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BACKGROUND & AIMS: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Here we aimed to reconcile these conflicting reports by repeating a key lineage tracing study from pericentral hepatocytes and characterised this Axin2CreERT2 model in detail.METHODS: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in depth phenotypic comparison including transcriptomics, metabolomics and analysis of protein through immunohistochemistry of Axin2CreERT2 mice to WT counterparts.RESULTS: We find that after careful definition of a baseline population there is marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We find substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/β-catenin signalling and related metabolomic disturbance.CONCLUSIONS: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci.

To test for evidence of statin-mediated effects in patients with castration-resistant prostate cancer (CRPC) as post-diagnosis use of statins in patients with prostate cancer is associated with favourable survival outcome.The SPECTRE trial was a 6-weeks-long proof-of-concept single-arm Phase II treatment trial, combining atorvastatin and androgen deprivation therapy in patients with CRPC (regardless of metastatic status), designed to test for evidence of statin-mediated effects in patients with CRPC. The primary study endpoint was the proportion of patients achieving a ≥50% drop from baseline in prostate-specific antigen (PSA) levels at any time over the 6-week period of atorvastatin medication (PSA response). Exploratory endpoints include PSA velocity and serum metabolites identified by mass spectrometry .At the scheduled interim analysis, one of 12 patients experienced a ≥50% drop in PSA levels (primary endpoint), with ≥2 patients satisfying the primary endpoint required for further recruitment. All 12 patients experienced substantial falls in serum cholesterol levels following statin treatment. While all patients had comparable pre-study PSA velocities, six of 12 patients showed decreased PSA velocities after statin treatment, suggestive of disease stabilization. Unbiased metabolomics analysis on serial weekly blood samples identified tryptophan to be the dominant metabolite associated with patient response to statin.Data from the SPECTRE study provide the first evidence of statin-mediated effects on CRPC and early sign of disease stabilization. Our data also highlight the possibility of altered tryptophan metabolism being associated with tumour response.

MYC is implicated in the development and progression of pancreatic cancer, yet the precise level of MYC deregulation required to contribute to tumor development has been difficult to define. We used modestly elevated expression of human MYC, driven from the Rosa26 locus, to investigate the pancreatic phenotypes arising in mice from an approximation of MYC trisomy. We show that this level of MYC alone suffices to drive pancreatic neuroendocrine tumors, and to accelerate progression of KRAS-initiated precursor lesions to metastatic pancreatic ductal adenocarcinoma (PDAC). Our phenotype exposed suppression of the type I interferon (IFN) pathway by the combined actions of MYC and KRAS, and we present evidence of repressive MYC–MIZ1 complexes binding directly to the promoters of the genes encodiing the type I IFN regulators IRF5, IRF7, STAT1, and STAT2. Derepression of IFN regulator genes allows pancreatic tumor infiltration by B and natural killer (NK) cells, resulting in increased survival.Significance:We define herein a novel mechanism of evasion of NK cell–mediated immunity through the combined actions of endogenously expressed mutant KRAS and modestly deregulated expression of MYC, via suppression of the type I IFN pathway. Restoration of IFN signaling may improve outcomes for patients with PDAC.This article is highlighted in the In This Issue feature, p. 747

6 supplementary Figures with legends under each; 3 Supplementary Tables; Supplementary Methods and References

Spreadsheet of genes co-regulated by MYC & Miz1 in KMC PDAC cells

Merged supplementary Figures & Figure legends and Tables & Table legends

Excel spreadsheet of NUAK1 inhibitor induced phospho-peptide alterations

Supplementary Methods

Supplementary Figure S2 shows additional data obtained during aphidicolin studies detailed in Fig 1 and additional data detailing expression of genes associated with ongoing and stalled replication forks from figure 3.

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to β-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of β-catenin activation in prostate cancer progression and treatment resistance. β-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with β-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of β-catenin activation, by mediating nuclear localization of NFκBp65 and β-catenin. Overall, WNT/β-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/β-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes.Significance:Targeting of both AR and WNT/β-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Legends for supplementary figures and tables

This file contains Supplementary Materials and Methods, Supplementary Figures, Legends to Supplementary Figures and Supplementary Tables. Figure S1 shows histopathological analysis of Ptenfl/fl and Ptenfl/fl Erk5fl/fl tumours at 3 and 9-10 month fixed time points and demonstrates that there is reduced %Ki67 positive nuclei in Ptenfl/fl Erk5fl/fl tumours at the 9-10 month time point. There is very little T lymphocyte infiltration at the 3 month time point but significant T lymphocyte infiltration is observed at the later time point. Figure S2 shows Western Blot and QPCR data to validate that P1 and PE cells are null for PTEN and ERK5 protein and Erk5 mRNA transcript where expected. Also shown is IHC staining demonstrating increased immunoreactivity for CCL5 and CXCL10 in Ptenfl/fl Erk5fl/fl compared to Ptenfl/fl tumours and that CCR5 and CXCR3 immunoreactivity is detected in the tumour epithelium and immune cells present in the stroma of Ptenfl/fl Erk5fl/fl tumours. Figure S3 shows IHC staining for CD45 demonstrating significant lymphocyte infiltration in Ptenfl/fl and Ptenfl/fl Erk5fl/fl tumours. Also shown is dual IHC staining for CD3/CD4 revealing a high degree of CD4 reactivity within the lymphoid aggregates of Ptenfl/fl Erk5fl/fl tumours.

Figures S1-S2-S3 + Suppl. Fig. legends + Suppl. Methods

All Supplementary data figures and legends. Supplementary Figure 1. In Silico analysis of RUNX1 expression in human kidney cancer. Supplementary Figure 2. Knockdown of RUNX1 with shRNA causes a growth defect in two ccRCC cell lines. Supplementary Figure 3. RUNX1 deletion in 786-O* cells and orthotopic xenograft. Supplementary Figure 4. Principle component analysis. Gene expression variances between control (786-O pX, Red) and RUNX1 CRISPR cells (786-O A1, yellow and 786-O A3, purple). Supplementary Figure 5. Heterozygous deletion of RUNX2 in a GEM model of kidney cancer does not affect survival.

RNAseq data set

Pathway Enrichment_RNASeq

Proteomics_CRPC

SLFN5_binding sites

RNASeq_SLFN5 KO

ATF4_binding sites

The recurring association of specific genetic lesions with particular types of cancer is a fascinating and largely unexplained area of cancer biology. This is particularly true of clear cell renal cell carcinoma (ccRCC) where, although key mutations such as loss of VHL is an almost ubiquitous finding, there remains a conspicuous lack of targetable genetic drivers. In this study, we have identified a previously unknown protumorigenic role for the RUNX genes in this disease setting. Analysis of patient tumor biopsies together with loss-of-function studies in preclinical models established the importance of RUNX1 and RUNX2 in ccRCC. Patients with high RUNX1 (and RUNX2) expression exhibited significantly poorer clinical survival compared with patients with low expression. This was functionally relevant, as deletion of RUNX1 in ccRCC cell lines reduced tumor cell growth and viability in vitro and in vivo. Transcriptional profiling of RUNX1-CRISPR–deleted cells revealed a gene signature dominated by extracellular matrix remodeling, notably affecting STMN3, SERPINH1, and EPHRIN signaling. Finally, RUNX1 deletion in a genetic mouse model of kidney cancer improved overall survival and reduced tumor cell proliferation. In summary, these data attest to the validity of targeting a RUNX1-transcriptional program in ccRCC.Significance:These data reveal a novel unexplored oncogenic role for RUNX genes in kidney cancer and indicate that targeting the effects of RUNX transcriptional activity could be relevant for clinical intervention in ccRCC.

List of antibodies

Androgen deprivation therapy (ADT) is the standard of care for treatment of nonresectable prostate cancer. Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we performed a comparative proteomic analysis of three in vivo, androgen receptor (AR)-responsive orthograft models of matched hormone-naïve prostate cancer and CRPC. Differential proteomic analysis revealed that distinct molecular mechanisms, including amino acid (AA) and fatty acid metabolism, are involved in the response to ADT in the different models. Despite this heterogeneity, Schlafen family member 5 (SLFN5) was identified as an AR-regulated protein in CRPC. SLFN5 expression was high in CRPC tumors and correlated with poor patient outcome. In vivo, SLFN5 depletion strongly impaired tumor growth in castrated conditions. Mechanistically, SLFN5 interacted with ATF4 and regulated the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreased intracellular levels of essential AA and impaired mTORC1 signaling in a LAT1-dependent manner. These results confirm that these orthograft models recapitulate the high degree of heterogeneity observed in patients with CRPC and further highlight SLFN5 as a clinically relevant target for CRPC.Significance:This study identifies SLFN5 as a novel regulator of the LAT1 amino acid transporter and an essential contributor to mTORC1 activity in castration-resistant prostate cancer.

Supplementary Table 1. The relationship between RUNX2 and clinico-pathological characteristics of kidney cancer in the TMA study.

Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem–like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133− non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with “replication factories” and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro. These data identify RS as a cancer stem cell–specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060–71. ©2018 AACR.

List of primers

Significantly down-reg genes in SLFN5 KO cells and tumours

Supplemental Table 1 Summary table of in vivo growth characteristics of E2, G7, R10, R15, R24, R9 and S2 GSC primary GBM cultures after intracranial injection in CD1 nude mice. Supplemental Table 2 List of primary antibodies utilised Supplemental Table 3 List of secondary antibodies utilised

Prostate cancer does not appear to respond to immune checkpoint therapies where T-cell infiltration may be a key limiting factor. Here, we report evidence that ablating the growth regulatory kinase Erk5 can increase T-cell infiltration in an established Pten-deficient mouse model of human prostate cancer. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared with control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T-cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T-cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease. Cancer Res; 77(12); 3158–68. ©2017 AACR.

The supplementary file contains details of the materials and methods used in the manuscript. File also contains supplementary figures S1 to S3 describing in details the murine model used to study role of activated ß-catenin in prostate cancer progression. Supplementary figure S4 describes the murine orthograft model generated to study mechanistic details. Supplementary figure S5 describes the metabolic profile of primary murine prostate cancer cells with Pten loss and ß-catenin activation and figures S6 and S7 show the effects of combining ADT with inhibition of WNT signalling.

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Selection for tumour cells that efficiently pass through narrow diameter microporous membranes reveals a prominent role for MAPK signalling in regulating cell elasticity.

BackgroundTracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs, and excess mortality. The underlying mechanisms facilitating adverse outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses.MethodsTracheal aspirates, tracheal cytology brushings, nasal swabs and stool samples were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic, and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome.ResultsChildren followed up serially from the time of tracheostomy up to three months post-procedure (n=9) were studied. A validation cohort of children with a long-term tracheostomy was also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Tracheostomy was associated with new, rapidly emergent and sustained airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. In contrast, reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter.ConclusionsChildhood tracheostomy is associated with rapidly emergent and persistent airway neutrophil recruitment and activation, with sustained proteolysis and superoxide generation. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients.Key messageThe effect tracheostomy has on children is not described. Tracheostomy in children results in persistent local airway neutrophilic inflammation, proteolysis, superoxide production and dysbiosis.

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production. ispartof: NATURE METABOLISM vol:4 issue:6 pages:693-+ ispartof: location:Germany status: published

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer related mortality worldwide. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulated progressive driver mutations, with hepatocytes the most likely cell of origin. However, the landscape of driver mutations in HCC is independent of the underlying aetiologies. Despite an increasing range of systemic treatment options for advanced HCC outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC. We generated over twenty-five new genetically-driven in vivo and in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance, and metastasis to distant organs. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with histopathology. In a proof-of-principle analysis, we verified response to standard of care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not been linked to HCC treatment before. Cladribine acts in a highly effective subtype-specific manner in combination with standard of care therapy.

Objective: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. Design Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. Results: neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1(+) dendritic cell activation and CD8(+) T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8(+) T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8(+) T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. Conclusion: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC., Cancer Research UK (CRUK); Newcastle Experimental Cancer Medicine Centre; Accelerator Award; Fondazione AIRC; MRC program; AstraZeneca; Wellcome Trust; Newcatle University Faculty of Medical Sciences; WE Harker Foundation; Ministry of Economy and Competitiveness (MINECO) (MICINN); Fundació Universitària Agustí Pedro i Pons; Instituto de Salud Carlos III (ISCIII); European Union (EU); European Social Fund; German Research Foundation (DFG); Fulbright España; National Institutes of Health (NIH); Samuel Waxman Cancer Research Foundation; Spanish National Health Institute; Fundación Científica de la Asociación Española Contra el Cáncer; Generalitat de Catalunya; Sara Borrell Fellowship

BackgroundTracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs and excess mortality. The underlying mechanisms facilitating adverse respiratory outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses.MethodsTracheal aspirates, tracheal cytology brushings and nasal swabs were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome.ResultsChildren followed up serially from the time of tracheostomy up to 3 months postprocedure (n=9) were studied. A further cohort of children with a long-term tracheostomy were also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Long-term tracheostomy was associated with airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. Reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter.ConclusionsLong-term childhood tracheostomy is associated with a inflammatory tracheal phenotype characterised by neutrophilic inflammation and the ongoing presence of potential respiratory pathogens. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2−/− Vav3−/− Tiam1−/−), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease., Loss of small GTPase RAC1 suppresses intestinal tumorigenesis caused by APC loss, but impacts normal intestinal homeostasis. Here, the authors provide an alternative method of reducing RAC1 activity by the combined targeting of three RAC-GEFs and show that this approach delays intestinal tumorigenesis without the detrimental effects on normal intestinal architecture.

Correction to: Nature Metabolism https://doi.org/10.1038/s42255-022-00582-0, published online 27 June 2022 In the version of this article initially published, the surname of the third author, Carla Riera-Domingo, was misspelled as Riero-Domingo. The error has been corrected in the HTML and PDF versions of the article.