TERT rare variant. According to ACMG/AMP guidelines and Sherloc criteria, 39 TERT rare variants were classified as VUS and one as likely pathogenic. Therefore, we cloned all rare missense variants and quantified their impact on telomere elongation in a cell-based assay. We found that 36 of 40 variants had severe or intermediate impairment in their capacity to elongate telomeres. Using a homology model of human TERT bound to the shelterin protein TPP1, we inferred that TERT rare variants disrupt domain-specific functions, including catalysis, protein-RNA interactions, and recruitment to telomeres. Our results indicate that the contribution of TERT rare variants to MDS pathogenesis and NRM risk is underrecognized and routine screening for TERT rare variants in MDS patients regardless of age or clinical suspicion could identify clinically inapparent telomere biology disorders and improve transplant outcomes through risk-adapted approaches.

genetics↗

bioRxiv2021

ARID1A-mutant and deficient bladder cancer is sensitive to EZH2 pharmacologic inhibition

Metastatic urothelial carcinoma of the bladder is generally incurable by current systemic therapy. Molecular characterization of bladder cancer (BLCa) has revealed multiple candidate driver genes for BLCa tumorigenesis. Epigenetic/chromatin modifiers have been shown to be frequently mutated in BLCa, with ARID1A mutations highly prevalent in nearly 20% of early and late stage tumors. EZH2 is a histone methyltransferase that acts as an oncogene. The data herein show that ARID1A deficient tumors, but not ARID1A wild-type tumors are sensitive to EZH2 inhibition. Specifically, EZH2 inhibitor-treated ARID1A deficient bladder cancer cells show significantly reduced cell viability, colony formation, and in vivo tumor growth relative to ARID1A-wild type bladder cancer cells. Thus, our study suggests that a specific subset of bladder cancer patients with ARID1A mutations can be therapeutically treated with pharmacologic inhibitors targeting EZH2.

cancer biology↗

bioRxiv2020

Separation and enrichment of sodium-motile bacteria using cost-effective microfluidics.

Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied example is the use of the sodium powered PomA-PotB chimeric stator unit in the naturally proton-powered E. coli. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive-force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eight-fold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement selection of highly-motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems.

microbiology↗

bioRxiv2020

Reprogramming of the FOXA1 cistrome in treatment-emergent neuroendocrine prostate cancer

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer1,2. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. To investigate the epigenomic basis of this resistance mechanism, we profiled histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identified a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium3,4, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying novel cancer dependencies through epigenomic profiling.

cancer biology↗

bioRxiv2020

Stagewise identification of significantly mutated gene clusters in major molecular classes of breast invasive carcinoma

Breast invasive carcinoma (BRCA) is most malignant and leading cause of death in women. The efforts are ongoing for improvement in early detection, prevention and treatment. Therefore, identification of biomarkers/candidate genes has become very important. The current work includes comprehensive analysis of RNA-sequencing data of 1097 BRCA samples and 114 normal adjacent tissues to identify dysregulated genes in major molecular classes of BRCA in various clinical stages. Huge number of dysregulated genes were found, some were stage-specific, and others were common. The pathways as interferon signaling, tryptophan degradation III, granulocyte adhesion & diapedesis and catecholamine biosynthesis were found in ER/PR+/HER-2- (p-value<0.010), pathways as RAR activation, adipogenesis, role of JAK1, JAK2 in interferon signaling, TGF-{beta} and STAT3 signaling (p-value<0.014) intricated in ER/PR-/HER-2+ and pathways as IL-1/IL-8 signaling, TNFR1/TNFR2 signaling, TWEAK and relaxin signaling (p-value<0.005) were found in triple negative breast cancer. The genes were clustered based on their mutation profile which revealed nine mutated clusters, some of which were known to be involved in well-known cancer signalling pathways while others were less characterized. Each cluster was analyzed in detail which led us to discover genes viz. NLGN3, MAML2, TTN, SYNE1, ANK2 as candidates in BRCA. The genes were found to be involved in important processes as chemotaxis, axon guidance, notch binding, cell-adhesion-molecule binding etc. They are central genes in the protein-protein-interaction network indicating they can have important regulatory roles. The qRT-PCR and western blot confirmed our findings in breast cancer cell-lines. Further, immunohistochemistry corroborated the results in ~100 tissue samples. The genes can be used as biomarker in BRCA.

bioinformatics↗

bioRxiv2020

LFA-1 signals to promote actin polymerization and upstream migration in T cells

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the PI3K and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, while VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl, failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior. Summary StatementInflammatory responses require leukocyte migration along the vascular wall. We show that signaling from {beta}2, but not {beta}1, integrins induces cytoskeletal changes needed for upstream migration under shear flow.

cell biology↗

bioRxiv2020

A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

Membrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. The phosphorylation site we identified is highly conserved in evolution and is regulated by the Ca2+-dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel mechanism by which Ykt6 conformation and activity is regulated by Ca2+ signaling with implications in Parkinsons Disease in which Ykt6 has been shown to play a role.

cell biology↗

bioRxiv2019

Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGF{beta}1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic TGF{beta} levels help ameliorate HO. Our data thus implicate CD47 activation as a novel therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.

immunology↗

bioRxiv2019

Targeting the PI3K/AKT pathway overcomes enzalutamide resistance by inhibiting induction of the glucocorticoid receptor

The PI3K-AKT pathway has pleiotropic effects, and its inhibition has long been of interest in the management of prostate cancer, where a compensatory increase in PI3K signaling has been reported following Androgen Receptor (AR) blockade. Prostate cancer cells can also bypass AR blockade through induction of other hormone receptors, in particular the glucocorticoid receptor (GR). Here we demonstrate that AKT inhibition significantly decreases cell proliferation through both cytostatic and cytotoxic effects. The cytotoxic effect is enhanced by AR inhibition and is most pronounced in models that induce compensatory GR expression. AKT inhibition increases canonical AR activity and remodels the chromatin landscape, decreasing enhancer interaction at the GR gene (NR3C1) locus. Importantly, it blocks induction of GR expression and activity following AR blockade. This is confirmed in multiple in vivo models, where AKT inhibition of established xenografts leads to increased canonical AR activity, decreased GR expression, and marked anti-tumor activity. Overall, our results demonstrate that inhibition of the PI3K/AKT pathway can block GR activity and overcome GR-mediated resistance to AR-targeted therapy. Ipatasertib is currently in clinical development, and GR induction may be a biomarker to identify responsive patients or a responsive disease state.\n\nSIGNIFICANCEInduced GR expression is compensatory for AR blockade and confers resistance to AR-targeted therapy. Here we show that inhibition of the PI3K/AKT pathway remodels the chromatin landscape, blocks the induction of GR expression and overrides enzalutamide resistance.

cancer biology↗