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Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

The inflammatory potential of the diet decreased significantly after one-year of nutrition counselling on the Mediterranean diet and low glycemic index in women with breast cancer living in Italy.

Cancer stem cells (CSCs) are immature, immortal cells within tumors, adept at resisting therapeutic pressure and responsible for local and distant disease recurrence. Non-genetic mechanisms of acquired resistance are increasingly being described, however molecular insights into this evolutionary process still lack. Here, we showed that Type I interferons (IFNs-I) act as molecular hubs of resistance during immunogenic chemotherapy, as they trigger the epigenetic regulator KDM1B, responsible for an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B pharmacological inhibition antagonizes the appearance of IFN-I-adapted CSCs, both in vitro and in vivo. Notably, IFN-I-adapted CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer patients receiving anthracycline-based chemotherapy, IFN-I and KDM1B signatures positively correlate with CSC and immune evasion markers. Our study identifies IFN-I→KDM1B axis as a potent engine of cancer cell reprogramming and recommends KDM1B targeting an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

Simple Summary Colorectal cancer can be subdivided into four distinct subtypes that are characterised by different clinical features and responses to therapies currently used in the clinic to treat this disease. One of those subtypes, called CMS4, is associated with a worse prognosis and poor response to therapies compared to other subtypes. We therefore set out to explore what proteins are differentially expressed and used in CMS4 to find potential new targets for therapy. We found that protein AKT3 is highly expressed in CMS4, and that active AKT3 inhibits a protein that stalls growth of cancer cells (p27KIP1). We can target AKT3 with inhibitors which leads to strongly reduced growth of cancer cell lines that are categorised as CMS4. Furthermore, our data suggests that high AKT3 expression in tumour cells may be used to identify poor prognosis colorectal cancer patients. Future research should point out if high AKT3 expression can be used to select colorectal cancer patients that have a poor prognosis but that could benefit from AKT3-targeted treatment. Abstract Colorectal cancer (CRC) is a heterogeneous disease that can currently be subdivided into four distinct consensus molecular subtypes (CMS) based on gene expression profiling. The CMS4 subtype is marked by high expression of mesenchymal genes and is associated with a worse overall prognosis compared to other CMSs. Importantly, this subtype responds poorly to the standard therapies currently used to treat CRC. We set out to explore what regulatory signalling networks underlie the CMS4 phenotype of cancer cells, specifically, by analysing which kinases were more highly expressed in this subtype compared to others. We found AKT3 to be expressed in the cancer cell epithelium of CRC specimens, patient derived xenograft (PDX) models and in (primary) cell cultures representing CMS4. Importantly, chemical inhibition or knockout of this gene hampers outgrowth of this subtype, as AKT3 controls expression of the cell cycle regulator p27KIP1. Furthermore, high AKT3 expression was associated with high expression of epithelial-mesenchymal transition (EMT) genes, and this observation could be expanded to cell lines representing other carcinoma types. More importantly, this association allowed for the identification of CRC patients with a high propensity to metastasise and an associated poor prognosis. High AKT3 expression in the tumour epithelial compartment may thus be used as a surrogate marker for EMT and may allow for a selection of CRC patients that could benefit from AKT3-targeted therapy.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

Simple Summary The ATR-CHK1 axis of the DNA damage response is crucial for the survival of most colorectal cancer stem cells (CRC-SCs), but a significant fraction of primary CRC-SCs either is resistant to ATR or CHK1 inhibitors or survives the abrogation of the ATR-CHK1 cascade despite an initial response. Here, we demonstrate that the targeting of RAD51 or MRE11 improves the sensitivity of primary CRC-SCs to the CHK1/2 inhibitor prexasertib by sequentially inducing replication stress, the abrogation of cell cycle checkpoints, and the emergence of mitotic defects. This results in the induction of mitotic catastrophe and CRC-SC killing via a caspase-dependent apoptosis. Abstract Cancer stem cells (CSCs) drive not only tumor initiation and expansion, but also therapeutic resistance and tumor relapse. Therefore, CSC eradication is required for effective cancer therapy. In preclinical models, CSCs demonstrated high capability to tolerate even extensive genotoxic stress, including replication stress, because they are endowed with a very robust DNA damage response (DDR). This favors the survival of DNA-damaged CSCs instead of their inhibition via apoptosis or senescence. The DDR represents a unique CSC vulnerability, but the abrogation of the DDR through the inhibition of the ATR-CHK1 axis is effective only against some subtypes of CSCs, and resistance often emerges. Here, we analyzed the impact of druggable DDR players in the response of patient-derived colorectal CSCs (CRC-SCs) to CHK1/2 inhibitor prexasertib, identifying RAD51 and MRE11 as sensitizing targets enhancing prexasertib efficacy. We showed that combined inhibition of RAD51 and CHK1 (via B02+prexasertib) or MRE11 and CHK1 (via mirin+prexasertib) kills CSCs by affecting multiple genoprotective processes. In more detail, these two prexasertib-based regimens promote CSC eradication through a sequential mechanism involving the induction of elevated replication stress in a context in which cell cycle checkpoints usually activated during the replication stress response are abrogated. This leads to uncontrolled proliferation and premature entry into mitosis of replication-stressed cells, followed by the induction of mitotic catastrophe. CRC-SCs subjected to RAD51+CHK1 inhibitors or MRE11+CHK1 inhibitors are eventually eliminated, and CRC-SC tumorspheres inhibited or disaggregated, via a caspase-dependent apoptosis. These results support further clinical development of these prexasertib-based regimens in colorectal cancer patients.

Biobanking of molecularly characterized colorectal cancer stem cells (CSCs) generated from individual patients and growing as spheroids in defined serum-free media offer a fast, feasible, and multi-level approach for the screening of targeted therapies and drug resistance molecular studies. By combining in vitro and in vivo analyses of cetuximab efficacy with genetic data on an ongoing collection of stem cell-enriched spheroids, we describe the identification and preliminary characterization of microsatellite stable (MSS) CSCs that, despite the presence of the KRAS (G12D) mutation, display epidermal growth factor (EGF)-dependent growth and are strongly inhibited by anti-EGF-receptor (EGFR) treatment. In parallel, we detected an increased resistance to anti-EGFR therapy of microsatellite instable (MSI) CSC lines irrespective of KRAS mutational status. MSI CSC lines carried mutations in genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results argue for a multifactorial origin of anti-EGFR resistance that emerges as the effect of multiple events targeting direct and indirect regulators of the EGFR pathway. An improved understanding of key molecular determinants of sensitivity/resistance to EGFR inhibition will be instrumental to optimize the clinical efficacy of anti-EGFR agents, representing a further step towards personalized treatments.

PTEN is one of the most frequently inactivated tumor suppressor genes in cancer. Loss or variation in PTEN gene/protein levels is commonly observed in a broad spectrum of human cancers, while germline PTEN mutations cause inherited syndromes that lead to increased risk of tumors. PTEN restrains tumorigenesis through different mechanisms ranging from phosphatase-dependent and independent activities, subcellular localization and protein interaction, modulating a broad array of cellular functions including growth, proliferation, survival, DNA repair, and cell motility. The main target of PTEN phosphatase activity is one of the most significant cell growth and pro-survival signaling pathway in cancer: PI3K/AKT/mTOR. Several shreds of evidence shed light on the critical role of PTEN in normal and cancer stem cells (CSCs) homeostasis, with its loss fostering the CSC compartment in both solid and hematologic malignancies. CSCs are responsible for tumor propagation, metastatic spread, resistance to therapy, and relapse. Thus, understanding how alterations of PTEN levels affect CSC hallmarks could be crucial for the development of successful therapeutic approaches. Here, we discuss the most significant findings on PTEN-mediated control of CSC state. We aim to unravel the role of PTEN in the regulation of key mechanisms specific for CSCs, such as self-renewal, quiescence/cell cycle, Epithelial-to-Mesenchymal-Transition (EMT), with a particular focus on PTEN-based therapy resistance mechanisms and their exploitation for novel therapeutic approaches in cancer treatment.

The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/β is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/β play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/β on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity.

ObjectiveCancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.DesignTo discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.ResultsThe drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.ConclusionsLY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]-targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)-driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC-enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC-enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti-EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance. Significance Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR-targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.

CD127 is the IL-7 receptor α-chain and its expression is tightly regulated during T-cell differentiation. We previously showed that the bone marrow (BM) is a key organ for proliferation and maintenance of both antigen-specific and CD44(high) memory CD8(+) T cells. Interestingly, BM memory CD8(+) T cells express lower levels of membrane CD127 than do the corresponding spleen and lymph node cells. We investigated the requirements for CD127 downmodulation by CD44(high) memory-phenotype CD8(+) T cells in the BM of C57BL/6 mice. By comparing genetically modified (i.e. CD127tg, IL-7 KO, IL-15 KO, IL-15Rα KO) with wild-type (WT) mice, we found that the key molecule regulating CD127 downmodulation was IL-15 but not IL-7, and that the intact CD127 gene was required, including the promoter. Indeed, CD127 mRNA transcript levels were lower in CD44(high) CD8(+) T cells from the BM than in those from the spleen of WT mice, indicating organ-specific regulation. Although levels of the CD127 transactivator Foxo1 were low in BM CD44(high) CD8(+) T cells, Foxo1 was not involved in IL-15-induced CD127 downmodulation. Thus, recirculating CD44(high) CD8(+) T cells passing through the BM transiently downregulate CD127 in response to IL-15, with implications for human therapies acting on the IL-7/CD127 axis, for example cytokine treatments in cancer patients.

International audience; CD127 is the IL-7 receptor α-chain and its expression is tightly regulated during T-cell differentiation. We previously showed that the bone marrow (BM) is a key organ for proliferation and maintenance of both antigen-specific and CD44(high) memory CD8(+) T cells. Interestingly, BM memory CD8(+) T cells express lower levels of membrane CD127 than do the corresponding spleen and lymph node cells. We investigated the requirements for CD127 downmodulation by CD44(high) memory-phenotype CD8(+) T cells in the BM of C57BL/6 mice. By comparing genetically modified (i.e. CD127tg, IL-7 KO, IL-15 KO, IL-15Rα KO) with wild-type (WT) mice, we found that the key molecule regulating CD127 downmodulation was IL-15 but not IL-7, and that the intact CD127 gene was required, including the promoter. Indeed, CD127 mRNA transcript levels were lower in CD44(high) CD8(+) T cells from the BM than in those from the spleen of WT mice, indicating organ-specific regulation. Although levels of the CD127 transactivator Foxo1 were low in BM CD44(high) CD8(+) T cells, Foxo1 was not involved in IL-15-induced CD127 downmodulation. Thus, recirculating CD44(high) CD8(+) T cells passing through the BM transiently downregulate CD127 in response to IL-15, with implications for human therapies acting on the IL-7/CD127 axis, for example cytokine treatments in cancer patients.

CD127 is the IL-7 receptor ?-chain and its expression is tightly regulated during T-cell differentiation. We previously showed that the bone marrow (BM) is a key organ for proliferation and maintenance of both antigen-specific and CD44(high) memory CD8(+) T cells. Interestingly, BM memory CD8(+) T cells express lower levels of membrane CD127 than do the corresponding spleen and lymph node cells. We investigated the requirements for CD127 downmodulation by CD44(high) memory-phenotype CD8(+) T cells in the BM of C57BL/6 mice. By comparing genetically modified (i.e. CD127tg, IL-7 KO, IL-15 KO, IL-15R? KO) with wild-type (WT) mice, we found that the key molecule regulating CD127 downmodulation was IL-15 but not IL-7, and that the intact CD127 gene was required, including the promoter. Indeed, CD127 mRNA transcript levels were lower in CD44(high) CD8(+) T cells from the BM than in those from the spleen of WT mice, indicating organ-specific regulation. Although levels of the CD127 transactivator Foxo1 were low in BM CD44(high) CD8(+) T cells, Foxo1 was not involved in IL-15-induced CD127 downmodulation. Thus, recirculating CD44(high) CD8(+) T cells passing through the BM transiently downregulate CD127 in response to IL-15, with implications for human therapies acting on the IL-7/CD127 axis, for example cytokine treatments in cancer patients.

Type I IFNs are central to a vast array of immunological functions. Their early induction in innate immune responses provides one of the most important priming mechanisms for the subsequent establishment of adaptive immunity. The outcome is either promotion or inhibition of these responses, but the conditions under which one or the other prevails remain to be defined. The main objective of the current study was to determine the involvement of IFN-α on murine CD4+CD25− Th cell activation, as well as to define the role played by this cytokine on CD4+CD25+ regulatory T (Treg) cell proliferation and function. Although IFN-α promotes CD4+CD25− Th cells coincubated with APCs to produce large amounts of IL-2, the ability of these cells to respond to IL-2 proliferative effects is prevented. Moreover, in medium supplemented with IFN-α, IL-2–induced CD4+CD25+ Treg cell proliferation is inhibited. Notably, IFN-α also leads to a decrease of the CD4+CD25+ Treg cell suppressive activity. Altogether, these findings indicate that through a direct effect on APC activation and by affecting CD4+CD25+ Treg cell-mediated suppression, IFN-α sustains and drives CD4+CD25− Th cell activation.