Stromal niche inflammation mediated by IL-1 signalling is a targetable driver of haematopoietic ageing.

Haematopoietic ageing is marked by a loss of regenerative capacity and skewed differentiation from haematopoietic stem cells (HSCs), leading to impaired blood production. Signals from the bone marrow niche tailor blood production, but the contribution of the old niche to haematopoietic ageing remains unclear. Here we characterize the inflammatory milieu that drives both niche and haematopoietic remodelling. We find decreased numbers and functionality of osteoprogenitors at the endosteum and expansion of central marrow LepR+ mesenchymal stromal cells associated with deterioration of the sinusoidal vasculature. Together, they create a degraded and inflamed old bone marrow niche. Niche inflammation in turn drives the chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors, which promotes myeloid differentiation and hinders haematopoietic regeneration. Moreover, we show how production of interleukin-1ß (IL-1ß) by the damaged endosteum acts in trans to drive the proinflammatory nature of the central marrow, with damaging consequences for the old blood system. Notably, niche deterioration, HSC dysfunction and defective regeneration can all be ameliorated by blocking IL-1 signalling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during ageing.

Disruption of stem cell niche-confined R-spondin 3 expression leads to impaired hematopoiesis.

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine.

Aged hematopoietic stem cells are refractory to bloodborne systemic rejuvenation interventions.

While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors.

Anti-VEGF Treatment Enhances CD8+ T-cell Antitumor Activity by Amplifying Hypoxia.

Antiangiogenic therapies that target the VEGF pathway have been used clinically to combat cancer for over a decade. Beyond having a direct impact on blood vessel development and tumor perfusion, accumulating evidence indicates that these agents also affect antitumor immune responses. Numerous clinical trials combining antiangiogenic drugs with immunotherapies for the treatment of cancer are ongoing, but a mechanistic understanding of how disruption of tumor angiogenesis may impact immunity is not fully discerned. Here, we reveal that blockade of VEGF-A with a mAb to VEGF augments activation of CD8+ T cells within tumors and potentiates their capacity to produce cytokines. We demonstrate that this phenomenon relies on the disruption of VEGFR2 signaling in the tumor microenvironment but does not affect CD8+ T cells directly. Instead, the augmented functional capacity of CD8+ T cells stems from increased tumor hypoxia that initiates a hypoxia-inducible factor-1α program within CD8+ T cells that directly enhances cytokine production. Finally, combinatorial administration of anti-VEGF with an immunotherapeutic antibody, anti-OX40, improved antitumor activity over single-agent treatments. Our findings illustrate that anti-VEGF treatment enhances CD8+ T-cell effector function and provides a mechanistic rationale for combining antiangiogenic and immunotherapeutic drugs for cancer treatment.

IFNγ-induced Chemokines Are Required for CXCR3-mediated T-Cell Recruitment and Antitumor Efficacy of Anti-HER2/CD3 Bispecific Antibody.

PURPOSE: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. EXPERIMENTAL DESIGN: The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. RESULTS: Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. CONCLUSIONS: In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.

Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis.

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1ß, G-CSF, TGFß and CXCL4) and activation of an inducible Irf8 and ß-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.

EGFR as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype.

Muscle-invasive bladder carcinoma (MIBC) constitutes a heterogeneous group of tumors with a poor outcome. Molecular stratification of MIBC may identify clinically relevant tumor subgroups and help to provide effective targeted therapies. From seven series of large-scale transcriptomic data (383 tumors), we identified an MIBC subgroup accounting for 23.5% of MIBC, associated with shorter survival and displaying a basal-like phenotype, as shown by the expression of epithelial basal cell markers. Basal-like tumors presented an activation of the epidermal growth factor receptor (EGFR) pathway linked to frequent EGFR gains and activation of an EGFR autocrine loop. We used a 40-gene expression classifier derived from human tumors to identify human bladder cancer cell lines and a chemically induced mouse model of bladder cancer corresponding to human basal-like bladder cancer. We showed, in both models, that tumor cells were sensitive to anti-EGFR therapy. Our findings provide preclinical proof of concept that anti-EGFR therapy can be used to target a subset of particularly aggressive MIBC tumors expressing basal cell markers and provide diagnostic tools for identifying these tumors.

PI3K/AKT pathway activation in bladder carcinogenesis.

The PI3K/AKT pathway is considered to play a major role in bladder carcinogenesis, but its relationships with other molecular alterations observed in bladder cancer remain unknown. We investigated PI3K/AKT pathway activation in a series of human bladder urothelial carcinomas (UC) according to PTEN expression, PTEN deletions and FGFR3, PIK3CA, KRAS, HRAS, NRAS and TP53 gene mutations. The series included 6 normal bladder urothelial samples and 129 UC (Ta n = 25, T1 n = 34, T2-T3-T4 n = 70). Expression of phospho-AKT (pAKT), phospho-S6-Ribosomal Protein (pS6) (one downstream effector of PI3K/AKT pathway) and PTEN was evaluated by reverse phase protein Array. Expression of miR-21, miR-19a and miR-222, known to regulate PTEN expression, was also evaluated. pAKT expression levels were higher in tumors than in normal urothelium (p < 0.01), regardless of stage and showed a weak and positive correlation with pS6 (Spearman coefficient RS = 0.26; p = 0.002). No association was observed between pAKT or pS6 expression and the gene mutations studied. PTEN expression was decreased in PTEN-deleted tumors, and in T1 (p = 0.0089) and T2-T3-T4 (p < 0.001) tumors compared to Ta tumors; it was also negatively correlated with miR-19a (RS = -0.50; p = 0.0088) and miR-222 (RS = -0.48; p = 0.0132), but not miR-21 (RS = -0.27; p = 0.18) expression. pAKT and PTEN expressions were not negatively correlated, and, on the opposite, a positive and moderate correlation was observed in Ta (RS = 0.54; p = 0.0056) and T1 (RS = 0.56; p = 0.0006) tumors. Our study suggests that PI3K/AKT pathway activation occurs in the entire spectrum of bladder UC regardless of stage or known most frequent molecular alterations, and independently of low PTEN expression.

HMCan: a method for detecting chromatin modifications in cancer samples using ChIP-seq data.

MOTIVATION: Cancer cells are often characterized by epigenetic changes, which include aberrant histone modifications. In particular, local or regional epigenetic silencing is a common mechanism in cancer for silencing expression of tumor suppressor genes. Though several tools have been created to enable detection of histone marks in ChIP-seq data from normal samples, it is unclear whether these tools can be efficiently applied to ChIP-seq data generated from cancer samples. Indeed, cancer genomes are often characterized by frequent copy number alterations: gains and losses of large regions of chromosomal material. Copy number alterations may create a substantial statistical bias in the evaluation of histone mark signal enrichment and result in underdetection of the signal in the regions of loss and overdetection of the signal in the regions of gain. RESULTS: We present HMCan (Histone modifications in cancer), a tool specially designed to analyze histone modification ChIP-seq data produced from cancer genomes. HMCan corrects for the GC-content and copy number bias and then applies Hidden Markov Models to detect the signal from the corrected data. On simulated data, HMCan outperformed several commonly used tools developed to analyze histone modification data produced from genomes without copy number alterations. HMCan also showed superior results on a ChIP-seq dataset generated for the repressive histone mark H3K27me3 in a bladder cancer cell line. HMCan predictions matched well with experimental data (qPCR validated regions) and included, for example, the previously detected H3K27me3 mark in the promoter of the DLEC1 gene, missed by other tools we tested.

A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12) and OR = 0.99 [0.37-2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

CDKN2A homozygous deletion is associated with muscle invasion in FGFR3-mutated urothelial bladder carcinoma.

The gene cyclin-dependent kinase inhibitor 2A (CDKN2A) is frequently inactivated by deletion in bladder carcinoma. However, its role in bladder tumourigenesis remains unclear. We investigated the role of CDKN2A deletion in urothelial carcinogenesis, as a function of FGFR3 mutation status, a marker for one of the two pathways of bladder tumour progression, the Ta pathway. We studied 288 bladder carcinomas: 177 non-muscle-invasive (123 Ta, 54 T1) and 111 muscle-invasive (T2-4) tumours. CDKN2A copy number was determined by multiplex ligation-dependent probe amplification, and FGFR3 mutations by SNaPshot analysis. FGFR3 mutation was detected in 124 tumours (43.1%) and CDKN2A homozygous deletion in 56 tumours (19.4%). CDKN2A homozygous deletion was significantly more frequent in FGFR3-mutated tumours than in wild-type FGFR3 tumours (p = 0.0015). This event was associated with muscle-invasive tumours within the FGFR3-mutated subgroup (p < 0.0001) but not in wild-type FGFR3 tumours. Similar findings were obtained for an independent series of 101 bladder carcinomas. The impact of CDKN2A deletions on recurrence-free and progression-free survival was then analysed in 89 patients with non-muscle-invasive FGFR3-mutated tumours. Kaplan-Meier survival analysis showed that CDKN2A losses (hemizygous and homozygous) were associated with progression (p = 0.0002), but not with recurrence, in these tumours. Multivariate Cox regression analysis identified CDKN2A loss as a predictor of progression independent of stage and grade. These findings highlight the crucial role of CDKN2A loss in the progression of non-muscle-invasive FGFR3-mutated bladder carcinomas and provide a potentially useful clinical marker for adapting the treatment of such tumours, which account for about 50% of cases at initial clinical presentation.

A novel epigenetic phenotype associated with the most aggressive pathway of bladder tumor progression.

BACKGROUND: Epigenetic silencing can extend to whole chromosomal regions in cancer. There have been few genome-wide studies exploring its involvement in tumorigenesis. METHODS: We searched for chromosomal regions affected by epigenetic silencing in cancer by using Affymetrix microarrays and real-time quantitative polymerase chain reaction to analyze RNA from 57 bladder tumors compared with normal urothelium. Epigenetic silencing was verified by gene re-expression following treatment of bladder cell lines with 5-aza-deoxycytidine, a DNA demethylating agent, and trichostatin A, a histone deacetylase inhibitor. DNA methylation was studied by bisulfite sequencing and histone methylation and acetylation by chromatin immunoprecipitation. Clustering was used to distinguish tumors with multiple regional epigenetic silencing (MRES) from those without and to analyze the association of this phenotype with histopathologic and molecular types of bladder cancer. The results were confirmed with a second panel of 40 tumor samples and extended in vitro with seven bladder cancer cell lines. All statistical tests were two-sided. RESULTS: We identified seven chromosomal regions of contiguous genes that were silenced by an epigenetic mechanism. Epigenetic silencing was not associated with DNA methylation but was associated with histone H3K9 and H3K27 methylation and histone H3K9 hypoacetylation. All seven regions were concordantly silenced in a subgroup of 26 tumors, defining an MRES phenotype. MRES tumors exhibited a carcinoma in situ-associated gene expression signature (25 of 26 MRES tumors vs 0 of 31 non-MRES tumors, P < 10⁻¹4), rarely carried FGFR3 mutations (one of 26 vs 22 of 31 non-MRES tumors, P < 10⁻¹6), and contained 25 of 33 (76%) of the muscle-invasive tumors. Cell lines derived from aggressive bladder tumors presented epigenetic silencing of the same regions. CONCLUSIONS: We have identified an MRES phenotype characterized by the concomitant epigenetic silencing of several chromosomal regions, which, in bladder cancer, is specifically associated with the carcinoma in situ gene expression signature.

Mosaicism for oncogenic G12D KRAS mutation associated with epidermal nevus, polycystic kidneys and rhabdomyosarcoma.

BACKGROUND: Epidermal nevus (EN) is a congenital disorder characterised by hyperpigmented epidermal thickening following a Blaschko's line. It is due to somatic mutations in either FGFR3 or PIK3CA in half of the cases, and remains of unknown genetic origin in the other half. EN is also seen as part of complex developmental disorders or in association with bladder carcinomas, also related to FGFR3 and PIK3CA mutations. Mosaic mutations of these genes have been occasionally found in syndromic EN. CASE REPORT: The co-occurrence of EN, rhabdomyosarcoma, polycystic kidneys and growth retardation in an infant is described. RESULTS: An oncogenic G12D KRAS mutation was detected in both the epidermal component of the EN and in the rhabdomyosarcoma but not in the dermal component of the EN lesion or in unaffected tissues, including normal skin or blood. CONCLUSION: This report shows for the first time that a KRAS mutation in epiderma causes EN. Observation of the same G12D KRAS mutation in two distinct regions of the body strongly suggests a somatic mosaicism. Finally, this report highlights the potentially underestimated importance of mosaic oncogene mutations in childhood cancers.

Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.

UNLABELLED: Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. We further investigated transcriptome-genotype-phenotype correlations in HCC. Global transcriptome analyses were performed on 57 HCCs and 3 hepatocellular adenomas and validated by quantitative RT-PCR using 63 additional HCCs. We determined loss of heterozygosity, gene mutations, promoter methylation of CDH1 and CDKN2A, and HBV DNA copy number for each tumor. Unsupervised transcriptome analysis identified 6 robust subgroups of HCC (G1-G6) associated with clinical and genetic characteristics. G1 tumors were associated with low copy number of HBV and overexpression of genes expressed in fetal liver and controlled by parental imprinting. G2 included HCCs infected with a high copy number of HBV and mutations in PIK3CA and TP53. In these first groups, we detected specific activation of the AKT pathway. G3 tumors were typified by mutation of TP53 and overexpression of genes controlling the cell cycle. G4 was a heterogeneous subgroup of tumors including TCF1-mutated hepatocellular adenomas and carcinomas. G5 and G6 were strongly related to beta-catenin mutations that lead to Wnt pathway activation; in particular, G6 tumors were characterized by satellite nodules, higher activation of the Wnt pathway, and E-cadherin underexpression. CONCLUSION: These results have furthered our understanding of the genetic diversity of human HCC and have provided specific identifiers for classifying tumors. In addition, our classification has potential therapeutic implications because 50% of the tumors were related to WNT or AKT pathway activation, which potentially could be targeted by specific inhibiting therapies.