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Cancer initiation is orchestrated by interplay between tumor-initiating cells and their stromal/immune environment. Here, by adapted scRNAsequencing, we decipher the predicted signaling between tissue-resident hematopoietic stem/progenitor cells (HSPCs) and their neoplastic counterparts with their native niches in the human bone marrow. LEPR+stromal cells are identified as central regulators of hematopoiesis through predicted interactions with all cells in the marrow. Inflammatory niche remodeling and the resulting deprivation of critical HSPC regulatory factors is predicted to repress distinct high-output HSC subsets inNPM1-mutated AML, with relative resistance of clonal cells. Stromal gene signatures reflective of niche remodeling are associated with reduced relapse rates and favorable outcome after chemotherapy, across all genetic risk categories. Elucidation of the intercellular signaling defining human AML, thus, predicts that inflammatory remodeling of stem cell niches drives tissue repression and clonal selection, but may pose a vulnerability for relapse-initiating cells in the context of chemotherapeutic treatment.statement of significanceTumor-promoting inflammation is considered an enabling characteristic of tumorigenesis, but mechanisms remain incompletely understood. By deciphering the predicted signaling between tissue-resident stem cells and their neoplastic counterparts with their environment, we identify inflammatory remodeling of stromal niches as a determinant of normal tissue repression and clinical outcome in human AML.Key pointsA comprehensive taxonomy of the predicted interactions between LEPR+stromal niches, HSPCs and adaptive/innate immune cells in the human NBM.Inflammation-associated decline of stromal niches in AML represses residual normal hematopoiesis with relative resistance of leukemic cells.Inflammatory decline of stromal niches is associated with reduced relapse risk and favorable outcome.

Supp Table 1: MDS Patient Disease Characteristics Supp Table 2: Pathways affected by hypermethylation in MDS stroma Supp Table 3: Genes that are methylated and underexpressed in MDS stroma Supp Table 4. MDS patient characteristics for primary MSC sample isolation Supp table 5: WNT Targets

Supplementary Methods

The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow–derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine–treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98–HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846–57. ©2017 AACR.

Supp Fig 1 : SFRP1 methylation with coculture Supp Fig 2 : Validation of methylation changes with stroma and leukemic cell coculture Supp Fig 3: FRZB upregulation in MDS stroma after 5-Azacytidine treatment SuppFig 4: FRZB knockdown leads to partial inhibition of erythroid differentiation induced by treatment of MDS stromawith 5-Aza SuppFig 5: The expression of WNT signature genes in MDS and control marrow derived CD34+ cells

SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction: For many cancers, adolescent and young adult (AYA) patients have a poorer prognosis than pediatric patients. This study compares long-term survival outcomes of children and AYAs diagnosed with acute myeloid leukemia (AML) in the Netherlands. This has not been done before despite the availability of high-quality data collected by the nationwide, population-based Netherlands Cancer Registry (NCR). Methods: Data on all AML patients aged 0-39 years, diagnosed between 1990-2015, were obtained from the NCR (N=2058). Relative (disease-specific) survival was estimated for all patients, children (0-17 years), and AYAs (18-39 years) using the traditional cohort approach. Early mortality was defined as death within 30 days of diagnosis. Multivariable analyses were performed using generalized linear models (excess mortality) and logistic regression (early mortality). Results: The prognosis of Dutch AML patients below the age of 40 years has improved considerably during the past decades with 5-year relative survival rates increasing from 39% in 1990-1999 to 61% in 2010-2015 (p trend When compared to children, the excess risk of dying due to AML was 70% higher in AYAs [95% confidence interval (95% CI), 47%-97%] after adjustment for follow-up time, sex, period of diagnosis, and stem cell transplantation. The excess hazard ratios (HRs) of mortality were 2.0 (95% CI, 1.6-2.4) for 18-29 year olds and 2.1 (95% CI, 1.7-2.6) for 30-39 year olds, using 1-9 year olds as reference. Early mortality declined over time from 9% to 4% (p trend Conclusions: AYAs diagnosed with AML in the Netherlands between 1990 and 2015 had a worse prognosis than pediatric patients. The survival disparity seemed most evident in recent years, suggesting that improvements in care resulting in better outcome for children have not led to equal benefits for AYAs yet. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Chemotherapy-induced bone marrow (BM) injury is a significant cause of morbidity and mortality in acute myeloid leukemia (AML). Time to hematologic recovery after standard (“7 + 3”) myeloablative chemotherapy can vary considerably among patients, but the factors that drive or predict BM recovery remain incompletely understood. Here, we assessed the composition of innate and adaptive immune subsets in the regenerating BM (day 17) after induction chemotherapy and related it to hematologic recovery in AML. T cells, and in particular the CD4 central memory (CD4CM) T-cell subset, were significantly enriched in the BM after chemotherapy, suggesting the relative chemoresistance of cells providing long-term memory for systemic pathogens. In contrast, B cells and other hematopoietic subsets were depleted. Higher frequencies of the CD4CM T-cell subset were associated with delayed hematopoietic recovery, whereas a high frequency of natural killer (NK) cells was related to faster recovery of neutrophil counts. The NK/CD4CM ratio in the BM after chemotherapy was significantly associated with the time to subsequent neutrophil recovery (Spearman's ρ = –0.723, p < 0.001, false discovery rate

RPS14, CSNK1A1, and miR-145 are universally co-deleted in the 5q- syndrome, but mouse models of each gene deficiency recapitulate only a subset of the composite clinical features. We analyzed the combinatorial effect of haploinsufficiency for Rps14, Csnk1a1, and miRNA-145, using mice with genetically engineered, conditional heterozygous inactivation of Rps14 and Csnk1a1 and stable knockdown of miR-145/miR-146a. Combined Rps14/Csnk1a1/miR-145/146a deficiency recapitulated the cardinal features of the 5q- syndrome, including (1) more severe anemia with faster kinetics than Rps14 haploinsufficiency alone and (2) pathognomonic megakaryocyte morphology. Macrophages, regulatory cells of erythropoiesis and the innate immune response, were significantly increased in Rps14/Csnk1a1/miR-145/146a deficient mice as well as in 5q- syndrome patient bone marrows and showed activation of the innate immune response, reflected by increased expression of S100A8, and decreased phagocytic function. We demonstrate that Rps14/Csnk1a1/miR-145 and miR-146a deficient macrophages alter the microenvironment and induce S100A8 expression in the mesenchymal stem cell niche. The increased S100A8 expression in the mesenchymal niche was confirmed in 5q- syndrome patients. These data indicate that intrinsic defects of the 5q- syndrome hematopoietic stem cell directly alter the surrounding microenvironment, which in turn affects hematopoiesis as an extrinsic mechanism.

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a strong propensity for leukemic evolution. Somatic mutations are critical early drivers of the disorder, but the factors enabling the emergence, selection, and subsequent leukemic evolution of these “leukemia-poised” clones remain incompletely understood. Emerging data point at the mesenchymal niche as a critical contributor to disease initiation and evolution. Disrupted inflammatory signaling from niche cells may facilitate the occurrence of somatic mutations, their selection, and subsequent clonal expansion. This review summarizes the current concepts about “niche-facilitated” bone marrow failure and leukemic evolution, their underlying molecular mechanisms, and clinical implications for future innovative therapeutic targeting of the niche in MDS.

Summary Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment., Graphical Abstract, Highlights • Reduction of endosteal BM and expansion of non-endosteal BM occurs with age • β2/β3-ARs exhibit opposite and niche-dependent regulation of myelopoiesis • β2-AR overriding β3-AR promotes myeloid expansion during physiological aging • Premature HSC aging in HGPS can be improved by targeting the microenvironment, Recent studies have suggested a microenvironmental contribution to stem-cell aging, but the mechanisms are largely unexplored. Méndez-Ferrer et al. report anatomical remodeling of blood-stem-cell-supporting niches and functional switch of β adrenergic signals, leading to myeloid expansion during aging. Targeting the microenvironment can improve pathological, premature, niche-dependent hematopoietic aging in mice.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for Rev1, a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.

The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow–derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine–treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98–HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846–57. ©2017 AACR.

Highlights Activation of NF-κB signaling in mesenchymal cells is common in LR-MDS.Activation of NF-κB in mesenchymal cells leads to transcriptional overexpression of inflammatory factors including negative regulators of hematopoiesis.Activation of NF-κB attenuates HSPC numbers and function ex vivo.

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs

Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.

Peptide receptor radionuclide therapy (PRRT) may induce long-term toxicity to the bone marrow (BM). The aim of this study was to analyze persistent hematologic dysfunction (PHD) after PRRT with 177Lu-DOTATATE in patients with gastroenteropancreatic neuroendocrine tumors (GEP NETs). Methods: The incidence and course of PHD were analyzed in 274 GEP NET patients from a group of 367 patients with somatostatin receptor-positive tumors. PHD was defined as diagnosis of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), myeloproliferative neoplasm (MPN), MDS/MPN, or otherwise unexplained cytopenia (for >6 mo). Using data from The Netherlands Cancer Registry, the expected number of hematopoietic neoplasms (MDS, AML, MPN, and MDS/MPN) was calculated and adjusted for sex, age, and follow-up period. The following risk factors were assessed: sex, age over 70 y, bone metastasis, prior chemotherapy, prior external-beam radiotherapy, uptake on the [111In-DTPA0]octreotide scan, tumor load, grade 3-4 hematologic toxicity during treatment, estimated absorbed BM dose, elevated plasma chromogranin A level, baseline blood counts, and renal function. Results: Eleven (4%) of the 274 patients had PHD after treatment with 177Lu-DOTATATE: 8 patients (2.9%) developed a hematopoietic neoplasm (4 MDS, 1 AML, 1 MPN, and 2 MDS/MPN) and 3 patients (1.1%) developed BM failure characterized by cytopenia and BM aplasia. The median latency period at diagnosis (or first suspicion of a PHD) was 41 mo (range, 15-84 mo). The expected number of hematopoietic neoplasms based on The Netherlands Cancer Registry data was 3.0, resulting in a relative risk of 2.7 (95% confidence interval, 0.7-10.0). No risk factors for PHD could be identified for the GEP NET patients, not even bone metastasis or estimated BM dose. Seven patients with PHD developed anemia in combination with a rise in mean corpuscular volume. Conclusion: The prevalence of PHD after PRRT with 177Lu-DOTATATE was 4% in our patient population. The median time at which PHD developed was 41 mo after the first PRRT cycle. The relative risk for developing a hematopoietic neoplasm was 2.7. No risk factors were found for the development of PHD in GEP NET patients.

Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of hematopoietic precursor cells, endothelial cells, as well as osteogenic differentiation. The identification and molecular characterization of these human regeneration-associated endothelial cells is thus anticipated to instruct the discovery of angiocrine factors driving bone marrow formation and recovery after injury.

Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn+ cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell–based adaptive immunity.

Summary Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2) negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL) model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway., Graphical Abstract, Highlights • UT2 is a transmembrane inhibitor of mTORC2 in hematopoietic progenitors • UT2 directly binds RICTOR, limiting the kinase activity of mTORC2 on pAKTS473 • UT2 modulates the outcome of an mTORC2-dependent hematopoietic cancer, In this article, Scadden and colleagues show that an undefined transmembrane molecule upstream of mTORC2 (UT2) negatively regulates AKT signaling via modulation of mTORC2. Their work points to the value of models that can be used to examine niche contributions to oncogenesis and reveals a previously unrecognized transmembrane modulator of a critical pathway with therapeutic implications for cancers such as T-ALL.

During embryogenesis, lymph nodes form through intimate interaction between lymphoid tissue inducer and lymphoid tissue organizer (LTo) cells. Shortly after birth in mice, specialized stromal cell subsets arise that organize microenvironments within the lymph nodes; however, their direct precursors have not yet been identified. In the bone marrow, mesenchymal stem cells are labeled with GFP in nestin-GFP mice, and we show that during all stages of development, nestin+ cells are present within lymph nodes of these mice. At day of birth, both mesenchymal CD31− and endothelial CD31+ LTo cells were GFP+, and only the population of CD31− LTo cells contained mesenchymal precursors. These CD31−nestin+ cells are found in the T and B cell zones or in close association with high endothelial venules in adult lymph nodes. Fate mapping of nestin+ cells unambiguously revealed the contribution of nestin+ precursor cells to the mesenchymal as well as the endothelial stromal populations within lymph nodes. However, postnatal tamoxifen induced targeting of nestin+ cells in nes-creER mice showed that most endothelial cells and only a minority of the nonendothelial cells were labeled. Overall our data show that nestin+ cells contribute to all subsets of the complex stromal populations that can be found in lymph nodes.

Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.

Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.

Myelodysplastic syndromes are a heterogeneous group of diseases characterized by ineffective hematopoiesis and the propensity to leukemic transformation. Their pathogenesis is complex and likely depends on interplay between aberrant hematopoietic cells and their microenvironment. How niche cells play a role in disease evolution is poorly defined, but the delineation of the hematopoietic stem cell niche and the ability to interrogate its role in hematopoietic disease in animal models have furthered our insights in recent years. The data support a view in which the microenvironment can play an active role in the evolution of myelodysplasia and myeloproliferative disorders, thus providing further rationale to explore therapeutic targeting of mesenchymal-hematopoietic interactions in these diseases.

Early events driving the initiation and evolution of neoplasms remain poorly defined but involvement of an instructive or permissive niche has been implicated. In this issue of Cell Stem Cell, Medyouf et al. (2014) provide insights into the role of the niche in myelodysplastic syndromes, the principle preleukemic disorder of the hematopoietic system.

Myeloid malignancies are driven by (epi)genetic aberrations in hematopoietic stem and/or progenitor cells (HSPC). HSPC in these disorders typically acquire a number of genetic lesions that, in concert, drive malignant transformation. How a single HSC, generally considered to be in a predominantly quiescent state in the postnatal bone marrow under homeostatic conditions, acquires multiple mutations, remains incompletely understood. Another central question remains how a mutated cell or 'preleukemic clone' persists and sometimes gains competitive advantage over non-mutated HSPC, while not always explained by hematopoietic cell-intrinsic capacities caused by the mutated state. Evolution principles predict that a cell's direct environment, or HSPC niche, may play critical roles in the induction and selection of genetic clones within a population. In earlier work, we postulated a concept of niche-induced oncogenesis in the hematopoietic system, wherein primary alterations in the mesenchymal stem cell niche can drive malignant transformation of supported, but distinct HSPC. Other emerging data support the idea of 'niche-facilitated' leukemogenesis, in which primary alterations in hematopoietic cells drive changes in their mesenchymal niche, facilitating leukemic evolution. Elucidation of the molecular signaling underlying these concepts holds great promise for novel avenues to attenuate leukemic evolution in preleukemic syndromes. Congenital, monogenic, bone marrow failure syndromes with leukemia predisposition, in which mutations in a single gene, present in both HSPC and niche cells, drives tissue failure, clonal evolution and malignant transformation, provide an unprecedented model system to study the molecular mechanisms and human relevance of these concepts. The lecture will address the findings of studies in Shwachman-Diamond Syndrome (SDS), caused by constitutive, bi-allelic, loss-of-function, mutations in the ribosome biogenesis gene SBDS, and characterized by bone abnormalities, neutropenia and a striking propensity to develop MDS/AML. Mouse models of global Sbds deficiency ('full knockouts') are embryonically lethal. In order to investigate the contributions of hematopoietic and niche cells to neutropenia and leukemogenesis, a 'deconstructing' approach, targeting deletion of Sbds to specific cell types in the bone marrow environment, needs to be taken. Targeted downregulation of Sbds in Cebpa -expressing HSPC and their downstream progeny results in profound neutropenia, caused by impaired lineage progression specifically at the myelocyte phase of myeloid development and associated with activation of the Tp53 tumor suppressor pathway. Myelodysplasia and leukemia, however, were not observed, suggesting that hematopoietic cell-non-autonomous processes may contribute to these processes in SDS. Mesenchymal-specific deletion of Sbds (Osterix -cre Sbdsfl/fl ) faithfully recapitulated both skeletal and myelodysplastic characteristics of human disease. The Sbds -deficient mesenchymal niche induces genotoxic stress in HSPC. Mechanistically, transcriptional activation of an inflammatory signature in niche cells, downstream of Tp53 activation, is implicated. Transcriptional activation of this signaling axis in the mesenchymal niche was found in a subset of MDS patients to predict leukemic evolution. The data support the idea that mesenchymal niche cells make a necessary contribution to disease pathogenesis in SDS. The findings complement emerging data from mouse models of niche-facilitated oncogenesis and human disease, pointing at inflammatory signaling in HSPC niches as a biologic commonality in preleukemia and myeloid neoplasm. A hypothetical model can be proposed of 'mesenchymal niche inflammation' promoting bone marrow failure, genotoxic stress, genetic instability and clonal evolution, of broader relevance to congenital bone marrow failure and leukemia predisposition syndromes. Recent data as well as controversies and uncertainties concerning this evolving new paradigm will be discussed. Disclosures Raaijmakers: Novartis: Consultancy.

Item does not contain fulltext PURPOSE OF REVIEW: The hematopoietic stem cell niche is critical for the maintenance and proliferation of hematopoietic stem cells and, as such, is not only essential for steady-state hematopoiesis but may also be relevant to hematologic disease. The present review discusses recent advances in the understanding of interactions within the niche, its potential role in disease pathogenesis and models of its use as a therapeutic target. RECENT FINDINGS: Recent studies have continued to provide important insights into the cellular and molecular components constituting the hematopoietic stem cell niche. Niche interactions have been shown to be involved in the pathogenesis of hematologic disease in animal models. Molecular components of the niche involved in these interactions have been identified, and proof of principle that their manipulation can result in therapeutic benefit is available. Finally, pharmacologic manipulation of the niche is now being tested in stem cell-based therapies. SUMMARY: Increasing insights into the molecular architecture of the hematopoietic stem cell niche have led to the exploitation of the niche as a target in stem cell therapies and offer the prospect of niche-targeted therapy as a new treatment modality in hematologic disease.

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein a (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.

Purpose: Acute myelogenous leukemia (AML) is a disease originating from normal hematopoietic CD34+CD38− progenitor cells. Modulation of the multidrug ATP-binding cassette transporter ABCB1 has not resulted in improved outcome in AML, raising the question whether leukemic CD34+CD38− cells are targeted by this strategy. Experimental Design: ABCB1-mediated transport in leukemic CD34+CD38− cells compared with their normal counterparts was assessed by quantitating the effect of specific ABCB1 modulators (verapamil and PSC-833) on mitoxantrone retention [defined as efflux index (EI), intracellular mitoxantrone fluorescence intensity in the presence/absence of inhibitor]. Results: ABCB1 was the major drug transporter in CD34+CD38− cells in normal bone marrow (n = 16), as shown by the abrogation of mitoxantrone extrusion by ABCB1 modulators (EI, 1.99 ± 0.08). Surprisingly, ABCB1-mediated drug extrusion was invariably reduced in CD34+CD38− cells in AML (n = 15; EI, 1.21 ± 0.05; P < 0.001), which resulted in increased intracellular mitoxantrone retention in these cells (mitoxantrone fluorescence intensity, 4.54 ± 0.46 versus 3.08 ± 0.23; P = 0.004). Active drug extrusion from these cells occurred in the presence of ABCB1 modulators in the majority of samples, pointing in the direction of redundant drug extrusion mechanisms. Residual normal CD34+CD38− cells could be identified by their conserved ABCB1-mediated extrusion capacity. Conclusion: ABCB1-mediated drug extrusion is reduced in leukemic CD34+CD38− progenitor cells compared with their residual normal counterparts. Redundant drug transport mechanisms confer mitoxantrone transport from leukemic progenitors. These data argue that ABCB1 modulation is not an effective strategy to circumvent drug extrusion from primitive leukemic progenitor cells and may preferentially target residual normal progenitors in AML.

Primary alterations of the mesenchymal niche can induce myelodysplasia and acute myeloid leukemia in mouse models, introducing a concept of niche-driven leukemogenesis (Raaijmakers et al, Nature 2010). The molecular mechanisms and human relevance of this concept, however, have remained elusive. We addressed these key questions by modelling Shwachman-Diamond-Syndrome (SDS), a human monogenic congenital disorder caused by loss-of function mutation in the SBDS gene and characterized by skeletal defects, bone marrow failure and a striking propensity for leukemic evolution. Targeted Sbds deletion from mesenchymal progenitor cells (MPCs) in mice (OsxCre/+Sbdsf/f; OCSf/f) resulted in bone abnormalities faithfully recapitulating human disease, including short stature and early-onset osteoporosis. Skeletal defects were associated with genotoxic stress in hematopoietic stem and progenitor cells (HSPCs) as demonstrated by mitochondrial membrane hyperpolarization, oxidative stress, DNA damage and cell cycle checkpoint activation (transcriptional modulation of DNA damage response/repair pathways and G0-G1 cell cycle arrest). DNA damage could be partially rescued by in vivo administration of the ROS scavenger N-acetylcysteine supporting the notion of niche induced DNA damage in HSPCs induced by mitochondria-derived superoxide radicals. Mechanistically, Sbds deficiency caused activation of the p53 tumor suppressorpathway in MPCs (upregulation of P53 and transcriptional activation of downstream targets (GSEA). Genetic deletion of Trp53 from MPCs (Osxcre/+Sbdsf/fTrp53f/f mice) rescued the skeletal phenotype and genotoxic stress in HSPCs. Comparison of the transcriptome of MPCs from OCSf/f mice to their highly FACS-purified mesenchymal (CD45-CD235-7AAD-CD31-CD271+CD105+) human equivalents from SDS patients (RNAseq; n=5) demonstrated a striking overlap in disrupted gene programs (GSEA), including ribosome biogenesis and significant overexpression of the proinflammatory molecules such as S100A8 and S100A9, bona fide p53 downstream targets. Activation of p53 and inflammatory molecules was an MPC-autonomous consequence of Sbds depletion as demonstrated by ex vivo knockdown of the gene in OP9 cells. S100A8/A9 overexpression and secretion from MPCs from OCSf/f mice was confirmed by FCM and serum ELISA. Exposure of HSPCs to recombinant murine S100A8/9 resulted in increased DNA damage and apoptosis associated with transcriptional activation of TLR4 downstream signaling, a bona fide S100A8A9 receptor. In vivo TLR4 blockade by neutralizing antibodies resulted in reduced γH2AX foci in HSPCs from OCSf/f mice, in support of the existence of a Tpr53-S100A8/A9-TLR4 axis driving genotoxic stress. Formal demonstration that niche-derived S100A8/9 is sufficient to drive genotoxic stress in HSPCs was provided by transplantation of wild-type hematopoietic cells into recipient S100A8/A9 transgenic mice (Cheng et al., 2008) resulting in accumulation of mitochondrial superoxide radicals and DNA-damage in wild-type HSPCs. Finally, to further define the clinical relevance of this inflammatory MPC-HSPC axis to human disease, we performed massive parallel RNA-sequencing of FACS purified mesenchymal cells from homogeneously treated low-risk MDS patients (n=45). Overexpression of S100A8 and S100A9 in MPCs(confirmed by IHC) was found in a considerable subset of patients (17/45; 38%). S100A8/9+ mesenchymal cells displayed transcriptional activation of p53 and TLR programs, in line with findings in the mouse model. Strikingly, patients in the niche-S100A8/9+ group displayed a higher frequency of leukemia evolution (29.4% vs. 14.2%) with significantly shorter evolution time (average 3.4 (1-7.5) vs 18.5 (7-40); p=.03) and progression-free survival (median 11.5 vs. 53 months, p=.03), independent of established prognostic factors and risk classification systems. Collectively, the data define niche-HSPC inflammatory signaling through the p53-S100A8/A9-TLR axis as an actionable determinant of genotoxic stress and disease outcome in human preleukemia, opening the way to niche-instructed, therapeutic targeting to attenuate leukemic evolution. Disclosures No relevant conflicts of interest to declare.

Mesenchymal cells contribute to the ‘stroma’ of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman–Bodian–Diamond syndrome—a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis. Although a series of genetic and epigenetic events in a single cell may be necessary for oncogenesis, it has been suggested that for malignancy to develop fully a permissive microenvironment or niche is required. Support for this view comes from a new mouse model in which haematopoietic malignancies are caused by genetic changes in the microenvironment of blood cells. Deletion in bone progenitor cells of Dicer1, a gene involved in microRNA processing, leads to a myelodysplastic syndrome-like phenotype that can progress to leukaemia. The progenitor cells have reduced levels of Sbds, the gene mutated in Schwachman–Bodian–Diamond syndrome, a bone marrow failure that predisposes to leukaemia. A new mouse model is developed in which haematopoietic malignancies are caused by genetic changes in the microenvironment of blood cells. Deletion in bone progenitor cells of Dicer1, a gene involved in microRNA processing, leads to a myelodysplastic syndrome-like phenotype which can progress to leukaemia. Deregulation of Sbds, which is mutated in human Schwachman–Bodian–Diamond syndrome, may be involved in this process.

Item does not contain fulltext In this issue, Wilson et al. (2008) demonstrate that there are two functional subsets of hematopoietic stem cells that have distinctive kinetics of cell cycling. They present evidence that cells may transition between the two kinetic states, establishing one subpopulation that is ready to proliferate and another that is a deeply quiescent reserve.

Shwachman-Diamond Syndrome (SDS) is a congenital bone marrow failure disorder characterized by neutropenia and predisposition to leukemia. SDS is associated with loss-of-function mutations in the SBDSgene, involved in ribosome biogenesis, but the cellular and molecular events driving neutropenia in SDS remain poorly defined, largely due to a lack of mammalian disease models recapitulating the hematopoietic features of SDS. To achieve deletion of Sbds in early hematopoietic/myeloid progenitors, we generated Cebpacre/+Sbdsf/f R26 EYFP/+ mice (Sbds Δ/Δ), which were non-viable. Analysis of E14.5 embryos demonstrated global conservation of the hematopoietic hierarchy in Sbds Δ/Δ embryos compared to Cebpacre/+ R26 EYFP/+ controls (Sbds +/+). Functional relevance of Sbds deletion in Cebpa+progenitors was tested by transplanting fetal liver cells from E14.5 Sbds Δ/Δ or Sbds +/+ embryos into lethally irradiated B6.SJL mice. Deficiency of Sbds in different EYFP+ hematopoietic subsets was confirmed by qPCR (log2 fold change: LKS -2.25, CMP -9.42, GMP -9.1 and neutrophils -7.29). Mice transplanted with Sbds Δ/Δ cells developed profound neutropenia (log2FC: -2.56; p=0.002, n=7), which was stable during the time of analysis (16 weeks). FACS and morphological studies of bone marrow EYFP+ cells demonstrated increased frequencies of early progenitor populations, recapitulating the left-shifted hematopoiesis observed in human SDS patients (Dror et al., Ann N Y Acad Sci 2011), with a pronounced accumulation of cKitint Gr1low EYFP+ myelocytes-metamyelocytes (MC-MMs) (frequency of EYFP+ cells: Sbds +/+ 15.3±1.7 %, Sbds Δ/Δ 39.4±1.1 %; p= 8.7x10-8) and a marked decrease in Gr1+ Mac1+ cells (Sbds +/+: 54.5±7.5 %, Sbds Δ/Δ: 22.3±2.8 %; p= 1.6x10-4), indicating that neutropenia was caused by disrupted lineage progression from MC-MMs to neutrophils. In line with this, whole transcriptome analysis of prospectively isolated EYFP+ MC-MMs (RNA-seq, n=4) revealed enrichment for hematopoietic stem and progenitor cell signatures in Sbds Δ/Δ recipients, while myeloid signatures were enriched in Sbds +/+ mice (GSEA). Transcript analysis further showed reduced expression of granule components produced at the MC-MM stage, such as Ltf, Mmp8, Mmp9. As expected, reduced expression of Sbds (FDR=6.6x10-9, log2FC=-2.67) and dysregulation of ribosome proteins (RP) production were observed, with increased expression of over 60 RP genes in SbdsΔ/Δ MC-MMs. To investigate the molecular events underlying impaired lineage progression, we focused on p53 activation, a common mechanism of tissue failure in ribosomopathies. P53 protein was significantly and selectively accumulated in EYFP+ MC-MMs of Sbds Δ/Δ recipients as measured by FACS (Mean Fluorescence Intensity FC: 1.8; p=0.01), with increased expression of p53 targets such as Cdkn1a (p21), Bbc3 (PUMA) and Bax. This was accompanied by increased apoptosis (annexin V FACS analysis) in both MC-MMs and mature neutrophils (FC: 1.36; p=0.02 and FC=2.44; p=1.6x10-4, respectively). However, pharmacological inhibition of the p53 pathway by pifithrin-α (2 months, i.p.) failed to rescue neutropenia, suggesting that alternative mechanisms, such as disrupted expression of transcription factors, may contribute to neutropenia in SDS. Transcript analysis of Sbds-deficient MC-MMs revealed downregulation of Rara (Retinoic acid receptor α) and its target Cdkn1b (p27), implicated in cell cycle arrest and terminal granulopoiesis (Walkley et al., Blood 2004). Congruent with this finding, Ki67 staining showed that end-stage granulocytes from Sbds Δ/Δ recipients fail to exit cell cycle. In conclusion, we established a mouse model of neutropenia caused by Sbds deficiency in the hematopoietic system, providing experimental support for a direct causative link between Sbds deficiency and neutropenia in mammals. The data reveal a previously unanticipated, selective dependency of late myeloid cells on this ubiquitous protein, while its deficiency spares the function of rapid cycling hematopoietic progenitors. Mechanistically, disrupted expression transcription factors governing terminal myeloid differentiation may be implicated. We anticipate the mouse model to be a valuable tool in further dissecting the molecular pathogenesis of SDS and enabling preclinical studies for disease modulation. Disclosures No relevant conflicts of interest to declare.

Contains fulltext : 47619.pdf (Publisher’s version ) (Closed access) The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding-cassette transporter involved in the transport of drugs used in the treatment of multiple myeloma (MM). Its expression, function and clinical significance in MM, however, are unknown. We report that BCRP is preferentially expressed and functionally active in normal plasma cells but that its function is significantly impaired in plasma cells in newly diagnosed MM. The data presented argue against a role for BCRP in primary drug resistance in MM and the utilisation as a molecular target as such but warrant research into its (patho)physiological role in normal and malignant plasma cells.

Purpose: Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38− stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. Experimental Design: BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38− cells. Results: BCRP was preferentially expressed in leukemic CD34+38− cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38− cells. Conclusions: These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.

Abstract 247 Introduction: Mesenchymal cells are a part of virtually every tissue in metazoans and are thought to participate in organ formation and homeostasis. In the hematopoietic system, mesenchymal cells of the osteoblast lineage have revealed their role as regulators of normal stem cell and hematopoietic physiology. Whether these cells, which have been relegated a relatively non-descript role of ‘stroma', participate in processes that result in disease is relatively understudied. Methods: To explore this, we conditionally deleted Dicer1, the endonuclease essential for miRNA biogenesis, from osteoprogenitor cells by intercrossing transgenic mice expressing a GFP-Cre recombinase under the transcriptional control of the osteoblastic lineage specific osterix promoter to mice containing conditional (floxed) Dicer1 alleles. Results: Deletion of Dicer1 from osteoprogenitor cells resulted in markedly disordered hematopoiesis, affecting multiple lineages and recapitulating key features of human myelodysplastic syndrome (MDS). These features included ineffective hematopoiesis with cytopenia, multilineage dysplasia, increased proliferation and intramedullary apoptosis of primitive hematopoietic cells, decreased B-cell progenitors, increased bone marrow vascularity and the propensity to develop hematopoietic neoplasms (myeloid sarcoma and acute monocytic leukemia-like disease). Comparative genomic hybridization of tumor and germline tissues revealed multiple genetic aberrancies in myeloid sarcomas induced by the Dicer1 deleted environment. The hematopoietic abnormalities were entirely microenvironment dependent with intact Dicer1 in hematopoietic cells. Transplantation of wild-type hematopoietic cells into the mutant environment recapitulated these abnormalities whereas, conversely, transplantation of hematopoietic cells from mutant mice into a wild-type environment resulted in complete normalization of hematopoiesis. In addition, hematopoietic abnormalities were not observed when Dicer1 was deleted from mature osteoblasts indicating a central role of osteoprogenitor cells in the regulation of hematopoiesis. Finally, gene expression profiling and cytokine arrays from osteoprogenitor cells identified candidate molecular effectors of the observed hematopoietic abnormalities. Conclusions: The data demonstrate that a distinctive, differentiation stage specific, stromal subset of osteolineage cells can induce a highly dysfunctional hematopoietic system, recapitulating key characteristics of human myelodysplastic syndrome. Further, they reveal that primary changes in a tissue microenvironment can initiate neoplastic disease. Disclosures: Scadden: Fate Therapeutics: Consultancy.

In most cases of acute myeloid leukemia (AML) CD34+CD38− cells are considered to be stem cells, responsible for the maintenance and relapse of AML. ATP binding cassette transporters function in the extrusion of xenobiotics and chemotherapeutical compounds, and may be involved in therapy resistance. Elucidation of mechanisms conferring drug resistance to CD34+CD38− cells is essential to provide novel targets for stem cell eradication in AML. We studied gene expression of all 45 transmembrane ABC transporters (the complete ABCA, B, C, D and G family) in human hematopoietic CD34+CD38− cells and more committed CD34+CD38+ progenitor cells, from healthy donors and patients with non-hematological diseases (N=11) and AML patients (N=11). Gene expression was assessed using a novel real-time RT-PCR approach with micro fluidic cards. In normal CD34+CD38− cells 36 ABC transporters were expressed, 22 of these displayed significant higher expression in the CD34+CD38− cell fraction compared to the CD34+CD38+ cell fraction. In addition to the known stem cell transporters (ABCB1, ABCC1 and ABCG2) these differential expressed genes included many members not previously associated with stem cell biology. In AML the ABC transporter expression profile was largely conserved, including expression of all 13 known drug transporters. These data suggest an important role for many ABC transporters in hematopoietic stem cell biology. In addition, the preferential expression of a high number of drug transport related transporters predicts that broad spectrum inhibition of ABC transporters is likely to be required for CD34+38− stem cell eradication in AML. This approach will, apart from affecting the leukemic stem cells, equally affect the normal stem cells.