An Early Neoplasia Index (ENI10), Based on Molecular Identity of CD10 Cells and Associated Stemness Biomarkers, is a Predictor of Patient Outcome in Many Cancers.

An accurate estimate of patient survival at diagnosis is critical to plan efficient therapeutic options. A simple and multiapplication tool is needed to move forward the precision medicine era. Taking advantage of the broad and high CD10 expression in stem and cancers cells, we evaluated the molecular identity of aggressive cancer cells. We used epithelial primary cells and developed a breast cancer stem cell­based progressive model. The superiority of the early-transformed isolated molecular index was evaluated by large-scale analysis in solid cancers. BMP2-driven cell transformation increases CD10 expression which preserves stemness properties. Our model identified a unique set of 159 genes enriched in G2­M cell-cycle phases and spindle assembly complex. Using samples predisposed to transformation, we confirmed the value of an early neoplasia index associated to CD10 (ENI10) to discriminate premalignant status of a human tissue. Using a stratified Cox model, a large-scale analysis (>10,000 samples, The Cancer Genome Atlas Pan-Cancer) validated a strong risk gradient (HRs reaching HR = 5.15; 95% confidence interval: 4.00­6.64) for high ENI10 levels. Through different databases, Cox regression model analyses highlighted an association between ENI10 and poor progression-free intervals for more than 50% of cancer subtypes tested, and the potential of ENI10 to predict drug efficacy. The ENI10 index constitutes a robust tool to detect pretransformed tissues and identify high-risk patients at diagnosis. Owing to its biological link with refractory cancer stem cells, the ENI10 index constitutes a unique way of identifying effective treatments to improve clinical care. SIGNIFICANCE: We identified a molecular signature called ENI10 which, owing to its biological link with stem cell properties, predicts patient outcome and drugs efficiency in breast and several other cancers. ENI10 should allow early and optimized clinical management of a broad number of cancers, regardless of the stage of tumor progression.

In Vitro Approaches to Determine the Potential Carcinogenic Risk of Environmental Pollutants.

One important environmental/health challenge is to determine, in a feasible way, the potential carcinogenic risk associated with environmental agents/exposures. Since a significant proportion of tumors have an environmental origin, detecting the potential carcinogenic risk of environmental agents is mandatory, as regulated by national and international agencies. The challenge mainly implies finding a way of how to overcome the inefficiencies of long-term trials with rodents when thousands of agents/exposures need to be tested. To such an end, the use of in vitro cell transformation assays (CTAs) was proposed, but the existing prevalidated CTAs do not cover the complexity associated with carcinogenesis processes and present serious limitations. To overcome such limitations, we propose to use a battery of assays covering most of the hallmarks of the carcinogenesis process. For the first time, we grouped such assays as early, intermediate, or advanced biomarkers which allow for the identification of the cells in the initiation, promotion or aggressive stages of tumorigenesis. Our proposal, as a novelty, points out that using a battery containing assays from all three groups can identify if a certain agent/exposure can pose a carcinogenic risk; furthermore, it can gather mechanistic insights into the mode of the action of a specific carcinogen. This structured battery could be very useful for any type of in vitro study, containing human cell lines aiming to detect the potential carcinogenic risks of environmental agents/exposures. In fact, here, we include examples in which these approaches were successfully applied. Finally, we provide a series of advantages that, we believe, contribute to the suitability of our proposed approach for the evaluation of exposure-induced carcinogenic effects and for the development of an alternative strategy for conducting an exposure risk assessment.

HERVs characterize normal and leukemia stem cells and represent a source of shared epitopes for cancer immunotherapy.

Human endogenous retroviruses (HERVs) represent 8% of the human genome. The expression of HERVs and their immune impact have not been extensively studied in Acute Myeloid Leukemia (AML). In this study, we used a reference of 14 968 HERV functional units to provide a thorough analysis of HERV expression in normal and AML bone marrow cells. We show that the HERV retrotranscriptome accurately characterizes normal and leukemic cell subpopulations, including leukemia stem cells, in line with different epigenetic profiles. We then show that HERV expression delineates AML subtypes with different prognoses. We finally propose a method to select and prioritize CD8+ T cell epitopes derived from AML-specific HERVs and we show that lymphocytes infiltrating patient bone marrow at diagnosis contain naturally occurring CD8+ T cells against these HERV epitopes. We also provide in vitro data supporting the functionality of HERV-specific CD8+ T-cells against AML cells. These results show that HERVs represent an important source of genetic information that can help enhancing disease stratification or biomarker identification and an important reservoir of alternative tumor-specific T cell epitopes relevant for cancer immunotherapy.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia.

Lipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL-AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.

Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

A minimal standardized human bone marrow microphysiological system to assess resident cell behavior during normal and pathological processes.

Bone marrow is a complex and dynamic microenvironment that provides essential cues to resident cells. We developed a standardized three-dimensional (3D) model to decipher mechanisms that control human cells during hematological and non-hematological processes. Our simple 3D-model is constituted of a biphasic calcium phosphate-based scaffold and human cell lines to ensure a high reproducibility. We obtained a minimal well-organized bone marrow-like structure in which various cell types and secreted extracellular matrix can be observed and characterized by in situ imaging or following viable cell retrieval. The complexity of the system can be increased and customized, with each cellular component being independently modulated according to the issue investigated. Introduction of pathological elements in this 3D-system accurately reproduced changes observed in patient bone marrow. Hence, we have developed a handy and flexible standardized microphysiological system that mimics human bone marrow, allowing histological analysis and functional assays on collected cells.

Deciphering Tumor Niches: Lessons From Solid and Hematological Malignancies.

Knowledge about the hematopoietic niche has evolved considerably in recent years, in particular through in vitro analyzes, mouse models and the use of xenografts. Its complexity in the human bone marrow, in particular in a context of hematological malignancy, is more difficult to decipher by these strategies and could benefit from the knowledge acquired on the niches of solid tumors. Indeed, some common features can be suspected, since the bone marrow is a frequent site of solid tumor metastases. Recent research on solid tumors has provided very interesting information on the interactions between tumoral cells and their microenvironment, composed notably of mesenchymal, endothelial and immune cells. This review thus focuses on recent discoveries on tumor niches that could help in understanding hematopoietic niches, with special attention to 4 particular points: i) the heterogeneity of carcinoma/cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs), ii) niche cytokines and chemokines, iii) the energy/oxidative metabolism and communication, especially mitochondrial transfer, and iv) the vascular niche through angiogenesis and endothelial plasticity. This review highlights actors and/or pathways of the microenvironment broadly involved in cancer processes. This opens avenues for innovative therapeutic opportunities targeting not only cancer stem cells but also their regulatory tumor niche(s), in order to improve current antitumor therapies.

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Altered BMP2/4 Signaling in Stem Cells and Their Niche: Different Cancers but Similar Mechanisms, the Example of Myeloid Leukemia and Breast Cancer.

Understanding mechanisms of cancer development is mandatory for disease prevention and management. In healthy tissue, the microenvironment or niche governs stem cell fate by regulating the availability of soluble molecules, cell-cell contacts, cell-matrix interactions, and physical constraints. Gaining insight into the biology of the stem cell microenvironment is of utmost importance, since it plays a role at all stages of tumorigenesis, from (stem) cell transformation to tumor escape. In this context, BMPs (Bone Morphogenetic Proteins), are key mediators of stem cell regulation in both embryonic and adult organs such as hematopoietic, neural and epithelial tissues. BMPs directly regulate the niche and stem cells residing within. Among them, BMP2 and BMP4 emerged as master regulators of normal and tumorigenic processes. Recently, a number of studies unraveled important mechanisms that sustain cell transformation related to dysregulations of the BMP pathway in stem cells and their niche (including exposure to pollutants such as bisphenols). Furthermore, a direct link between BMP2/BMP4 binding to BMP type 1 receptors and the emergence and expansion of cancer stem cells was unveiled. In addition, a chronic exposure of normal stem cells to abnormal BMP signals contributes to the emergence of cancer stem cells, or to disease progression independently of the initial transforming event. In this review, we will illustrate how the regulation of stem cells and their microenvironment becomes dysfunctional in cancer via the hijacking of BMP signaling with main examples in myeloid leukemia and breast cancers.

Single-cell analysis of autophagy activity in normal and de novo transformed human mammary cells.

Assessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli.

Long-Term Exposure of Early-Transformed Human Mammary Cells to Low Doses of Benzo[a]pyrene and/or Bisphenol A Enhances Their Cancerous Phenotype via an AhR/GPR30 Interplay.

It is of utmost importance to decipher the role of chronic exposure to low doses of environmental carcinogens on breast cancer progression. The early-transformed triple-negative human mammary MCF10AT1 cells were chronically (60 days) exposed to low doses (10-10 M) of Benzo[a]pyrene (B[a]P), a genotoxic agent, and/or Bisphenol A (BPA), an endocrine disruptor. Our study revealed that exposed MCF10AT1 cells developed, in a time-dependent manner, an acquired phenotype characterized by an increase in cancerous properties (anchorage independent growth and stem-like phenotype). Co-exposure of MCF10AT1 cells to B[a]P and BPA led to a significantly greater aggressive phenotype compared to B[a]P or BPA alone. This study provided new insights into the existence of a functional interplay between the aryl hydrocarbon receptor (AhR) and the G protein-coupled receptor 30 (GPR30) by which chronic and low-dose exposure of B[a]P and/or BPA fosters the progression of MCF10AT1 cells into a more aggressive substage. Experiments using AhR or GPR30 antagonists, siRNA strategies, and RNAseq analysis led us to propose a model in which AhR signaling plays a "driver role" in the AhR/GPR30 cross-talk in mediating long-term and low-dose exposure of B[a]P and/or BPA. Retrospective analysis of two independent breast cancer cohorts revealed that the AhR/GPR30 mRNA expression signature resulted in poor breast cancer prognosis, in particular in the ER-negative and the triple-negative subtypes. Finally, the study identified targeting AhR and/or GPR30 with specific antagonists as a strategy capable of inhibiting carcinogenesis associated with chronic exposure to low doses of B[a]P and BPA in MCF10AT1 cells. Altogether, our results indicate that the engagement of both AhR and GPR30 functions, in particular in an ER-negative/triple-negative context of breast cells, favors tumor progression and leads to poor prognosis.

Targeting BMP signaling in the bone marrow microenvironment of myeloid leukemia.

The bone morphogenetic protein (BMP) pathway regulates the fate and proliferation of normal hematopoietic stem cells (HSC) as well as interactions with their niche. While BMP2 and BMP4 promote HSC differentiation, only BMP4 maintains HSC pool and favors interactions with their niche. In myeloid leukemia, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in Chronic Myeloid Leukemia (CML) and Acute Myeloid leukemia (AML) responsible for leukemic stem cells (LSC) survival. In AML, BMP pathway alterations sustain and promote resistant immature-like leukemic cells by activating a new signaling cascade. Binding of BMP4 to BMPR1A leads to ΔNp73 expression, which in turn induces NANOG, altogether associated with a poor patient's prognosis. Despite efficient targeted therapies, like Tyrosine Kinase Inhibitors (TKI) in CML, many patients retain LSCs. Our laboratory demonstrated that the BMP pathway sustains a permanent pool of LSCs expressing high levels of BMPR1B receptor, that evolve upon treatment to progressively implement a BMP4 autocrine loop, leading to TKI-resistant cells. Single cell RNA-Seq analysis of TKI-persisting LSCs showed a co-enrichment of BMP with Jak2-signaling, quiescence and stem cell (SC) signatures. Using a new model of persisting LSCs, we recently demonstrated that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways and could be targeted by blocking BMPR1B/Jak2 signal. Lastly, a specific BMPR1B inhibitor impaired BMP4-mediated LSC protection against TKIs. Altogether, data based on various studies including ours, indicate that BMP targeting could eliminate leukemic cells within a protective bone marrow microenvironment to efficiently impact residual resistance or persistence of LSCs in myeloid leukemia.

Bone marrow niche-derived extracellular matrix-degrading enzymes influence the progression of B-cell acute lymphoblastic leukemia.

Specific and reciprocal interactions with the bone marrow microenvironment (BMM) govern the course of hematological malignancies. Matrix metalloproteinase-9 (MMP-9), secreted by leukemia cells, facilitates tumor progression via remodeling of the extracellular matrix (ECM) of the BMM. Hypothesizing that leukemias may instruct the BMM to degrade the ECM, we show, that MMP-9-deficiency in the BMM prolongs survival of mice with BCR-ABL1-induced B-cell acute lymphoblastic leukemia (B-ALL) compared with controls and reduces leukemia-initiating cells. MMP-9-deficiency in the BMM leads to reduced degradation of proteins of the ECM and reduced invasion of B-ALL. Using various in vivo and in vitro assays, as well as recipient mice deficient for the receptor for tumor necrosis factor (TNF) α (TNFR1) we demonstrate that B-ALL cells induce MMP-9-expression in mesenchymal stem cells (MSC) and possibly other cells of the BMM via a release of TNFα. MMP-9-expression in MSC is mediated by activation of nuclear factor kappa B (NF-κB) downstream of TNFR1. Consistently, knockdown of TNF-α in B-ALL-initiating cells or pharmacological inhibition of MMP-9 led to significant prolongation of survival in mice with B-ALL. In summary, leukemia cell-derived Tnfα induced MMP-9-expression by the BMM promoting B-ALL progression. Inhibition of MMP-9 may act as an adjunct to existing therapies.

The current paradigm and challenges ahead for the dormancy of disseminated tumor cells.

Disseminated tumor cells (DTCs) are known to enter a state of dormancy that is achieved via growth arrest of DTCs and/or a form of population equilibrium state, strongly influenced by the organ microenvironment. During this time, expansion of residual disseminated cancer is paused and DTCs survive to fuel relapse, sometimes decades later. This notion has opened a new window of opportunity for intervening and preventing relapse. Here we review recent data that have further augmented the understanding of cancer dormancy and discuss how this is leading to new strategies for monitoring and targeting dormant cancer.

Early Steps of Mammary Stem Cell Transformation by Exogenous Signals; Effects of Bisphenol Endocrine Disrupting Chemicals and Bone Morphogenetic Proteins.

Estrogens are major regulators of the mammary gland development, notably during puberty, via estrogen receptor (ER) activation, leading to the proliferation and differentiation of mammary cells. In addition to estrogens, the bone morphogenetic proteins (BMPs) family is involved in breast stem cell/progenitor commitment. However, these two pathways that synergistically contribute to the biology of the normal mammary gland have also been described to initiate and/or promote breast cancer development. In addition to intrinsic events, lifestyle habits and exposure to environmental cues are key risk factors for cancer in general, and especially for breast cancer. In the latter case, bisphenol A (BPA), an estrogen-mimetic compound, is a critical pollutant both in terms of the quantities released in our environment and of its known and speculated effects on mammary gland biology. In this review, we summarize the current knowledge on the actions of BMPs and estrogens in both normal mammary gland development and breast cancer initiation, dissemination, and resistance to treatment, focusing on the dysregulations of these processes by BPA but also by other bisphenols, including BPS and BPF, initially considered as safer alternatives to BPA.

Downregulation of the histone methyltransferase SETD2 promotes imatinib resistance in chronic myeloid leukaemia cells.

OBJECTIVES: Epigenetic modifiers were important players in the development of haematological malignancies and sensitivity to therapy. Mutations of SET domain-containing 2 (SETD2), a methyltransferase that catalyses the trimethylation of histone 3 on lysine 36 (H3K36me3), were found in various myeloid malignancies. However, the detailed mechanisms through which SETD2 confers chronic myeloid leukaemia progression and resistance to therapy targeting on BCR-ABL remain unclear. MATERIALS AND METHODS: The level of SETD2 in imatinib-sensitive and imatinib-resistant chronic myeloid leukaemia (CML) cells was examined by immunoblotting and quantitative real-time PCR. We analysed CD34+ CD38- leukaemic stem cells by flow cytometry and colony formation assays upon SETD2 knockdown or overexpression. The impact of SETD2 expression alterations or small-molecule inhibitor JIB-04 targeting H3K36me3 loss on imatinib sensitivity was assessed by IC50, cell apoptosis and proliferation assays. Finally, RNA sequencing and ChIP-quantitative PCR were performed to verify putative downstream targets. RESULTS: SETD2 was found to act as a tumour suppressor in CML. The novel oncogenic targets MYCN and ERG were shown to be the direct downstream targets of SETD2, where their overexpression induced by SETD2 knockdown caused imatinib insensitivity and leukaemic stem cell enrichment in CML cell lines. Treatment with JIB-04, an inhibitor that restores H3K36me3 levels through blockade of its demethylation, successfully improved the cell imatinib sensitivity and enhanced the chemotherapeutic effect. CONCLUSIONS: Our study not only emphasizes the regulatory mechanism of SETD2 in CML, but also provides promising therapeutic strategies for overcoming the imatinib resistance in patients with CML.

A new signaling cascade linking BMP4, BMPR1A, ΔNp73 and NANOG impacts on stem-like human cell properties and patient outcome.

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients' bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients' outcome.