Lineage Restriction Research Articles

Developmental compartments in the larval trachea of Drosophila.

The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 revealed that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein-expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks.

Gene-Regulatory Logic to Induce and Maintain a Developmental Compartment.

Gene-Regulatory Logic to Induce and Maintain a Developmental Compartment.

Abstract PR05: IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation

Abstract Myeloid cells are a heterogenous and abundant population of haematopoietic cells that are virtually present in all mammalian tissues, where they monitor local microenvironment to maintain homeostasis. All myeloid cells originate from the pluripotent hematopoietic stem cells that undergo progressive restriction in their lineage potential to give rise to mature granulocytes and macrophages. Lineage restriction and differentiation are regulated by timely activation of specific set of lineage-specific transcription factors in concert with down-regulation of other set(s) of transcription factors that are important for alternative cell lineage potential. Altered expression of these lineage-specific transcription factors often leads to deregulation of myelopoiesis and resultant hematopoietic disorders. Therefore, lineage-specific transcription factors are essential for myeloid cell lineage differentiation and maturation. Mice with a null mutation of irf8, the gene that encodes IFN regulatory factor 8 (IRF8), exhibit massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs). Therefore, IRF8 is a myeloid cell lineage-specific transcription factor that plays an essential function in the regulation of myelopoiesis. Particularly, IRF8 may determine differentiation, lineage commitment, and immune function of monocytes versus granulocytes under physiological conditions. A hallmark of cancer-bearing mice is the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Interestingly, IRF8 is silenced in MDSCs from tumor-bearing mice. Therefore, IRF8 is apparently a key transcription factor that mediates MDSC differentiation. However, the molecular mechanism underlying IRF8 regulation of MDSCs is largely unknown. Because MDSCs is induced by inflammation, we therefore hypothesized that IRF8 may repress the expression of proinflammatory factors to mediate differentiation of MDSCs/IMCs under physiological and pathological conditions. To test this hypothesis, we made use of conventional IRF8 KO mice, mice with IRF8 deficiency only in myeloid cells, mice with IRF8 deficiency only in T cells, and tumor-bearing mouse models. Here we report an intriguing finding that although IRF8 conventional mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b+Gr1+ IMCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit normal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and IMC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased IMC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased IMC accumulation in the recipient chimera mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. These data thus determine that IRF8 functions in T cells to repress GM-CSF expression to suppress IMCs. However, in tumor-bearing mice, IRF8 is silenced in MDSCs but not in T cells, suggesting a different mechanism of MDSC regulation by IRF8. We observed that silencing IRF8 using IRF8-specific siRNA dramatically increase GM-CSF expression in tumor cells. Therefore, IRF8 represses GM-CSF expression in tumor cells to mediate MDSC differentiation. In summary, we determine that IRF8 regulates GM-CSF expression in T cells and tumor cells, respectively, to mediate myelopoiesis under physiological and pathological conditions. This abstract is also presented as Poster A84. Citation Format: Amy Paschall, Ruihua Zhang, Kankana Bardhan, Chen-Feng Qi, Liang Peng, Geming Lu, Jianjun Yang, Miriam Merad, Mary Zimmerman, Tracy McGaha, Gang Zhou, Andrew Mellor, Scott I. Abrams, Herbert Morse, Keiko Ozato, Huabao Xiong, Kebin Liu. IRF8 regulates GM-CSF expression in T cells and tumor cells to mediate myeloid-derived suppressor cell differentiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR05.

Two Effective Routes for Removing Lineage Restriction Roadblocks: From Somatic Cells to Hepatocytes.

The conversion of somatic cells to hepatocytes has fundamentally re-shaped traditional concepts regarding the limited resources for hepatocyte therapy. With the various induced pluripotent stem cell (iPSC) generation routes, most somatic cells can be effectively directed to functional stem cells, and this strategy will supply enough pluripotent material to generate promising functional hepatocytes. However, the major challenges and potential applications of reprogrammed hepatocytes remain under investigation. In this review, we provide a summary of two effective routes including direct reprogramming and indirect reprogramming from somatic cells to hepatocytes and the general potential applications of the resulting hepatocytes. Through these approaches, we are striving toward the goal of achieving a robust, mature source of clinically relevant lineages.

A control-theoretical approach to the identification of a commitment switch in B lymphopoiesis cell fate determination.

Cellular differentiation is continuously orchestrated by complex networks of transcription factors, signaling molecules and genetic and epigenetic events, a fundamental prerequisite for the design of strategies for reprogramming differentiated cells to immature stem/progenitor cells is a thorough understanding of such complex regulatory machinery. Therefore, mathematical models, along with the associated analysis and control methods, are highly needed in this research field. In the present work, we provide a first model of the genetic regulatory network driving the cellular fate determination at the stage of lymphoid lineage commitment, in particular during lineage restriction of multipotent progenitors to early B-cell committed precursors.

Abstract 4232: CAPE (caffeic acid phenethyl ester) induces a mammary stem cell lineage restriction to a luminal phenotype via chromatin remodeling

Abstract Propolis, and its active component, CAPE, is a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE, inhibits 1) growth of ER+/PR+, ER-/PR- and Her2 + breast cancer cells, 2) the tumorigenic potential of breast cancer stem cells, and 3) mediates its effects through epigenetic modifications via HDAC inhibition, resulting among others, in increased ER in ER- cells. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC). The cell-of-origin hypothesis states that these cancers arise from transformation of mammary stem cells (SC). We tested the ability of CAPE to inhibit SC self-renewal in murine mammary epithelial cells (MEC) and a human breast epithelial cell line (MCF10A). MEC isolated from BALB/c mice were cultured as mammospheres (MMS) for 7 days in ULA 96 well plates using media containing 5% FBS and 5% Matrigel. MMS were dissociated into single cells, recultured in the presence of CAPE for 7 days and passaged in secondary and tertiary passages in media alone. Alternatively, MCF10A transfected with a Let7c-miRNA reporter were sorted for Let7c-negative cells, and similarly cultured with CAPE. Murine MMS were analyzed for luminal marker, K18, basal marker K14, or progesterone receptor (PR) using immunofluorescence. Open chromatin states were identified using ATAC-seq and peaks were independently called using PeakDeck. Open chromatin areas unique to CAPE treated murine MMS were used for pathway analysis performed by Ingenuity Pathway Analysis and confirmed by Integrative Genome Viewer. CAPE treatment resulted in a dose dependent decrease in mammosphere forming efficiency that persisted in secondary and tertiary passages, indicating reduced self-renewal. CAPE shifted the cells within murine MMS from predominantly K14 to K18-positive and increased PR expression. Interestingly, ATAC-seq showed enrichment of open chromatin of the SMARCA4 gene in the exonic region. SMARCA4 is a chromatin remodeling gene that is involved in SWI/SNF complex and alters the positioning of the nucleosome, it is thought to regulate transcription of certain genes by altering the chromatin structure around those genes. In addition, SMARCA4 can bind BRCA1, and regulate the expression of the tumorigenic breast cancer SC marker, CD44. These data indicate that CAPE both inhibited mammary SC self-renewal and shifted the lineage commitment to a luminal lineage. ATAC-seq demonstrated effects at the chromatin level that may regulate the SMARCA4 gene, which controls the transcription of many genes involved in stem cell renewal, pluripotency, and embryonic development and is differentially expressed in the luminal phenotype of human MEC. Thus, CAPE may have an effect on lineage commitment in agreement with our chemoprevention strategy to reduce TNBC breast cancer development. Citation Format: Coral O. Omene, Manan Patel, Kasthuri Kannan, Adriana Heguy, Mary Helen Barcellos-Hoff. CAPE (caffeic acid phenethyl ester) induces a mammary stem cell lineage restriction to a luminal phenotype via chromatin remodeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2015-4232

Genes Left Behind: Climate Change Threatens Cryptic Genetic Diversity in the Canopy-Forming Seaweed Bifurcaria bifurcata.

The global redistribution of biodiversity will intensify in the coming decades of climate change, making projections of species range shifts and of associated genetic losses important components of conservation planning. Highly-structured marine species, notably brown seaweeds, often harbor unique genetic variation at warmer low-latitude rear edges and thus are of particular concern. Here, a combination of Ecological Niche Models (ENMs) and molecular data is used to forecast the potential near-future impacts of climate change for a warm-temperate, canopy forming seaweed, Bifurcaria bifurcata. ENMs for B. bifurcata were developed using marine and terrestrial climatic variables, and its range projected for 2040-50 and 2090-2100 under two greenhouse emission scenarios. Geographical patterns of genetic diversity were assessed by screening 18 populations spawning the entire distribution for two organelle genes and 6 microsatellite markers. The southern limit of B. bifurcata was predicted to shift northwards to central Morocco by the mid-century. By 2090-2100, depending on the emission scenario, it could either retreat further north to western Iberia or be relocated back to Western Sahara. At the opposing margin, B. bifurcata was predicted to expand its range to Scotland or even Norway. Microsatellite diversity and endemism were highest in Morocco, where a unique and very restricted lineage was also identified. Our results imply that B. bifurcata will maintain a relatively broad latitudinal distribution. Although its persistence is not threatened, the predicted extirpation of a unique southern lineage or even the entire Moroccan diversity hotspot will erase a rich evolutionary legacy and shrink global diversity to current (low) European levels. NW Africa and similarly understudied southern regions should receive added attention if expected range changes and diversity loss of warm-temperate species is not to occur unnoticed.

Computational Modeling of a Transcriptional Switch Underlying B-Lymphocyte Lineage Commitment of Hematopoietic Multipotent Cells.

Despite progresses in identifying the cellular mechanisms at the basis of the differentiation of hematopoietic stem/progenitor cells, little is known about the regulatory circuitry at the basis of lineage commitment of hematopoietic multipotent progenitors. To address this issue, we propose a computational approach to give further insights in the comprehension of this genetic mechanism. Differently from T lymphopoiesis, however, there is at present no mathematical model describing lineage restriction of multipotent progenitors to early B-cell precursors. Here, we provide a first model—constructed on the basis of current experimental evidence from literature and of publicly available microarray datasets—of the genetic regulatory network driving the cellular fate determination at the stage of lymphoid lineage commitment, with particular regard to the multipotent-B-cell progenitor transition. By applying multistability analysis methods, we are able to assess the capability of the model to capture the experimentally observed switch-like commitment behavior. These methods allow us to confirm the central role of zinc finger protein 521 (ZNF521) in this process, that we had previously reported, and to identify a novel putative functional interaction for ZNF521, which is essential to realize such characteristic behavior. Moreover, using the devised model, we are able to rigorously analyze the mechanisms underpinning irreversibility of the physiological commitment step and to devise a possible reprogramming strategy, based on the combined modification of the expression of ZNF521 and EBF1.

Ets Factors Regulate Neural Stem Cell Depletion and Gliogenesis in Ras Pathway Glioma.

As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.

Oxidative DNA demethylation mediated by Tet enzymes

AbstractDNA modification, methylation of cytosine (5mC), and oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) can have profound effects on genome function in animals. These modifications are intricately involved in DNA methylation reprograming dynamics during mammalian development. Together, they contribute to cell lineage restriction and maintenance, while also undergoing dynamic changes during cellular transitions and induced reprograming. The last five years have seen an intense research focus on enzymatic DNA demethylation, triggered by the discovery of 5hmC and Tet dioxygenases. In this review, we evaluate recent findings that have provided new insights into the mechanisms underlying DNA demethylation and its effect on developmental regulation.

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

Dual embryonic origin and patterning of the pharyngeal skeleton in the axolotl (Ambystoma mexicanum).

The impressive morphological diversification of vertebrates was achieved in part by innovation and modification of the pharyngeal skeleton. Extensive fate mapping in amniote models has revealed a primarily cranial neural crest derivation of the pharyngeal skeleton. Although comparable fate maps of amphibians produced over several decades have failed to document a neural crest derivation of ventromedial elements in these vertebrates, a recent report provides evidence of a mesodermal origin of one of these elements, basibranchial 2, in the axolotl. We used a transgenic labeling protocol and grafts of labeled cells between GFP+ and white embryos to derive a fate map that describes contributions of both cranial neural crest and mesoderm to the axolotl pharyngeal skeleton, and we conducted additional experiments that probe the mechanisms that underlie mesodermal patterning. Our fate map confirms a dual embryonic origin of the pharyngeal skeleton in urodeles, including derivation of basibranchial 2 from mesoderm closely associated with the second heart field. Additionally, heterotopic transplantation experiments reveal lineage restriction of mesodermal cells that contribute to pharyngeal cartilage. The mesoderm-derived component of the pharyngeal skeleton appears to be particularly sensitive to retinoic acid (RA): administration of exogenous RA leads to loss of the second basibranchial, but not the first. Neural crest was undoubtedly critical in the evolution of the vertebrate pharyngeal skeleton, but mesoderm may have played a central role in forming ventromedial elements, in particular. When and how many times during vertebrate phylogeny a mesodermal contribution to the pharyngeal skeleton evolved remain to be resolved.

High-resolution determination of human immune cell signatures from fine-needle liver aspirates.

High-resolution determination of human immune cell signatures from fine-needle liver aspirates.

Identification of a human clonogenic monocyte/macrophage progenitor in fetal and adult hematopoiesis (HEM5P.232)

Abstract During steady-state hematopoiesis, the monocytic lineage, which includes macrophages, osteoclasts, monocytes, and monocyte-derived dendritic cells (moDCs), arises from a progenitor hierarchy in bone marrow. In humans, multipotent myeloid progenitors such as the granulocyte-monocyte progenitor (GMP) are proximal in this hierarchy, however the distal stages of monocytopoieis are unclear. We report here identification of a human clonogenic, monocytic-committed progenitor with a lineage negative, CD34+ CD38+ CD45RA+ CD123int CD115+ phenotype, which was developmentally conserved across bone marrow, cord blood, and fetal liver. Ex vivo analysis demonstrated a high cloning efficiency, and monocytic but not granulocytic CFU potential. Differentiation in the presence of M-CSF resulted in exclusive production of functional M2-like macrophages, whereas cultures modified with RANKL and GM-CSF/IL-4 resulted in highly enriched cultures of functional osteoclasts and moDCs, respectively. Transplantation into immunodeficient mice reconstituted bone marrow, splenic, pulmonary, and hepatic macrophage subsets, as well as circulating CD14+ monocytic cells. Of note, GMP depleted of CD115+ cells could regenerate the monocytic progenitor phenotype in vitro and in vivo, consistent with a step-wise program of lineage restriction during monocytopoiesis. Identification of the steady-state human monocytic progenitor offers opportunities for therapeutic expansion or modification of the monocytic lineage.

Extreme population subdivision despite high colonization ability: contrasting regional patterns in intertidal tardigrades from the west coast of North America

AbstractAimTo determine whether microscopic marine organisms experience more extreme population subdivision than co‐distributed macroscopic organisms.LocationThe intertidal zone of the west coast of North America.MethodsWe used phylogenetic methods, haplotype networks and analyses of molecular variance (AMOVAs) to examine geographical subdivision in tardigrades (Echiniscoides) between the northern tip of Vancouver Island and San Diego.ResultsAnalyses of 330 individuals from 19 populations revealed a genetically diverse set of tardigrades probably comprising between four and fifteen species. Southern populations displayed extreme population subdivision and contained many deeply divergent lineages that are fully or nearly restricted to single sampling sites. In contrast, northern populations exhibited lower levels of phylogeographical structure and few geographically restricted lineages. AMOVAs showed that inter‐population variation was maximized when nearly all southern populations were kept distinct and all northern populations were treated as a single population.Main conclusionsStrong patterns of population subdivision on fine spatial scales in tardigrades stand in sharp contrast to previous studies of macroscopic organisms across this same range, suggesting that geographical subdivision in microscopic organisms is stronger than in co‐occurring macroscopic organisms.

Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst.

The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst-stage embryo. The EPI lineage isdistinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur invivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages invivo. A period of apoptosis occurred concomitantly with ICM cell-fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early mammalian embryonic development prevents fluctuations in cell fate.

Lineage-dependent effects of aryl hydrocarbon receptor agonists contribute to liver tumorigenesis.

Rodent cancer bioassays indicate that the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD), causes increases in both hepatocytic and cholangiocytic tumors. Effects of AHR activation have been evaluated on rodent hepatic stem cells (rHpSCs) versus their descendants, hepatoblasts (rHBs), two lineage stages of multipotent, hepatic precursors with overlapping but also distinct phenotypic traits. This was made possible by defining the first successful culture conditions for ex vivo maintenance of rHpScs consisting of a substratum of hyaluronans and Kubota's medium (KM), a serum-free medium designed for endodermal stem/progenitor cells. Supplementation of KM with leukemia inhibitory factor elicited lineage restriction to rHBs. Cultures were treated with various AHR agonists including TCDD, 6-formylindolo-[3,2-b]carbazole (FICZ), and 3-3'-diindolylmethane (DIM) and then analyzed with a combination of immunocytochemistry, gene expression, and high-content image analysis. The AHR agonists increased proliferation of rHpSCs at concentrations producing a persistent AHR activation as indicated by induction of Cyp1a1. By contrast, treatment with TCDD resulted in a rapid loss of viability of rHBs, even though the culture conditions, in the absence of the agonists, were permissive for survival and expansion of rHBs. The effects were not observed with FICZ and at lower concentrations of DIM. Conclusion: Our findings are consistent with a lineage-dependent mode of action for AHR agonists in rodent liver tumorigenesis through selective expansion of rHpSCs in combination with a toxicity-induced loss of viability of rHBs. These lineage-dependent effects correlate with increased frequency of liver tumors. (Hepatology 2015;61:548-560)

Dual Roles of Histone H3 Lysine 9 Acetylation in Human Embryonic Stem Cell Pluripotency and Neural Differentiation

Early neurodevelopment requires cell fate commitment from pluripotent stem cells to restricted neural lineages, which involves the epigenetic regulation of chromatin structure and lineage-specific gene transcription. However, it remains unclear how histone H3 lysine 9 acetylation (H3K9Ac), an epigenetic mark representing transcriptionally active chromatin, is involved in the neural commitment from pluripotent embryonic stem cells (ESCs). In this study, we demonstrate that H3K9Ac gradually declines during the first 4 days of in vitro neural differentiation of human ESCs (hESCs) and then increases during days 4-8. Consistent with this finding, the H3K9Ac enrichment at several pluripotency genes was decreased, and H3K9Ac occupancies at the loci of neurodevelopmental genes increased during hESC neural commitment. Inhibiting H3K9 deacetylation on days 0-4 by histone deacetylase inhibitors (HDACis) promoted hESC pluripotency and suppressed its neural differentiation. Conversely, HDACi-elicited up-regulation of H3K9 acetylation on days 4-8 enhanced neural differentiation and activated multiple neurodevelopmental genes. Mechanistically, HDACis promote pluripotency gene transcription to support hESC self-renewal through suppressing HDAC3 activity. During hESC neural commitment, HDACis relieve the inhibitory activities of HDAC1/5/8 and thereby promote early neurodevelopmental gene expression by interfering with gene-specific histone acetylation patterns. Furthermore, p300 is primarily identified as the major histone acetyltransferase involved in both hESC pluripotency and neural differentiation. Our results indicate that epigenetic modification plays pivotal roles during the early neural specification of hESCs. The histone acetylation, which is regulated by distinct HDAC members at different neurodevelopmental stages, plays dual roles in hESC pluripotency maintenance and neural differentiation.

Hematopoietic Stem Cells Are Intrinsically Protected Against MLL-ENL Mediated Transformation

Studies on the developmental pathways of hematopoietic stem cells (HSCs) have led to roadmaps of differentiation and resulted in key information concerning lineage relationships and restriction points in the blood system. This knowledge is also central to understand the etiology of acute myeloid leukemia (AML), where recent work has proposed that the heterogeneity and aggressiveness of AML can associate with the developmental stage of transformation.Balanced chromosomal translocations that result in fusion proteins with aberrant transcriptional regulatory activities are frequent initiating events in acute myeloid leukemia, and a prototype family of such chimeric transcription factors is represented by fusions involving the mixed lineage leukemia-1 (MLL1) gene. Previous work using mouse models have suggested that at some stage of normal differentiation there is a loss of competence to induce AML. However discrepancies exists between these mouse models concerning the target cells of MLL fusion genes. While it is clear that cells can lose competence for leukemic transformation as part of their normal differentiation, the question remains whether the most primitive HSCs are always imbued with leukemogenic competency as part of their normal biology.To address this, we developed a Doxycycline inducible transgenic mouse model of the human chimeric transcription factor Mixed Lineage Leukemia-Eleven Nineteen Leukemia (MLL-ENL). Prospective isolations of candidate leukemia-initiating cells followed by adoptive transfers allowed us to detail leukemia-initiation and competence throughout the hematopoietic hierarchy. We show that AML can origin from multiple HPC subsets with intrinsic granulocytic/monocytic potential. Closely related myeloid progenitors displayed distinct leukemic- and functional capacity in response to physiological levels of MLL-ENL, highlighting the importance of a careful prospective isolation of progenitor populations. AML could also develop efficiently from common lymphoid progenitors, supporting a latent myeloid potential of these cells. By contrast, early commitment to the megakaryocytic/erythroid lineages was incompatible with leukemic development. By contrast, disease failed to arise from the most primitive progenitor subsets, including HSCs. Investigations of the immediate transcriptional responses to MLL-ENL showed evidence for a block in differentiation in both myeloid progenitors and HSCs, while MLL-ENL restricted cell cycle progression uniquely in HSCs. Our study highlights how an oncogene can exert unique functions depending on the developmental position of its cellular targets and demonstrate the existence of a mechanism, operational at the level of immature HSCs/progenitors, which act to prevent leukemic development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitative Analysis of Patient-Specific Lesions in Primary and Xenografted Myelodysplastic Syndromes Reveals Complex Hierarchies and Subclonal Diversity That Evolve during Disease Progression

BackgroundMyelodysplastic Syndromes (MDS) likely arise from an evolutionary process involving accumulation of somatic mutations and selection that occurs at the level of hematopoietic stem cells. Using next generation sequencing (NGS), several groups found recurrent somatic mutations to be associated with MDS. However, the history of stepwise molecular progression is still poorly defined. Likewise, little is known about patient-specific subclonal compositions, which may well contribute to the tremendous heterogeneity observed both in terms of clinical manifestations and response to treatment. Therefore, we sought to reconstruct patient-specific clonal hierarchies and decipher their dynamic evolution during long-term disease monitoring in order to better understand MDS pathogenesis and aid in adapting targeted therapeutic options in the future.MethodsBone marrow (BM) or CD34+ cells from patients with MDS were subjected to mutational screening by whole exome sequencing (WES, n=44) or targeted NGS (n=28) interrogating up to 17 recurrently mutated genes. Mesenchymal stromal cells (MSCs) were used as germline control. Allelic burdens were quantified with custom pyrosequencing assays and interphase-FISH in FACS purified myeloid, erythroid, lymphoid and stem cells isolated from both patients’ BM and corresponding xenografts in NSG mice. For unbiased follow up analysis, BM or CD34+ cells from two distant time points (median: 3 years, range 0.5-4.0 years) were analyzed for 13 patients using WES and these data were used to design patient-specific mutational panels. These panels contained amplicons representing mutational events uniquely present at one time point as well as others present at both time points. The panels were then subjected to ultra-deep-sequencing (UDS) to accurately quantify mutational burdens in all follow up (FU) samples.ResultsIntegrative mutational data allowed us to reconstruct patient-specific mutational hierarchies in 35 cases, which revealed both linear and branching evolution in MDS. The data is also in support of the notion that potential founder lesions are highly enriched in genes involved in RNA splicing and epigenetic regulators, which we further show to be frequently detected in primary and/or xenografted lymphocytes. In contrast, we clearly demonstrate that large scale cytogenetic lesions (e.g. monosomy 7, trisomy 8, del(5q)) occur as late mutational events in at least 85% of the cases analyzed (n=17/20, 3 unresolved cases). Moreover, we could readily demonstrate the existence of subclonal heterogeneity in the patients’ BM, with variable contribution to different lineages and also cases showing lineage restriction of specific sub-clones. Most importantly, UDS analysis of FU samples from 13 independent patients (median FU 3.3 years, range FU 0.5-11.8 years and median of 5 samples per patient) with detailed clinical data revealed a highly dynamic clonal evolution during the course of the disease and dramatic shifts in the composition of mutational (sub-)clusters during therapy. Treatment often resulted in complete disappearance of specific sub-clones and most importantly, simultaneous outgrowth of previously undetectable subclones that subsequently dominated the marrow.ConclusionBy reconstructing patient-specific mutational hierarchies we gained invaluable insights into the dynamic clonal composition and the molecular progression of human MDS. Our study shows that acquisition of large scale cytogenetic lesions appears to be a rather late event, which likely indicates that such lesions might not be tolerated by healthy stem cells. Most importantly, WES and subsequent deep sequencing analysis of FU samples demonstrated that patient-specific clonal diversity is highly dynamic and modulated during the course of the disease. This is especially true upon response to treatment, where concomitant disappearance and emergence of new pathogenic subclones was observed. Our work unravels two major paths of mutational acquisition by which MDS cells evade therapeutic pressure: (1) linear evolution of the previously sensitive subclone or (2) by branching evolution of an earlier independent founder clone. Our findings therefore strongly emphasize the importance of a genetically unbiased disease monitoring and the development of novel therapeutic strategies aiming at targeting founder lesions that are present in all ensuing subclones. DisclosuresNolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.