Biology subjects

Hill, C. S.

Publications and source records attributed to Hill, C. S..

11 recordsLinked to original sources

bioRxiv2026

The coumarin derivative X6632 is a pan-ID protein inhibitor that suppresses tumor growth by targeting cancer cells and the tumor-associated microvasculature

Inhibitor of DNA binding (ID) proteins are key regulators of tumor cell stemness, therapy resistance and pathological angiogenesis in multiple cancer types and other diseases. Here, we characterize the coumarin-derived compound X6632 as a pan-ID inhibitor with dual activity against tumor cells and the tumor-associated microvasculature in a number of human and murine models. X6632 efficiently suppressed ID protein expression, inhibited the proliferation, migration, invasion of melanoma cells, and impaired multiple endothelial cell functions, including proliferation, migration, invasion, tube formation and sprouting in vitro. In back-to-back comparisons, X6632 exhibited an approximately ten-fold higher efficacy compared to the first-generation ID antagonist AGX51. In vivo, X6632 potently reduced pathological (neo)vascularization in established angiogenesis models, including oxygen-induced retinopathy and in Matrigel plug assays. It also significantly decreased blood vessel density in syngeneic melanoma models, delayed tumor growth and, when combined with immune checkpoint blockade, achieved superior tumor control compared with either monotherapy. Moreover, X6632 inhibited clonogenic growth in several breast cancer models, and robustly suppressed the growth of triple negative breast cancer in vivo, both in the highly aggressive 4T1 syngeneic model and in patient-derived xenografts. Collectively, these data establish X6632 as a second-generation, pan-ID protein inhibitor that can simultaneously target malignant cells and the tumor-supporting vasculature, and support the further pre-clinical development of the compound for the treatment of melanoma, breast cancer and potentially additional ID-dependent malignancies, as well as diseases driven by pathological neoangiogenesis.

cancer biology↗

bioRxiv2026

Pro-domain-dependent folding and co-receptor-mediated targeting to optimize an antagonistic TGF-β monomer for gene-based delivery

Transforming growth factor-beta (TGF-{beta}), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-{beta} inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-{beta} (mmTGF-{beta}), which potently and specifically inhibits TGF-{beta} activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-{beta} was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-{beta} pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-{beta}. Furthermore, we show that fusion of mmTGF-{beta} to a CD44 binding domain enhances the inhibitory potential of mmTGF-{beta} on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-{beta} for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

bioengineering↗

bioRxiv2025

Anti-activin treatment increases T cell infiltration in breast and pancreatic tumours and promotes survival in a SMAD4-null mouse pancreatic cancer model

Activin A and B share the same downstream signalling pathway (activation of SMAD2/3) as TGF-{beta} and consequently elicit many of the same functional responses as TGF-{beta}, including immune suppression, activation of cancer-associated fibroblasts (CAFs) and extracellular matrix production and remodelling. However, activins role in tumourigenesis has been relatively overlooked compared to TGF-{beta}s. We generated and characterized a dual specificity human antibody that recognizes both activin A and B and compared its activity in syngeneic mouse models of breast cancer and pancreatic ductal adenocarcinoma (PDAC) with an activin A-specific antibody. We demonstrate that activin A and B are central to the function of CAFs and therapeutic inhibition of activin results in a reduction of collagen rich desmoplastic barriers, enabling the infiltration of cytotoxic T cells. This is correlated with an upregulation of the T cell chemoattractant CXCL10, which is normally repressed by activin signalling. Interestingly, despite greater T cell infiltration, activin A inhibition resulted in poorer survival in the KPC mouse model of PDAC and slightly larger tumours in the breast cancer model, indicating a tumour suppressive role of activin A-rich CAFs. Strikingly, however, treatment with the same anti-activin A antibody of PDAC tumours where SMAD4 is deleted in the tumour cells, resulted in increased survival, which was potentiated with additional treatment with immune checkpoint inhibitors. These results suggest that anti-activin therapy has potential for the cohort of PDAC patients exhibiting inactivation of SMAD4.

cancer biology↗

bioRxiv2024

Structures of TGF-beta with betaglycan and the signaling receptors reveal the mechanism whereby betaglycan potentiates receptor complex assembly and signaling

Betaglycan (BG) is a transmembrane co-receptor of the transforming growth factor-{beta} (TGF-{beta}) family of signaling ligands. It is essential for embryonic development and tissue homeostasis and fertility in adults. It functions by enabling binding of the three TGF-{beta} isoforms to their signaling receptors and is additionally required for inhibin A (InhA) activity. Despite its requirement for the functions of TGF-{beta}s and InhA in vivo, structural information explaining BG ligand selectivity and its mechanism of action is lacking. Here, we determine the structure of TGF-{beta} bound both to BG and the signaling receptors, TGFBR1 and TGFBR2. We identify key regions responsible for ligand engagement, which has revealed novel binding interfaces that differ from those described for the closely related co-receptor of the TGF-{beta} family, endoglin, thus demonstrating remarkable evolutionary adaptation to enable ligand selectivity. Finally, we provide a structural explanation for the hand-off mechanism underlying TGF-{beta} signal potentiation.

biophysics↗

bioRxiv2023

N4-Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N4-hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.

microbiology↗

bioRxiv2022

An improved Erk biosensor reveals oscillatory Erk dynamics driven by mitotic erasure during early development

Erk signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics in vivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by Fgf signaling at the blastula embryonic margin. Here we describe an improved Erk-specific biosensor which we term modified Erk Kinase Translocation Reporter (modErk-KTR). We demonstrate the utility of this biosensor in vitro and in developing zebrafish and Drosophila embryos. Moreover, we show that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Signaling is rapidly extinguished just prior to mitosis, which we refer to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. Our modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics in vivo.

developmental biology↗

bioRxiv2022

Nodal signaling establishes a competency window for stochastic cell fate switching

Specification of the germ layers by Nodal signaling has long been regarded as an archetype of how graded morphogens induce different cell fates. However, this deterministic model cannot explain why only a subset of cells at the margin of the early zebrafish embryo adopt the endodermal fate, while their immediate neighbours, experiencing similar signaling profiles, become mesoderm. Combining pharmacology, quantitative imaging and single cell transcriptomics, we demonstrate that sustained Nodal signaling establishes a bipotential progenitor state where cells initially fated to become mesoderm can switch to an endodermal fate. Switching is a random event, the likelihood of which is modulated by Fgf signaling. This inherently imprecise mechanism nevertheless leads to robust endoderm formation because of buffering at later stages. Thus, in contrast to previous deterministic models of morphogen action, Nodal establishes a temporal window when cells are competent to undergo a stochastic cell fate switch, rather than determining fate itself.

developmental biology↗

bioRxiv2020

Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-{beta} signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional corepressor SKI, which is a negative regulator of TGF-{beta} signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-{beta} responses, in both knockin cells expressing an SGS mutation, and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-{beta}-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

biochemistry↗

bioRxiv2019

Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses

AbstractThe signalling pathways initiated by members of the transforming growth factor-{beta} (TGF{beta}) family of cytokines control many metazoan cellular processes, including proliferation and differentiation, epithelial-mesenchymal transition (EMT), and apoptosis. TGF{beta} signalling is therefore strictly regulated to ensure appropriate context-dependent physiological responses. In an attempt to identify novel regulatory components of the TGF{beta} signalling pathway, we performed a pharmacological screen using a cell line engineered to report the endogenous transcription of the TGF{beta}-responsive target gene PAI-1. The screen revealed that small-molecular inhibitors of salt-inducible kinases (SIKs) attenuate TGF{beta}-mediated transcription of PAI-1 without affecting receptor-mediated SMAD phosphorylation, SMAD complex formation or nuclear translocation. We provide evidence that genetic inactivation of SIK isoforms also attenuates TGF{beta}-dependent transcriptional responses. Pharmacological inhibition of SIKs using multiple small-molecule inhibitors potentiated apoptotic cell death induced by TGF{beta} stimulation. Our data therefore provides evidence for a novel function of SIKs in modulating TGF{beta}-mediated transcriptional and cellular responses.

cancer biology↗

bioRxiv2019

Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration

Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinsons disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.

neuroscience↗

bioRxiv2019

TGF-β family ligands exhibit distinct signaling dynamics that are driven by receptor localization

Growth factor-induced signal transduction pathways are tightly regulated at multiple points intracellularly, but how cells monitor levels of extracellular ligand and translate this information into appropriate downstream responses remains unclear. Understanding signaling dynamics is thus a key challenge in determining how cells respond to external cues. Here, we demonstrate that different TGF-{beta} family ligands, namely Activin A and BMP4, signal with distinct dynamics, which differ profoundly from those of TGF-{beta} itself. The distinct signaling dynamics are driven by differences in the localization and internalization of receptors for each ligand, which in turn determine the capability of cells to monitor levels of extracellular ligand. Using mathematical modeling, we demonstrate that the distinct receptor behaviors and signaling dynamics observed may be primarily driven by differences in ligand-receptor affinity. Furthermore, our results provide a clear rationale for the different mechanisms of pathway regulation found in vivo for each of these growth factors.

cell biology↗