Summary Cancer-related inflammation impacts significantly on cancer development and progression. From early stages, neutrophils and macrophages are drawn to pre-neoplastic cells in the epidermis, but before directly interacting, they must first breach the underlying extracellular matrix barrier layer that includes the basement membrane. Using several different skin cancer models and a collagen I-GFP transgenic zebrafish line, we have undertaken correlative light and electron microscopy (CLEM) to capture the moments when immune cells traverse the basement membrane. We show evidence both for active proteolytic burrowing and for the opportunistic use of pre-existing weak spots in the matrix layer. We show that these small holes, as well as much larger, cancer cell-generated or wound-triggered gaps in the matrix barrier, provide portals for immune cells to access cancer cells in the epidermis and thus are rate limiting in cancer progression.
Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease.
Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a pre-activated nucleoside released. 3'-deoxyadenosine (3'-dA) is a naturally-occurring adenosine analogue with established anti-cancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design:We describe the synthesis of NUC-7738. We determine the IC 50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in cancer patients. Results:We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide binding protein 1. PK and tumour samples obtained from the ongoing first-in-human Phase 1 clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective pro-apoptotic agent in cancer cells with effects on the NF-B pathway.Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and support its further clinical evaluation as a novel cancer treatment within the growing pantheon of anti-cancer ProTides. Statement of translational relevanceProTide modification of nucleoside analogues is designed to overcome the cellular resistance mechanisms that limit their efficacy. This consists of the chemical addition of a protective phosphoramidate moiety onto the parent nucleoside, in this case the natural nucleoside analogue 3'-deoxyadenosine (3'-dA) to form the ProTide NUC-7738. The mode of action of 3'-dA has been the subject of speculation as its major limitation is its short plasma half-life due to rapid enzymatic deamination by adenosine deaminase (ADA). We demonstrate that NUC-7738 is resistant to deamination by ADA and cleaved by the intracellular phosphoramidase HINT1 into 3'-dAMP for conversion to the active metabolites 3'-dADP and 3'-dATP. We show that NUC-7738 promotes proapoptotic pathways and attenuates NF-B. These findings are validated in tumour samples from patients in an ongoing first-in-human trial of NUC-7738 from which we have identified biomarkers to enrich our understanding of NUC-7738 and patients most likely benefiting from it.
LARP1 is an oncogenic RNA-binding protein required for ribosome biogenesis and cancer cell survival. From published in vitro studies, there is disparity over which of two different LARP1 protein isoforms (termed the long LI-LARP1 and short SI-LARP1) is the canonical. Here, after conducting a series of biochemical and cellular assays, we conclude that LI-LARP1 (NM_033551.3 > NP_056130.2) is the dominantly expressed form. We observe that SI-LARP1 (NM_015315.5> NP_056130.2) is epigenetically repressed and that this repression is evolutionarily conserved in all but a small subclade of mammalian species. As with other LARP family members, there are multiple potential LARP1 mRNA isoforms that appear to be censored within the nucleus. The capacity of the cell to modulate splicing and expression of these apparently 'redundant' mRNAs hints at contextually specific mechanisms of LARP1 expression.
Background: 3'-deoxyadenosine (3'-dA; also known as cordycepin) is a nucleoside analog that has shown potent anti-cancer activity in non-clinical studies but has not been clinically developed because of its vulnerability to rapid degradation by the circulating enzyme adenosine deaminase (ADA) and its poor uptake into cancer cells. The ProTide NUC-7738 is a pre-activated and protected nucleotide analog (3'-dA 5'monophosphate; 3'-dAMP) specifically designed to overcome the limitations of 3'-dA. NUC-7738's phosphoramidate moiety renders it resistant to ADA degradation. Here we compared NUC-7738 to 3'-dA in several model systems prior to conducting a first-in-class dose-escalation/expansion study of NUC-7738 in patients with advanced cancers. Materials and Methods: To determine the potency of NUC-7738, IC50 values were measured in multiple cancer cell lines and compared to the parent compound, 3'-dA. Chemical inhibitors of ADA and other 3'-dA processing enzymes were applied to assess the relative ability of NUC-7738 to bypass these pathways. Using genome-wide gene-trap screens and RNA sequencing we compared mechanisms of action (MOA) for NUC-7738 and 3'-dA. Results: NUC-7738 demonstrated up to 185x greater anti-cancer potency than 3'-dA across a variety of cancer cells lines. Gene trap experiments showed that the intracellular activating enzyme adenosine kinase (ADK) and the hENT1 transporter were amongst the highest enriched genes for 3'-dA, whilst no enrichments for these genes were observed in NUC-7738 treated cells. In support of this, in vitro inhibition assays showed that unlike 3'-dA, NUC-7738 is resistant to ADA breakdown, is not reliant on hENT1 transport for its cellular uptake, and is independent of ADK for its activity. As expected, RNA sequencing analysis demonstrated overlap between the MOA of NUC-7738 and 3'-dA; both cause cancer cell death via the intrinsic apoptosis pathway and suppression of pro-survival signaling. Further investigation of gene candidates was employed in ex-vivo cancer kidney cancer samples. Conclusion: Phosphoramidate chemistry was used to transform the nucleoside analog 3'-dA into NUC-7738, rendering it resistant to degradation by ADA and enabling it to enter cancer cells independent of nucleoside transporters, both of which contribute to NUC-7738's substantially greater in vitro potency compared to 3'-dA. The gene trap approach allowed a sophisticated comparison of the MOA of NUC-7738 with 3'-dA. By overcoming the resistance mechanisms associated with 3'-dA, NUC-7738 generates higher levels of the active anti-cancer metabolite in cancer cells. These data supported the initiation of NuTide:701, a first-in-human Phase I study assessing the safety, tolerability, pharmacokinetics and pharmacodynamics of NUC-7738 in patients with advanced solid tumors that are resistant to conventional treatment. Citation Format: Hagen Schwenzer, Michaela Serpi, Valentina Ferrari, James Chettle, Josephine Morris, Ruud van Stiphout, Erica de Zan, Sebastian Nijman, Mustafa Elshani, Mary Kudsy, David Harrison, Gareth Bond, Sarah P. Blagden. From bench to bedside: Using ProTide chemistry to transform 3'-deoxyadenosine into the novel anti-cancer agent Nuc-7738 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 931.
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