Primary Murine Hematopoietic Cells Research Articles

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

SMARCA5 interacts with NUP98-NSD1 oncofusion protein and sustains hematopoietic cells transformation

BackgroundAcute myeloid leukemia (AML) is characterized by accumulation of aberrantly differentiated hematopoietic myeloid progenitor cells. The karyotyping-silent NUP98-NSD1 fusion is a molecular hallmark of pediatric AML and is associated with the activating FLT3-ITD mutation in > 70% of the cases. NUP98-NSD1 fusion protein promotes myeloid progenitor self-renewal in mice via unknown molecular mechanism requiring both the NUP98 and the NSD1 moieties.MethodsWe used affinity purification coupled to label-free mass spectrometry (AP-MS) to examine the effect of NUP98-NSD1 structural domain deletions on nuclear interactome binding. We determined their functional relevance in NUP98-NSD1 immortalized primary murine hematopoietic stem and progenitor cells (HSPC) by inducible knockdown, pharmacological targeting, methylcellulose assay, RT-qPCR analysis and/or proximity ligation assays (PLA). Fluorescence recovery after photobleaching and b-isoxazole assay were performed to examine the phase transition capacity of NUP98-NSD1 in vitro and in vivo.ResultsWe show that NUP98-NSD1 core interactome binding is largely dependent on the NUP98 phenylalanine-glycine (FG) repeat domains which mediate formation of liquid-like phase-separated NUP98-NSD1 nuclear condensates. We identified condensate constituents including imitation switch (ISWI) family member SMARCA5 and BPTF (bromodomain PHD finger transcription factor), both members of the nucleosome remodeling factor complex (NURF). We validated the interaction with SMARCA5 in NUP98-NSD1+ patient cells and demonstrated its functional role in NUP98-NSD1/FLT3-ITD immortalized primary murine hematopoietic cells by genetic and pharmacological targeting. Notably, SMARCA5 inhibition did not affect NUP98-NSD1 condensates suggesting that functional activity rather than condensate formation per se is crucial to maintain the transformed phenotype.ConclusionsNUP98-NSD1 interacts and colocalizes on the genome with SMARCA5 which is an essential mediator of the NUP98-NSD1 transformation in hematopoietic cells. Formation of NUP98-NSD1 phase-separated nuclear condensates is not sufficient for the maintenance of transformed phenotype, which suggests that selective targeting of condensate constituents might represent a new therapeutic strategy for NUP98-NSD1 driven AML.

Manipulation of Fatty Acid Metabolism Impairs Megakaryocyte Differentiation and Platelet Production

Background: Obesity is associated with an altered plasma lipid composition that impacts platelet activation, indicating that lipids directly affect platelet function and reactivity. However, very little is known about which essential lipids are important to enable platelet production from their precursors, megakaryocytes (MKs), and whether obesity influences lipid composition and consequently affects thrombopoiesis.Aim: We aimed to determine which lipids and associated pathways play a significant role in MK differentiation and platelet production in health and upon obesity. Our long-term goal is to determine if we can manipulate MK maturation and platelet production through lipid incorporation or inhibition.Methods: In order to determine the lipid profile of MKs through maturation, we performed an extensive lipidomics screen on primary MK progenitors, mature MKs, and platelets. Based on our findings, we then targeted de novo fatty acid (FA) synthesis in MKs to determine whether manipulation of this pathway attenuates MK differentiation and platelet formation. In parallel, we investigated megakaryo- and thrombopoiesis in an experimental setting of diet-induced obesity (DIO) in mice to examinate how altered lipid compositions within the plasma affect MK maturation.Results: Lipidomic data revealed increased polyunsaturated FA and plasmalogen content in the late stages of MKs maturation, suggesting that lipid composition is undergoing changes that may enable platelet production. To validate and extend these results, we inhibited multiple enzymes in de novo lipogenesis and FA synthesis. Using two different sources of primary murine hematopoietic stem cells (HSCs), we found that MK differentiation and maturation from HSCs was significantly decreased up to 60% after inhibition of acetyl-coA carboxylase (ACC) and 80% after fatty acid synthetase (FASN) inhibition, both enzymes from the de novo lipogenesis pathway. Moreover, we also found that MK differentiation and maturation were highly decreased (up to 70%) after the inhibition of acyl-coA synthetase (ACS). To further examine the role of fatty acids incorporation on platelet production, we next treated mature MKs with an ACS inhibitor to investigate the direct effect of FA incorporation on proplatelet formation; we observed a significant, multifoldreduction in proplatelet area. While dihydroceramides were altered in the lipidomic data, impairment of the de novo ceramide synthesis pathway did not affect either MK maturation or platelet production. Moreover, we observed an increased abundance of HSCs, myeloid cells, and MK progenitors in the bone marrow of DIO mice compared to control mice on a chow diet. This further supports an important role for lipids in megakaryopoiesis and suggests that differences in platelet reactivity during obesity may be caused by altered MK maturation and/or platelet production.Conclusions: Our results elucidate an important and previously unrecognized role for fatty acid synthesis in megakaryopoiesis and platelet production. The findings imply that an altered lipid content, as observed in patients with obesity, may not only impact platelet reactivity but also MK maturation and function. Consequently, platelets with an altered lipid content might originate from pathogenic MKs in obesity, which makes lipid-regulating proteins in MKs novel and viable therapeutic targets. DisclosuresMachlus: KEROS Therapeutics: Consultancy, Honoraria; STRM.BIO: Consultancy, Honoraria.

A Novel CSF3R Activating Mutation Identified in a Patient with Chronic Neutrophilic Leukemia

Mutations in CSF3R, the growth factor receptor that signals to drive neutrophil production, are commonly found in Chronic Neutrophilic Leukemia (CNL). There are two main types of CSF3R mutations in CNL: membrane-proximal point mutations and cytoplasmic truncating mutations. The membrane proximal point mutations-notably T618I-are the most common in CNL. These point mutations lead to the loss of glycosylation sites, causing ligand-independent dimerization of the receptor and enhanced activation of downstream signaling pathways. Previously, these glycan-altering mutations were thought to reside in a relatively restricted region near the membrane spanning 610-618 that contains an N-linked glycosylation site and three putative O-linked glycosylation sites.A 55-year old man presented with a WBC of 50,000 and a predominant neutrophilia with 84% neutrophils and bands and no immature cells. A bone marrow examination showed a markedly hypercellular bone marrow exhibiting myeloid-predominant maturing trilineage hematopoiesis with left-shifted myeloid maturation, mild megakaryocytic atypia (small, hypolobated forms), mild reticulin fibrosis (grade 1 of 3). He had multi-station small volume lymphadenopathy and a spleen that measured 16.6 cm craniocaudally. The findings were most consistent with chronic neutrophilic leukemia.Targeted NGS analysis revealed three mutations in CSF3R: Q739* (VAF 26%), Q741* (VAF 24%) and N579Y (VAF 52%). ASXL1 G646fs (38% VAF) and ETNK1 H243Q (VAF 12%) mutations were also present. The truncating mutations in CSF3R were reported as pathogenic, but the N579Y mutation was listed as a variant of uncertain significance. When truncating mutations occur in CNL, they most frequently occur alongside an activating point mutation. This, combined with the knowledge that N579 was previously mapped as a site of receptor glycosylation prompted us to assess the oncogenicity of this variant.We undertook several parallel approaches to characterize this variant. First, a cytokine independent growth assay was performed in Ba/F3 cells. The CSF3R N579Y variant allowed these cells to proliferate in the absence of cytokine support, similarly to the well characterized CSF3R T618I variant. Furthermore, it enabled growth independent from the ligand for CSF3R (GCSF), indicating that this mutation likely also confers ligand-independent dimerization. In an orthogonal approach, we found that CSF3R N579Y enables primary murine hematopoietic cells to grow in cytokine-free methylcellulose to a similar extent as CSF3R T618I.To understand the downstream consequences of receptor activation we undertook immunoblot analysis and discovered that CSF3R N579Y robustly enhances JAK/STAT and MAPK signaling downstream of the receptor. Because of the know activation of JAK by CSF3R mutations, a JAK1/2 inhibitor (ruxolitinib) has been under investigation for treatment of CSF3R-mutant CNL. We find that CSF3R N579Y confers sensitivity to ruxolitinib in both Ba/F3 cells and murine colony forming unit assays. Furthermore, ruxolitinib inhibited colony formation of CD34 positive cells isolated from a bone marrow aspirate from the patient whose leukemia harbored CSF3R N579Y, CSF3R Q739* and CSF3R Q741* mutations. Collectively, our data suggests that N579Y is a pathogenic and potentially actionable variant and that N-linked glycosylation outside of the membrane-proximal region is necessary to maintain the receptor in an inactive state. [Display omitted] DisclosuresSanz-Altamira: Kura Oncology: Consultancy. DeAngelo: Autolus: Consultancy; Forty-Seven: Consultancy; Incyte: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Servier: Consultancy; Takeda: Consultancy; Abbvie: Research Funding; Blueprint: Research Funding; Amgen: Consultancy; Agios: Consultancy; Glycomimetrics: Research Funding. Maxson: Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Heme Regulates the Expression in Erythroblasts of Placenta Growth Factor, a Contributor to Pulmonary Hypertension in Sickle Cell Disease, through the Oxidant Response Transcription Factor NRF2

Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PGF), which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PGF levels, leading us to the investigation of a mechanism where excessive heme induces PGF expression. Our lab's published work has demonstrated a significant heme-bound iron (hemin) stimulation of the PGF promoter in human K562 cells and in primary human erythroid cells. Our initial experiments with N-acetylcysteine and the nonspecific oxidant hydrogen peroxide, suggested to us a role of oxidant stress in PGF regulation, hypothetically through the NRF2 antioxidant response mechanism.We conducted gene expression analysis by quantitative real time PCR for PGF in human K562 erythroblastoid cells, erythroid-differentiated human and mouse primary bone marrow cells before and after exposure to hemin. We tested the effect of Nrf2 agonists and an antagonist. We analyzed expression of heme oxygenase-1 as a positive control, and expression of several Nrf2 family members, as well as baseline genes. We performed siRNA gene knockdown experiments in cultured cells, chromatin immunoprecipitation experiments and intravenous injection of hemin (120 μmol/kg) in the tail veins of wild type and Nrf2 null mice, followed by assay of plasma PGF by ELISA and qPCR of bone marrow.We find that heme regulates the expression of several members of the Nrf2 family, which in turn may appear to affect PGF expression. More specifically, the antioxidant transcription factory family members NRF2, MafF, MafG and BACH1 are transcriptionally activated in a specific temporal pattern after heme exposure. NFE2 and MafK transcripts were simultaneously repressed by heme treatment. The well-characterized non-oxidative NRF2 agonist sulforaphane activated the endogenous PGF promoter and the NRF2 inhibitor brusatol inhibited heme induction of the endogenous PGF promoter. Specific gene knockdown results using siRNA supports additive roles for NRF2 and its heterodimeric partner MafG in activating the PGF promoter in response to heme, presumably as a heterodimer. We verified direct binding of NRF2 on the PGF promoter by chromatin immunoprecipitation experiments. While PGF regulation shows quite many common aspects to HO-1 regulation (heme and oxidative stress response), we find that protoporphyrin induces HO-1 (known to require BACH-1), but not PGF.In addition to the experimental data from our human K562 cell culture models, we have found corroborating results in primary erythroid-differentiated human and murine hematopoietic cells. In vivo, intravenous injection of heme in wild type mice produces robust secretion of PGF protein into plasma within 3 hours (192 ± 16 vs. 527 ± 62 pg/mL, P <0.0001, mean ± SEM, one-way ANOVA with Sidak's multiple comparison test) and this heme induction of plasma PGF protein is blunted in the Nrf2 null mice (527 ± 62 vs. 343 ± 21 pg/mL, P=0.004).Our results support a mechanism in which accelerated heme exposure in SCD promotes robust expression of PGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG, culminating in expression of the vascoconstrictor endothelin-1. This mechanism helps to explain the clinical observation that adults with SCD heavily exposed to red cell transfusion are more likely to develop high endothelin levels and pulmonary hypertension, as a potential consequence of excess heme trafficking from the turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; MAST Therapeutics: Research Funding; Novartis: Consultancy.

Transcriptional Repression of RASSF2 Impairs Hippo Kinase-Dependent p53 Activation and Tumor Suppression in t(8;21) AML

The t(8;21) chromosomal translocation is a recurring cytogenetic abnormality in acute myeloid leukemia (AML), found in ~10% of AML patients. This translocation results in the stable fusion of the RUNX1 (AML1) and RUNX1T1 genes, and formation of the oncofusion protein RUNX1-ETO (RE). One function of RE in promoting leukemia development is the recruitment of aberrant transcription factor complexes to regulatory regions of RUNX1 target genes known to be critical for myeloid differentiation, such as CEBPA, SPI1, NFE2, and CSF1R . Despite this knowledge, additional RE target genes remain poorly characterized, and the complete molecular mechanism through which RE leads to leukemic transformation remains to be elucidated.Previously, we used a mouse model of t(8;21) AML to compare gene expression in RE-expressing leukemia cells to the normal mouse hematopoietic progenitor (lin-cKit+) compartment (Lo et al, 2012). Among the most significantly downregulated genes was Ras-association domain family member 2 (Rassf2). RASSF genes are frequently epigenetically silenced in human tumors, making them highly relevant to study in the context of cancer development. Using public gene expression data, RT-qPCR, and western blotting, we found that RASSF2 expression is specifically downregulated in t(8;21) AML cells compared to non-t(8;21) AML cells and normal CD34+ controls. Furthermore, RE knockdown in a t(8;21) AML cell line led to marked upregulation of RASSF2 mRNA. Based on ChiP-seq data analysis this repression appears to be mediated by direct RE binding and HDAC recruitment to the promoter region of both alternative RASSF2 transcriptional start sites in the genomic locus.To investigate the significance of this repression we used retroviral vectors to re-express RASSF2 in t(8;21) AML cell lines and RE-transduced primary murine hematopoietic cells in vivo and in vitro . Re-expression of RASSF2 significantly improved survival and reduced the total leukemic burden in a primary mouse model of RE-driven AML in vivo . RASSF2 inhibited leukemic transformation and the self-renewal ability of RE-transduced mouse bone marrow cells in a serial replating/colony formation assay, without a noticeable effect on normal hematopoietic progenitors. This inhibition correlated with increased apoptotic cells in the context of RE and RASSF2 co-expression.Given that RASSF2 is a non-enzymatic scaffolding protein, we hypothesized that it mediates tumor suppressive functions via its Ras-association or SARAH effector domains. We saw no detectable effect of RASSF2 expression on oncogenic Ras signaling in t(8;21) AML cells. This led us to investigate the SARAH domain-mediated interaction of RASSF2 with the pro-apoptotic Hippo kinases, MST1 and MST2. RASSF2 expression in t(8;21) AML cells led to an increased protein amount of both MST1 and MST2 in a SARAH domain-dependent manner. Importantly, use of either a RASSF2 SARAH domain deletion mutant, or Mst1(-/-)Mst2(-/-) mouse bone marrow cells, completely abrogated the ability of RASSF2 to inhibit RE leukemia cell self-renewal in the serial replating/colony formation assay. We found this inhibition to be independent of canonical Hippo pathway signaling, and therefore utilized a proximity-dependent biotin identification approach (BioID, Roux et al, 2012) to map the protein interactome of RASSF2-MST1/2 in a t(8;21) AML cell line. Interestingly, this revealed an interaction between RASSF2 and the protein deacetylase SIRT1. This highlighted a role for RASSF2 in promoting p53 acetylation in response to cellular stress, which is dependent on MST1/2-mediated negative regulation of SIRT1. Overall, our study revealed a critical tumor suppressive role of RASSF2-MST1/2 signaling via the SIRT1-p53 axis in t(8;21) AML. DisclosuresNo relevant conflicts of interest to declare.

Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy-controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb-dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase-deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy-induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.

Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo

AbstractChemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate–mediated intracellular Ca2+ flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest.

Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy-controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb-dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase-deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy-induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.

PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear.Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.

Functional Dissection and Targeting Of Bmi1 Independence Of MN1 Leukemia

While PcG protein, Bmi1, plays a critical role in development of leukemic stem cells (LSCs), we have recently shown a differential Bmi1 dependency for LSCs initiated by different oncogenic transcription factors associated with distinct prognostic outcomes. PML-RARA and AML1-ETO leukemias associated with good prognosis are dependent on Bmi1, whereas poor prognostic leukemia induced by MLL fusion that is capable of activating multiple Hox genes is Bmi1 independent. However the role of Bmi1 and the mechanisms of over-coming Bmi1 dependency in other acute myeloid leukemia (AML) subtypes are still largely unknown. Aberrations of the MN1 gene mostly as an over-expression or rarely as a fusion partner of TEL in patients carrying the translocation t(12;22)(p13;q11) are frequently found in AML and myelodysplastic syndrome (MDS). Forced expression of MN1 induces aggressive AML with a 100% penetrance in mouse models within a few weeks, a latency significantly shorter than myeloid malignancy induced by MN1-TEL. Consistently, MN1 over-expression correlates with reduced drug response and poor prognosis of AML patients as well as high level of Bmi1 expression. In spite of these striking experimental and clinical features, the molecular mechanisms underlying MN1 leukemia are still largely unknown, and little progress has been made in targeting this leukemia. In the current study, we sought out to investigate the role of Bmi1 for MN1 mediated leukemic transformation and the mechanisms underlying this aggressive leukemia. In contrast to MN1-TEL that is dependent on Bmi1 for transformation of murine primary hematopoietic cells, MN1 over-expression could transform Bmi1-/- hematopoietic progenitor cells (HPCs) and induced serially transplantable AML in mice with the same latency as MN1 transformed wild type (wt) HPCs. Further molecular analyses revealed a significant up-regulation of p16Ink4a and cellular senescence in MN1-TEL Bmi1-/- HPCs, which was absent in MN1 transformed cells. Senescence in MN1-TEL Bmi1-/- cells could be rescued by re-expression of Bmi1 or suppression of p16 respectively, consistently suggesting an inherent functional difference between MN1 and MN1-TEL reflected by contrasting Bmi1 dependences. To identify the functional domain critical for the Bmi1 independency, structure/function analysis revealed that the removal of DNA binding domain (DBD) in the TEL moiety conferred Bmi1 independent transformation to MN1-TEL (MN1-TELΔDBD). MN1-TELΔDBD was able to induce transplantable leukemia in both the wild type and Bmi1 deficient cells. Conversely, fusion of TEL DBD to full length MN1 abolished its Bmi1 independence, and failed to transform Bmi1 deficient cells. These results strongly suggest that MN1 regulated molecules/pathways critical for Bmi1 independent transformation are misguided by the TEL DBD in MN1-TEL leukemia, providing a unique platform to dissect the pathways for Bmi1 independent transformation in AML. By performing global gene expression analyses on MN1, MN1-TEL and MN1-TELΔDBD transformed cells, we identified 1727 genes differentially expressed in MN1 transformed cells compared with MN1-TEL transformed cells; whereas only 44 genes were differentially expressed in MN1-TEL versus MN1-TELΔDBD transformed cells. When we overlapped these two gene sets together, we generated a unique gene set containing 34 genes associated with Bmi1 independence, including metabolic enzymes, signaling molecules and transcription factors such as Hoxa gene that has previously implicated in Bmi1 independent leukemic transformation. To assess the functional significance and the potential of targeting the candidates in this gene list in overcoming Bmi1 independent transformation, we performed functional analyses using shRNA approaches with a focus on those pharmacologically tractable candidates to suppress Bmi1 independent leukemic transformation. As a result, we were able to identify and demonstrate two different classes of enzymes with rigid catalytic domains that are required for Bmi1 independent transformation by MN1. Together, we dissect the mechanisms underlying Bmi1-independent leukemic transformation, and provide promising novel targets for MN1 leukemia. Disclosures: No relevant conflicts of interest to declare.

The Oncogenic Transcription Factor STAT5 Triggers Aberrant Expression Of CD25 (IL-2RA) In Neoplastic Stem Cells In Ph+ CML

Chronic myeloid leukemia (CML) is a stem cell neoplasm characterized by the Philadelphia (Ph) chromosome and the related oncoprotein, BCR/ABL. Despite the availability of novel BCR/ABL-targeting tyrosine kinase inhibitors (TKI), many patients relapse which may be due to resistance of neoplastic stem cells (NSC) against TKI. So far, little is known about specific markers and targets expressed on CML NSC. We here report that CML NSC express the interleukin-2R alpha-chain CD25 in a specific (aberrant) manner. Whereas normal bone marrow (BM) stem cells were found to express only low amounts or did not express CD25, CD34+/CD38− NSC in CML were found to express CD25 in almost all patients examined (31/33=95%), independent of the phase of disease. CML NSC were also found to express IL-1RAP, DPPIV (CD26), Siglec-3 (CD33), Campath-1 (CD52), KIT (CD117) and IL-3RA (CD123). We were also able to show that highly purified CD25+ NSC express BCR/ABL and engraft irradiated NOD-SCID-IL-2Rg-/- (NSG) mice with BCR/ABL+ cells, whereas CD34+/CD38-/CD25-/CD26- cells from the same patients failed to express BCR/ABL, and engrafted NSG mice with BCR/ABL-negative cells. To define signalling-molecules contributing to the expression of CD25 in CML NSC, we employed primary murine hematopoietic cells infected with bcr/abl-p210 in combination with a retrovirus encoding for STAT5A or STAT5B. Enforced expression of either STAT5A or STAT5B resulted in enhanced expression of CD25 in Lin-/Sca-1+/Kit+ mouse BM stem cells (LSK cells), regardless of the presence or absence of bcr/abl. In fact, the CD25-promoting effect of STAT5 was seen in normal stem cells as well as in bcr/abl-transformed LSK cells. Correspondingly, shRNA against STAT5 was found to downregulate expression of CD25 in the human CML cell line KU812. The BCR/ABL-targeting TKI imatinib, nilotinib and ponatinib were also found to inhibit expression of CD25 on KU812 cells. In primary CML NSC, ponatinib was found to induce marked suppression of CD25, whereas imatinib and nilotinib showed only weak effects. In consecutive experiments, we found that the MEK inhibitors RDEA119 and PD032509, the STAT5-targeting drug pimozide and the dual PI3 kinase/mTOR blocker BEZ235 all inhibit 3H-thymidine uptake and thus proliferation in KU812 cells. However, whereas RDEA119 and PD032509 as well as pimozide were found to downregulate expression of CD25 in KU812 cells, BEZ235 was found to even upregulate expression of CD25 on KU812 cells. We next applied an shRNA against CD25. The shRNA-induced knock-down of CD25 in KU812 resulted in an increased proliferative capacity, suggesting that CD25 may act as a potential tumor suppressor in CML stem- and progenitor cells. Together, our data show that CD25 is a novel functionally relevant marker-antigen and potential tumor suppressor of CML NSC. Expression of CD25 in CML cells appears to be triggered by MEK and STAT5 activity, and may be negatively regulated by the PI3 kinase/mTOR pathway. Whether modulation of CD25 can be exploited clinically and used as basis for the development of novel NSC-targeting treatment concepts in CML remains to be determined in future studies. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

The transcription factors Runx1 and c-Myc have individually been shown to regulate important gene targets as well as to collaborate in oncogenesis. However, it is unknown whether there is a regulatory relationship between the two genes. In this study, we investigated the transcriptional regulation of endogenous c-Myc by Runx1 in the human T cell line Jurkat and murine primary hematopoietic cells. Endogenous Runx1 binds to multiple sites in the c-Myc locus upstream of the c-Myc transcriptional start site. Cells transduced with a C-terminally truncated Runx1 (Runx1.d190), which lacks important cofactor interaction sites and can block C-terminal-dependent functions of all Runx transcription factors, showed increased transcription of c-Myc. In order to monitor c-Myc expression in response to early and transiently-acting Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Murine splenocytes treated with TAT-Runx1.d190 showed an increase in the transcription of c-Myc within 2 hours, peaking at 4 hours post-treatment and declining thereafter. This effect is dependent on the ability of Runx1.d190 to bind to DNA. The increase in c-Myc transcripts is correlated with increased c-Myc protein levels. Collectively, these data show that Runx1 directly regulates c-Myc transcription in a C-terminal- and DNA-binding-dependent manner.

Receptor Tyrosine Kinases, Notch Mutations and PTEN Loss Converge On mTOR in T-ALL and Cause Addiction to Cap Dependent mRNA Translation.

Abstract 2415 Introduction.High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL) and has been linked to c-myc independence, as well as resistance of T-ALL to Glucocorticoids and Notch inhibition. The master regulator of PI3K-AKT signaling is the lipid phosphatase PTEN. Despite impaired or lost PTEN activity in T-ALL experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is needed to sustain high mTOR activation. Here we investigated how RTK signaling contributes to the pathogenesis of T-ALL. To mimic RTK signaling we expressed ΔTrkA, a constitutively active nerve growth factor RTK in primary murine hematopoietic cells. Injection of ΔTrkA-transduced cells into C57BL6 mice induced transplantable T-ALL with a latency of 120 days. Results.Signaling studies showed that ΔTRKA activates mTORC1 over the MAPK pathway, but not mTORC2. In contrast, ΔTRKA+ T-ALL showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of mTORC1 and mTORC2 and absent MAPK signaling. To understand the rewired signaling network we first ruled out contribution of insertional mutagenesis by sequencing vector integration sites. Array-CGH revealed hetero- or homozygous loss of PTEN in 4 of 6 T-ALL lines. In the remaining 2 T-ALL lines, which showed normal PTEN protein levels, we detected two PTEN mutations in the phosphatase domain, D92G and G165E, the latter of which has not been described before. Re-expression of the mutants in PTEN−/− cells revealed complete loss of function for D92G and a partial loss for the G165E mutant. Restoration of wt PTEN expression in the T-ALLs resulted in reversion of the signaling pattern. Sequencing of Notch1 revealed both, PEST domain mutations and 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein. Notch inhibition in PTEN−/− cell lines resulted in robust inhibition of mTORC2, showing that Notch contributes to mTORC2 activation independently of the Hes1-PTEN axis. Despite the loss of PTEN most T-ALLs remained addicted to the input of the leukemia initiating RTK, ΔTRKA. Importantly, the cooperation of three oncogenic pathways converging on mTOR culminated in an oncogenic addiction to mTOR. We screened several inhibitors of the mTOR pathway and found that ΔTRKA+ PTEN− Notch+ T-ALL are highly susceptible to an inhibition of cap dependent translation by 4EGI-1, whereas primary hematopoietic cells and freshly isolated thymocytes were far less susceptible. Polysome profiling indicated a profound decrease in ribosome occupancy upon treatment with 4EGI-1. Microarray analysis of polysome fractions revealed that mRNAs belonging to genes that were strongly upregulated between T-ALL and normal thymocytes were specifically shifted out of the actively translated transcriptome by 4EGI-1. These genes included members of the translational apparatus itself, mitochondrial matrix proteins, cell cycle regulators such as CyclinD1 and c-myc as well as Bcl-2. Western blots confirmed early loss of these proteins after cessation of cap dependent translation. Conclusions.Biochemical and genetic dissection of T-ALLs induced by constitutive RTK signaling uncovered activating Notch mutations and PTEN loss as cooperating events and revealed a strong, druggable addiction to cap-dependent translation downstream of oncogenic mTOR. The striking overlap of genes defining the molecular difference between T-ALL blasts and normal thymocytes and genes that preferentially lost ribosomal occupancy upon 4EGI-1 treatment suggests that this drug targets oncogenic core pathways in T-ALL and deserves further investigation. Disclosures:No relevant conflicts of interest to declare.

Sudemycin Selectively Inhibits Growth of Primary Murine Hematopoietic Cells Expressing Mutant U2AF1

Abstract 554Core components of the pre-mRNA splicing complex (the “spliceosome”) are targets of recurrent somatic mutation in patients with myelodysplastic syndrome (MDS). Using whole genome and targeted resequencing, we previously identified recurrent missense mutations in U2AF1 (encoding U2AF35, the 35 kDa U2 accessory factor) at serine 34 (S34F or S34Y) or glutamine 157 (Q157R) in 8.7% of de novo MDS patients. The mechanism by which mutations in U2AF1 and other recurrently mutated spliceosome components contribute to MDS pathogenesis is unknown, but recurrent mutations in this pathway suggest that it may provide a novel therapeutic target. Pladienolide B and FR901464 are two bacterial products that modulate activity of the spliceosome. The derivatives, E7107 and spliceostatin A, and synthetic analogs (sudemycins), bind SF3B, a complex that includes SF3B1 and six other subunits of the spliceosome. Treatment of cell lines with these agents causes accumulation of aberrantly spliced gene products. Sudemycins have favorable pharmacokinetic properties (improved solubility, chemical stability, and plasma half-life) and induce cell cycle arrest and apoptosis in low nanomolar concentrations in cell lines in vitro. They also inhibit growth in xenograft models in vivo, with no evidence of toxicity in mice treated with pharmacologically active doses. We hypothesize that splicing gene mutations cause alterations in splice isoforms that provide a selective advantage to tumor cells, and that perturbation of this environment with small molecule spliceosome modulators may cause a selective disadvantage for cells with these mutations. To test this hypothesis, we generated MSCV-based retroviruses encoding the wildtype (MSCV-WT-ires-GFP) or S34F mutant (MSCV-MT- ires-GFP) U2AF1 alleles. We infected c-kit+ primary C57BL/6J murine bone marrow cells with these recombinant retroviruses (or empty vector) and cultured them in vitro with cytokine-supplemented media. Consistent with previous reports, vehicle-treated cells (0.1% DMSO) expressing the mutant allele had reduced growth over 5 days, compared to cells expressing the wildtype allele or empty vector (P<0.0001, n=5 technical replicates, two experiments). At doses between 10–150 nM, sudemycin (but not an inactive sudemycin analog) induced a reproducible dose-dependent decrease in cell growth, most significant at 75 nM (p<0.0001, n=5 technical replicates, two experiments), and an increase in apoptosis (p=<0.05, n=3 technical replicates), as measured by annexin V positivity, in cells expressing the mutant, compared to the wildtype allele or empty vector. To study the effect of the U2AF1 mutation and sudemycin exposure on splicing, we analyzed RNA isoforms of Mdm2 and Fmr1, two genes known to undergo alternative splicing. After retroviral transduction, GFP+ cells were sorted and RNA was isolated for RT-PCR analysis at baseline or 2h–6h after exposure to sudemycin (75 nM) or vehicle. The U2AF1 S34F mutation increased the abundance of Fmr1 transcripts utilizing a cryptic splice acceptor, compared to cells transduced with empty vector or wildtype U2AF1 (P<0.05, n=3 technical replicates). Sudemycin treatment decreased Fmr1 cryptic splice site utilization (p<0.05, n=3 technical replicates) and increased Mdm2 exon skipping (p<0.05, n=3 technical replicates) at 2h and 6hr, compared to vehicle-treated cells, with the most significant differences in cells expressing mutant U2AF1. These experiments provide proof-of-concept that cells expressing mutant U2AF1 have increased sensitivity to spliceosome modulators and warrant further investigation as a possible treatment for patients with these mutations. Ongoing experiments include in vivo experiments in mice transplanted with transduced cells, and in vitro treatment of primary clinical samples. Small molecule modulators of the spliceosome may provide a novel therapeutic strategy for MDS/AML patients with spliceosome mutations. Disclosures:No relevant conflicts of interest to declare.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Discovery of Novel Recurrent Mutations in Childhood Early T-Cell Precursor Acute Lymphoblastic Leukemia by Whole Genome Sequencing - a Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Abstract 68Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown.Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort.We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1).We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations.Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR.Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures:Evans:St. Jude Children’s research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity’s Board of Directors or advisory committees.

Genetic correction of X-linked chronic granulomatous disease with novel foamy virus vectors

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the CYBB gene encoding gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). Affected individuals suffer from recurrent life-threatening infections due to impaired superoxide production by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Novel foamy virus vectors expressing the human codon-optimized gp91(phox) were evaluated for the genetic correction of the disease in the X-CGD cell line and in X-CGD mouse model. The vectors were evaluated in vitro, in the human X-CGD PLB-985 cell line and in the X-CGD bone marrow Lin(-) cells. Transplantation of transduced Lin(-) cells was performed in X-CGD mice after busulfan conditioning. Real-time polymerase chain reaction was used for chimerism and vector copy number determination. Restoration of reduced NADPH oxidase production was assessed by nitrobluetetrazolium and dihydrorhodamine assays. High and stable gp91(phox) expression, as well as reconstitution of reduced NADPH activity, was achieved in the human X-CGD PLB-985 cell line and in primary murine X-CGD hematopoietic stem cells ex vivo. Transplantation of transduced bone marrow hematopoietic stem cells in the murine model of X-CGD, even with low multiplicities of infection (MOI), reconstituted the levels of oxidase-producing neutrophils and provided enzymatic activity that reached 70% of normal. Foamy virus vectors expressing the human gp91(phox) transgene constitute potential candidates for the gene therapy of CGD because they combine lack of pathogenicity with efficacy even at low MOI.

Cytokinesis Failure In Fanconi Anemia Pathway Deficient Hematopoietic Cells

AbstractAbstract 878Fanconi Anemia (FA) is a human genomic instability disorder characterized by progressive bone marrow failure, congenital abnormalities and high predisposition to cancer. Bone marrow failure in FA children is attributed partly to the excessive apoptosis and subsequent failure of the hematopoietic stem cell compartment. Understanding the mechanisms of bone marrow failure may allow better diagnosis and treatment for FA and other aplastic anemia patients. There are fourteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O). The FA pathway, regulated by these FA gene products, mediates DNA repair and promotes normal cellular resistance to DNA crosslinking agents. Recent studies suggest that besides maintaining genomic stability, the FA pathway may also play a role in mitosis since FANCD2 and FANCI, the two key FA proteins, are localized to the extremities of ultra-fine DNA bridges (UFBs) linking sister chromatids during cell division (Chan et al, Nat Cell Biol, 11:753-760, 2009; Naim and Rosselli, Nat Cell Biol, 11:761-768, 2009). Whether FA proteins play a direct role in cell division is still unclear. To dissect the mechanisms of bone marrow failure in FA, we have investigated the requirement of FA pathway during mitosis. Initially, we investigated the number of DNA bridges occurring during mitosis in FA-deficient and proficient cells by immunofluorescence and Hoechst staining. FA-deficient patient cell lines (FANCG-deficient and FANCD1/BRCA2-deficient cells) as well as Hela cells with shRNA-mediated knockdown of the FA pathway, displayed an increase in UFBs compared to the FA proficient cells during mitosis. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein, FANCM, was recruited to the bridges at a later stage. Since the DNA bridges occluding the cleavage furrow potentially induce cytokinesis failure, we assessed FA-deficient cells for multinucleation. The increased number of DNA bridges correlated with a higher rate of binucleated cells in FA deficient Hela cell lines and FA patient-derived fibroblast cells. Moreover, an increase in binucleated cells was also detectable in FA-deficient primary murine bone marrow hematopoietic stem cells (Fancd2-/- cells and Fancg-/- cells) compared to the wild-type cells undergoing proliferation and in FA patient-derived bone marrow stroma cells compared to the stroma cells from normal human bone marrow. Interestingly, the increase in binucleated cells in FA-deficient murine hematopoietic stem cells correlated with the increase in apoptotic cells. Binuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Therefore, we investigated whether the FA-deficient cells are sensitive to the cytokinesis inhibitors. FA-deficient murine bone marrow lineage negative cells (Fancd2-/- cells) or FA human fibroblast cells were exposed to VX-680 (an inhibitor of Aurora kinases regulating cytokinesis) in culture for 72 hrs and cell survival was assessed. VX-680 caused increased toxicity (reduced cell viability and increased apoptosis) on FA-deficient cells in comparison to the wild-type cells. Enhanced inhibition of clonogenic growth of murine FA-deficient bone marrow cells (Fancd2-/- cells) compared to the wild-type cells was also observed by exposure to VX-680. These data indicated that FA pathway-deficient hematopoietic cells are hypersensitive to cytokinesis inhibitors. Collectively, our results underscore the importance of the FA pathway in mitosis and suggest that the cytokinesis failure observed in FA deficient hematopoietic cells could contribute to bone marrow failure in Fanconi anemia patients. Disclosures:No relevant conflicts of interest to declare.

Cytokinesis failure occurs in Fanconi anemia pathway–deficient murine and human bone marrow hematopoietic cells

Fanconi anemia (FA) is a genomic instability disorder characterized by bone marrow failure and cancer predisposition. FA is caused by mutations in any one of several genes that encode proteins cooperating in a repair pathway and is required for cellular resistance to DNA crosslinking agents. Recent studies suggest that the FA pathway may also play a role in mitosis, since FANCD2 and FANCI, the 2 key FA proteins, are localized to the extremities of ultrafine DNA bridges (UFBs), which link sister chromatids during cell division. However, whether FA proteins regulate cell division remains unclear. Here we have shown that FA pathway-deficient cells display an increased number of UFBs compared with FA pathway-proficient cells. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein FANCM was recruited to the UFBs at a later stage. The increased number of bridges in FA pathway-deficient cells correlated with a higher rate of cytokinesis failure resulting in binucleated cells. Binucleated cells were also detectable in primary murine FA pathway-deficient hematopoietic stem cells (HSCs) and bone marrow stromal cells from human patients with FA. Based on these observations, we suggest that cytokinesis failure followed by apoptosis may contribute to bone marrow failure in patients with FA.