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.

Immature CML cells implement a BMP autocrine loop to escape TKI treatment.

The BCR-ABL specific tyrosine kinase inhibitors (TKI) changed the outcome of chronic myeloid leukemia (CML), turning a life-threatening disease into a chronic illness. However, TKI are not yet curative, because most patients retain leukemic stem cells (LSC) and their progenitors in bone marrow and relapse following treatment cessation. At diagnosis, deregulation of the bone morphogenetic protein (BMP) pathway is involved in LSC and progenitor expansion. Here, we report that BMP pathway alterations persist in TKI-resistant patients. In comparison with patients in complete cytogenetic remission, TKI-resistant LSC and progenitors display high levels of BMPR1b expression and alterations of its cellular localization. In vitro treatment of immature chronic phase CML cells with TKI alone, or in combination with interferon-α, results in the preferential survival of BMPR1b+ cells. We demonstrated persistent and increasing BMP4 production by patients' mesenchymal cells with resistance. Patient follow-up revealed an increase of BMPR1b expression and in BMP4 expression in LSC from TKI-resistant patients in comparison with diagnosis, while remaining unchanged in sensitive patients. Both leukemic and nonleukemic cells exhibit higher BMP4 levels in the bone marrow of TKI-resistant patients. Exposure to BMP2/BMP4 does not alter BCR-ABL transcript expression but is accompanied by the overexpression of TWIST-1, a transcription factor highly expressed in resistant LSC. By modulating BMP4 or BMPR1b expression, we show that these elements are involved in TKI resistance. In summary, we reveal that persistence of BMP alterations and existence of an autocrine loop promote CML-primitive cells' TKI resistance.

Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and on BMPRIb overexpression.

Leukemic stem cells in chronic phase chronic myelogenous leukemia (CP-CML) are responsible for disease persistence and eventual drug resistance, most likely because they survive, expand, and are sustained through interactions with their microenvironment. Bone morphogenetic proteins 2 (BMP2) and 4 (BMP4) regulate the fate and proliferation of normal hematopoietic stem cells, as well as interactions with their niche. We show here that the intrinsic expression of members of the BMP response pathway are deregulated in CML cells with differences exhibited in mature (CD34(-)) and immature (CD34(+)) compartments. These changes are accompanied by altered functional responses of primitive leukemic cells to BMP2 and BMP4 and strong increases in soluble BMP2 and BMP4 in the CML bone marrow. Using primary cells and a cell line mimicking CP-CML, we found that myeloid progenitor expansion is driven by the exposure of immature cells overexpressing BMP receptor Ib to BMP2 and BMP4. In summary, we demonstrate that deregulation of intracellular BMP signaling in primary CP-CML samples corrupts and amplifies their response to exogenous BMP2 and BMP4, which are abnormally abundant within the tumor microenvironment. These results provide new insights with regard to leukemic stem cell biology and suggest possibilities for the development of novel therapeutic tools specifically targeting the CML niche.

Deregulation of TWIST-1 in the CD34+ compartment represents a novel prognostic factor in chronic myeloid leukemia.

The mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) often remain obscure. Analysis of patient samples during disease progression revealed the up-regulation of the oncogene TWIST-1, also measured in primary samples from TKI-resistant patients. Moreover, we found that TWIST-1 was overexpressed in CML diagnostic samples of patients who later developed cytogenetic resistance to imatinib, even those without any detectable resistance mechanism. We confirmed the up-regulation of TWIST-1 at both RNA and protein levels in imatinib-resistant cell lines, irrespective of any other resistance mechanism. Analysis with specific small interfering RNA suggested TWIST-1 involvement in the resistance phenotype. Finally, the kinetics of TWIST-1 expression during the individual medical histories of CML patients indicated that TWIST-1 expression is down-regulated by TKIs and up-regulated with TKI resistance. We hypothesize that the overexpression of the TWIST-1 oncogene represents a novel key prognostic factor potentially useful for optimizing CML management in the TKI era.

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.

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.

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling.

Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-beta family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34(+) progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor pathways. Inhibition of the BMP signaling pathway with blocking antibodies, natural soluble inhibitors (FLRG or follistatin), or soluble BMP receptors reveals that thrombopoietin uses the BMP4 pathway to induce megakaryopoiesis, whereas the inverse is not occurring. Finally, we show that thrombopoietin up-regulates the BMP4 autocrine loop in megakaryocytic progenitors by inducing their production of BMP4 and up-regulating BMP receptor expression. In summary, this work indicates that BMP4 plays an important role in the control of human megakaryopoiesis.

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.

A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin.

FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.

Regulation of human erythropoiesis by activin A, BMP2, and BMP4, members of the TGFbeta family.

Activin A, BMP2, and BMP4, members of the TGFbeta family, have been implicated in the regulation of hematopoiesis. Here we explore and compare, for the first time in human primary cells, the role of activin A, BMP2, and BMP4 during erythropoiesis. Using in vitro erythroid differentiation of CD34(+) primary cells, we obtained the main stages of early erythropoiesis, characterized at the molecular, biochemical, and functional levels. Our results indicate that BMP2 acts on early erythroid cells and activin A on a more differentiated population. We report an insight into the mechanism of commitment of erythropoiesis by activin A and BMP2 involving two key events, increase in EPO-R and decrease in GATA2 expression. Simultaneous addition of activin A with BMP molecules suggests that BMP2 and BMP4 differently affect activin A induction of erythropoiesis. Follistatin and FLRG proteins downmodulate the effects of activin A and BMP2 on erythroid maturation.