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IntroductionVasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supportingde novovessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs.MethodsWe analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX and GEMMs. VM-proficient cell subpopulations inex vivocultures were molecularly profiled by RNA sequencing and mass spectrometry. We evaluated their 3D structure and defined collagen-integrin interactions.ResultsWe show that VM vessels are present in 23/25 CDX models and in 2 GEMMs. Perfused VM vessels support tumor growth and only Notch-active non-NE cells are VM-competentin vivoandex vivo, expressing pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization signatures. On Matrigel, VM-primed non-NE cells re-model ECM into hollow tubules in an integrin β1-dependent process.ConclusionsWe identify VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take significant steps towards understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve SCLC patient outcomes in future.

The Notch pathway is a conserved cell-cell communication pathway that controls cell fate decisions. Here we sought to determine how Notch pathway activation inhibits the neuroendocrine cell fate in the lungs, an archetypal process for cell fate decisions orchestrated by Notch signaling that has remained poorly understood at the molecular level. Using intratumoral heterogeneity in small-cell lung cancer as a tractable model system, we uncovered a role for the transcriptional regulators REST and YAP as promoters of the neuroendocrine to non-neuroendocrine transition. We further identified the specific neuroendocrine gene programs repressed by REST downstream of Notch in this process. Importantly, we validated the importance of REST and YAP in neuroendocrine to non-neuroendocrine cell fate switches in both developmental and tissue repair processes in the lungs. Altogether, these experiments identify conserved roles for REST and YAP in Notch-driven inhibition of the neuroendocrine cell fate in embryonic lungs, adult lungs, and lung cancer.

Introduction Recent consensus defines four SCLC subtypes on the basis of transcription factor expression: ASCL1, NEUROD1, POU2F3, and YAP1. The rare YAP1 subtype is associated with “neuroendocrine (NE)-low” cells among SCLC cell lines and patient samples. We evaluated YAP1 in 39 patients with phenotypically diverse circulating tumor cell–derived explant (CDX) models and revisited YAP1 in terms of prevalence, cell phenotype, and intertumor and intratumor heterogeneity. Methods YAP1 transcript and protein expression were assessed by RNA sequencing and immunohistochemistry or multiplexed immunofluorescence of NE and non-NE CDX subpopulations. Physically separated NE and non-NE CDX ex vivo culture lysates were Western blotted for YAP1, NE marker SYP, and AXL. Results RNA sequencing normalized for the four subtype transcription factors identified YAP1 expression in 14 of 39 CDX. A total of 10 CDX expressed YAP1 protein, and eight had strong YAP1 expression confined to rare non-NE cell clusters. This was confirmed in ex vivo CDX cultures in which adherent non-NE cells lacking SYP expression expressed YAP1. However, in two CDX, weaker cellular YAP1 expression was observed, widely dispersed in SYP-positive NE cells. Conclusions YAP1 was predominantly expressed in non-NE cell clusters in SCLC CDX, but two of 39 CDX expressed YAP1 in NE cells. CDX22P, with relatively high YAP1 expression, is an ASCL1 NE subtype with a low NE score and an outlier within this subtype in our CDX biobank. These descriptive data reveal subtly different YAP1 expression profiles, paving the way for functional studies to compare YAP1 signaling in non-NE and low NE cell contexts for potentially personalized therapeutic approaches.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine (NE) cancer with rapid and widespread metastasis and a dismal prognosis measurable only in months. The inherent lack of tumor biopsies has limited our understanding of SCLC phenotypic heterogeneity, and except for the addition of Immune Checkpoint Inhibitors the therapeutic strategy has remained largely unchanged for decades. We are investigating Vascular Mimicry (VM) in SCLC, which is linked to poor prognosis and may provide a novel therapeutic opportunity1. VM describes the ability of aggressive tumor cells to undergo trans-endothelial differentiation, enabling angiogenesis-independent tumor vascularization. We hypothesize that, via extracellular matrix (ECM) remodeling, VM provides a favorable tumor microenvironment (TME) to support tumor progression and metastasis. We explored SCLC VM competency and molecular mechanisms underpinning this using histopathology and transcriptomic and proteomic analyses of NE and non-NE SCLC cells in patient faithful Circulating Tumor Cell (CTC)-Derived eXplant (CDX)2,3 models and genetically-engineered mouse models (GEMMs). We demonstrate that within human and murine SCLC tumors in vivo and ex vivo the non-NE cells, unlike NE cells, are VM competent. NE SCLC cells in an ASCL1-expressing CDX model undergo a Notch-driven transition to a non-NE phenotype. We show that non-NE cells are transcriptionally primed to undergo VM and express gene signatures consistent with pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization. These cells undergo post-translational modification of proteins involved in cell-cell and cell-ECM adhesion including a VM-specific glycosylation of integrin β1. On Matrigel VM-primed non-NE cells remodeled the ECM forming tubular networks, an established surrogate assay for VM competence. Integrin β1 blockade impedes VM in vitro by inhibiting non-NE cell collagen adhesion and thus ECM remodeling. This previously unrecognized functional intratumoral heterogeneity in SCLC supports therapeutic co-targeting of NE and non-NE cells to curtail VM-supported tumor growth and metastasis. The ultimate aim is to identify targetable therapeutic options with which to inhibit VM to improve outcomes for patients with SCLC. References 1S. C. Williamson et al., Vasculogenic mimicry in small cell lung cancer. Nat Commun 7, 1 (2016). 2C. L. Hodgkinson et al., Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nature medicine 20, 897 (2014). 3K. L. Simpson et al., A biobank of small cell lung cancer CDX models elucidates inter-and intratumoral phenotypic heterogeneity. Nature Cancer 1, 4 (2020). Citation Format: Sarah M. Pearsall, Stuart C. Williamson, Fernando García Marqués, Sam Humphrey, Ellyn Hughes, Ian Cannell, Kristopher K. Frese, Melanie Galvin, Mathew Carter, Lynsey Priest, Alastair Kerr, Martin J. Humphries, Jonathon D. Humphries, Fiona Blackhall, Gregory Hannon, Julien Sage, Sharon J. Pitteri, Kathryn L. Simpson, Caroline Dive. Lineage plasticity in small cell lung cancer generates non- neuroendocrine cells primed for vascular mimicry [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 2653.

Biocatalysts provide an efficient, inexpensive and environmentally friendly alternative to traditional organic synthesis, especially for compounds with complex stereochemistries. The past decade has seen a significant rise in the use of biocatalysts for the synthesis of compounds in an industrial setting; however, the incorporation of single enzymatically catalysed steps into organic synthesis schemes can be problematic. The emerging field of synthetic biology has sparked interest in the development of whole-cell factories that can convert simple, common metabolites into complex, high-value molecules with a range of applications such as pharmaceuticals and biofuels. This Review summarises conventional methods and recent advances in metabolic engineering of pathways in microorganisms for the synthesis of natural products.

Review: [recent developments in metabolic pathway engineering and synthetic biology towards the use of whole-cell microbial factories for the synthesis of useful natural compounds; 100 refs.