The Role of microRNA-155 in Mouse Models of MLL -AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2446-2446
Author(s):  
Anna Staffas ◽  
Edith Schneider ◽  
Linda Fogelstrand ◽  
Linda Röhner ◽  
Michael Heuser ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Micro Rna ◽  
Blood Analysis ◽  
Leukemic Cells ◽  
Mouse Bone Marrow ◽  
Fusion Genes

Abstract Background: Genetic rearrangements that fuse the mixed lineage leukemia (MLL) gene, now termed KMT2A, to one of a variety of partners are seen in 5% - 20% of acute myeloid leukemia (AML). MLL -fusions are especially common in childhood AML and many of them are associated with poor prognosis. The MLL -fusions perturb transcription through different mechanisms and they are often associated with high expression of the transcription factors HOXA9 and MEIS1. Based on a micro-RNA screen in an AML mouse model mimicking the step-wise development of aggressive AML we have found that concurrent Hoxa9 and Meis1 overexpression is associated with upregulation of micro-RNA-155 (miR-155). Expression of miR-155 was also found to be higher in bone marrow samples from patients with MLL- AML compared with bone marrow from healthy donors (p <0.001), as were the expression of HOXA9 and MEIS1 (p <0.05). In lymphomas, miR-155 plays a pivotal role as an oncogene. It is frequently upregulated in samples from lymphoma patients and a mouse model of lymphoma showed a certain degree of miR-155-addiction which could be targeted by miR-155 inhibitors. Despite the differences in the pathobiology of AML and lymphoma, the upregulation of miR-155 in AML with high HOXA9 and MEIS1 expression may indicate miR-155 as a relevant therapeutic target also in MLL -AML. Methods: To test the importance of miR-155 and its potency as a drug target in MLL -AML we used a miR-155 knock-out mouse model (miR-155-/-) (Thai et al, Science, 2007). MLL -fusion genes of varying leukemic potential; MLL-AF5 (KMT2A-AFF4), MLL-ENL (KMT2A-MLLT1), MLL-AF9 (KMT2A-MLLT3) were retrovirally expressed in miR-155-/- mouse bone marrow (mbm) cells and in wild-type mbm cells (miR-155+/+). Results: In concordance with the previous findings in human AML patient samples, miR-155+/+ cells expressing MLL-AF5, MLL-ENL, or MLL-AF9 showed upregulation of miR-155 (p < 0.05). Also, Hoxa9 and Meis1 transcripts were increased (p<0.05). Interestingly, the magnitude of upregulation of both miR-155 and Meis1 correlated with the degree of aggressiveness based on disease latency and survival observed in these leukemia models with highest upregulation in MLL-ENL and MLL-AF9 and lowest in MLL-AF5 (p<0.05). Expression of the MLL-fusion genes in miR-155-/- mbm cells resulted in similar induction of Hoxa9 and Meis1 expression as in miR-155+/+ mbm cells, indicating that miR-155 is downstream of the Hoxa9/Meis1 axis. To determine the leukemic potential in vivo, we transplanted recipient mice with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-ENL or MLL-AF9. Engraftment of leukemic cells, based on peripheral blood analysis, did not differ between mice transplanted with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-fusions. Also, disease development induced by MLL-AF9 and MLL-ENL (4-8 weeks and 10-32 weeks, respectively) was similar in mice transplanted with miR-155-/- mbm cells and mice transplanted with miR-155+/+ mbm cells. In accordance with the in vivo results, functional studies in vitro showed that the proliferative capacity and colony forming ability of MLL -fusion expressing cells were similar in miR-155+/+ mbm cells and miR-155-/- mbm cells, indicating that miR-155 is not essential for MLL-ENL- or MLL-AF9-induced leukemic transformation. Conclusions: In summary, miR-155 is upregulated in MLL-AML in both mice and man, seemingly through an MLL>HOXA9/MEIS1>miR-155 axis. Since absence of miR-155 does not alter the leukemic potential induced by MLL-AF9 or MLL-ENL, miR-155 may contribute to, but is not pivotal for MLL leukemogenesis. We therefore conclude that miR-155 is not a therapeutic target in MLL- AML. Disclosures No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Distinct Metabolic Dependency of Normal and Leukemic Cells in a Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 759-759
Author(s):  
Rushdia Z. Yusuf ◽  
Sanket S. Acharya ◽  
Vionnie Yu ◽  
Borja Saez ◽  
Mildred Duvet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Tca Cycle ◽  
Leukemic Cells ◽  
Mouse Bone Marrow ◽  
Metabolomic Profiling ◽  
Glycolysis Pathway ◽  
Rate Limiting

Abstract Abstract 759 We hypothesized that metabolic differences between leukemia initiating cells and their normal counterparts represent a vulnerability in the leukemia initiating cell, which can be therapeutically exploited. To test this hypothesis, we used the MLL-AF9 acute myeloid leukemia (AML) model in mice. Actin-DsRed mouse bone marrow transduced with MLL-AF9 expressing retrovirus was used to produce serially transplantable leukemia. Leukemic granulocyte-monocyte precursors (L-GMPs), defined by others to be the leukemia initiating cells were flow sorted from secondary recipient mice and compared with normal GMPs (N-GMPs) from actin Ds-Red mice. Gene expression profiling, metabolomic profiling via liquid chromatography- mass spectrometry and an in vitro shRNA screen were used to identify metabolic pathways preferentially activated in leukemia initiating cells. Of 1574 defined metabolic enzymes, 44 were found to be differentially expressed between L-GMPs and their normal counterparts (N-GMPs). These together with 117 classic rate limiting enzymes were subjected to shRNA knockdown in vitro. Metabolomic profiling of both cell populations was used to corroborate findings from shRNA knockdowns. L-GMPs and N-GMPs were transduced with lentivirus expressing shRNAs of interest (5 shRNAs per gene) in a 384 well format, selected with puromycin and cultured for 72–96 hours in the presence of GFP-positive primary bone marrow stroma. The number of cells in each well at the end of this experiment was quantitated using an Image Xpress microscope. Genes, the knockdown of which by at least two independent shRNAs produced a two fold or more decrease in L-GMPs as compared to control wells and did not similarly decrease N-GMPs, were chosen for in vivo validation. Ten genes in the glycolysis pathway and TCA cycle, fatty acid metabolism and detoxification, and ketohexokinase were identified. Ketohexokinase, a rate-limiting enzyme in fructose metabolism was particularly potent and of interest given its potential to be exploited therapeutically. In vivo assessment of its relative ability to inhibit malignant versus normal hematopoietic cells is ongoing. These studies provide preliminary support for the hypothesis that specific metabolic circuits are differentially active in leukemia initiating cells in MLL-AF9 AML and may represent unique points of vulnerability that can be targeted therapeutically. Authors 1 and 2 contributed equally. Authors 3 and 4 contributed equally. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 778-778
Author(s):  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Twee Tsao ◽  
Yihua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Signaling Pathways ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Mtor Signaling ◽  
Leukemic Cells ◽  
Survival Signaling

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

PML-RARα Deregulates an Unexpectedly Small Number of Genes in Pre-Leukemic Promyelocytes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3526-3526
Author(s):  
Coline M Gaillard ◽  
Taku A Tokuyasu ◽  
Emmanuelle Passegué ◽  
Scott C. Kogan
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Leukemic Cells ◽  
Cytokine Signaling ◽  
Proliferation Capacity ◽  
Number Of Genes

Abstract Abstract 3526 Background: Acute Promyelocytic Leukemia (APL) is characterized by the accumulation in the blood and bone marrow of abnormal promyelocytes, which have the ability to transfer the disease to secondary recipients in animal models. The PML-RARα fusion protein is thought to be the primary abnormality implicated in the pathology, and is believed to prevent transcription of genes necessary for normal myeloid development and differentiation. Identifying PML-RARα targets is critical for understanding the road to leukemic transformation. However, such targets have so far been identified using cell line assays in vitro, murine cells differentiated into promyelocytes in vitro, or fully transformed murine or human leukemic cells. Focusing on the cell population in which the transforming potential is acquired, we describe here a novel strategy to identify the transcriptomic dysregulation induced by PML-RARα expression in maturing myeloid populations in vivo. Methods: We utilize a murine model of human APL in which the human PML-RARα fusion gene is expressed under the control of the MRP8 promoter, driving its expression in maturing myeloid populations. Those animals can be described as pre-leukemic since they eventually develop leukemia when additional mutations occur. Fresh bone marrows from normal (Fvb/n) or pre-leukemic (PML-RARα) animals were harvested. Using an improved cell surface antigen staining strategy and fluorescence-activated cell sorting, three populations of increasingly differentiated myeloid populations have been sorted (Granulocyte Macrophage Progenitor, Early promyelocyte and Late promyelocyte). RNA was extracted and submitted for whole-genome microarray analysis. In addition, we are using a variety of bioinformatics approaches to decipher the network of novel interactions driven by PML-RARα expression. Results: Markers used in our sorting strategy were validated in the dataset, including CD34 and Gr1. In the normal samples, markers of neutrophil maturation increased, largely as expected, and a number of early transcription factors decreased in an expected manner including Hoxa9 and Meis1. One remarkable finding was that despite the previously described ability of PML-RARα to regulate transcription from multiple sites in the genome, only a small number of genes were differentially impacted by the expression of this protein. Surprisingly, well-known regulators of myeloid differentiation that have been implicated in the retinoic acid responsiveness of APL including Sfpi1 (PU.1) and Cebpa were not differentially expressed. However, in pre-leukemic samples PML-RARα did cause decreased expression of multiple neutrophilic granule genes including Ltf, Mmp9 and Ngp. The gene most upregulated in the pre-leukemic samples was Spp1 which encodes the osteopontin phosphoprotein. Of interest, we identified the myeloid tumor suppressor Irf8 to be downregulated 5 fold in the presence of PML-RARα. To investigate the importance of IRF8 levels in APL initiation, we transplanted Irf8+/+ PML-RARα or Irf8+/− PML-RARα bone marrow into irradiated recipients. Despite the potential for decreased expression of IRF8 to contribute to APL, we observed no difference. This result does not confirm a role for IRF8 in APL pathogenesis, but further investigations are needed to exclude such a role. Bioinformatics studies highlighted enrichment in cell cycle-related genes upon PML-RARα expression, suggesting a possible difference in the proliferation capacity of the pre-leukemic cells, which is currently under investigation. Conclusions: We found that in vivo the transcriptome was only modestly dysregulated by the presence of PML-RARα. These observations open up new questions on the role of the fusion protein in pathogenesis: How does PML-RARα prime pre-leukemic cells for full transformation? How do secondary events allow an initiated cell to advance to a fully transformed state? Such questions are currently being investigated, with a special interest on looking at the cooperation between PML-RARα and activated cytokine signaling in leukemia initiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1648-1648 ◽  
Author(s):  
Yaser Heshmati ◽  
Gözde Turköz ◽  
Aditya Harisankar ◽  
Sten Linnarsson ◽  
Marios Dimitriou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Loss Of Function ◽  
Potential Therapeutic Target ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment

Blood ◽  
2012 ◽  
Vol 120 (13) ◽  
pp. 2679-2689 ◽  
Author(s):  
Zhihong Zeng ◽  
Yue Xi Shi ◽  
Twee Tsao ◽  
YiHua Qiu ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Leukemic Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Mtor Kinase ◽  
Phosphorylated Akt ◽  
Induced Apoptosis

Abstract The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

Modeling Growth Of Pediatric T-ALL In Vivo and In Vitro: Clinical Meaning and Activation Of The NFkB Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2571-2571
Author(s):  
Sandrine Poglio ◽  
Xavier Cahu ◽  
Benjamin Uzan ◽  
Hélène Lapillonne ◽  
Thierry Leblanc ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Stromal Cells ◽  
Leukemic Cells ◽  
Mouse Bone Marrow ◽  
Nsg Mice ◽  
Nfkb Activation ◽  
Two Systems

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the proliferation of T-cell precursors in various sites, such as thymus, bone marrow, blood, lymph nodes or central nervous system. As T-ALL cells alone do not successfully grow in vitro, xenografts of T-ALL cells into NOD/scid/IL-2R null (NSG) mice and long-term co-cultures of T-ALL cells with stromal cells have been developed to study the biology of T-ALL cells (Armstrong et al, Blood, 2009). However, the growth of T-ALL cells in these two systems is highly variable across T-ALL samples. Moreover, the clinical relevance of both assays and, except for NOTCH pathway activation, the molecular pathways involved in successful in vivo and in vitro growths are still elusive. The aim of this work was to determine the relationships between clinical, biological and molecular characteristics of human T-ALL at diagnosis and the growth of T-ALL in these two systems. Human T-ALL blood samples were collected at diagnosis from pediatric or young adult patients with T-ALL. 50,000 T-ALL cells were intravenously injected into NSG mice. Mouse bone marrow samples were collected every 3-4 weeks from day 35 to day 210 post-transplant. Leukemic engraftment was monitored using flow cytometry measuring the % of human CD45+CD7+ leukemic cells. Time to leukemic engraftment (TTL) was defined as the time between T-ALL injection and the detection of ≥20% leukemic cells in at least one mouse. In vitro co-culture growth assay consisted in plating 200,000 cells on MS5 or MS5-DL1 (Armstrong, Blood, 2009) and count every 7 days up to 28 days. A total of 36 samples were tested of which 22 (61%) engrafted into mice. Global median TTL was 82 days (range, 36-121) defining short (TTL<82 days) and long or no engraftment (TTL>82 days) TTL groups. Patient gender, age, mediastinal involvement or abnormal karyotype had no significant impact on TTL. A trend for a shorter TTL was observed for T-ALL samples with a white blood cell count (WBC) > median WBC = 146 G/L (p =0.06). Samples containing more than 20% of TCRαβ or CD8 positive cells exhibited increased incidence of engraftment (p = 0.049 and p=0.04 respectively) whereas CD34, CD1a, CD4 or sCD3 markers were not significantly correlated with TTL. Unlike samples with TLX1, TLX3 overexpression or NOTCH/FBXW7 mutations, samples with SIL-TAL1 deletion exhibited a shorter TTL (p = 0.0004). The 2-year progression free survival of “short TTL” patients was 72% vs 70% for patients with “longer TTL” or no engraftment (p=0.38). T-ALL samples for which growth could be achieved on MS5 cells also displayed a shorter TTL. To unravel molecular mechanisms involved in the growth of leukemic cells in these two systems, micro-arrays were performed for 8 “short TTL” T-ALL versus 8 “long TTL or no engraftment” T-ALL. 346 genes were differentially express in short TTL samples compared to long/no TTL samples (P<0.05, fold change: 1.5). As expected, most of genes up-regulated in short TTL group were implicated in cell cycle function enhancing the commitment of cells to S/M phases. Analysis of regulated networks revealed that several indirect modulators of NFkB (MAL, AhR and CYLD) were significantly up/down regulated in short TTL patient samples resulting in NFkB activation. Overall, T-ALL with SIL-TAL1 deletion display an increased ability to engraft into NSG mice, in accordance with increased WBC in T-ALL patients. Contrary to B-ALL, shorter TTL is not associated with poor prognosis in T-ALL. Moreover, NSG engraftment and co-culture on stromal cells are well correlated. A shorter TTL seems to be associated with an increased leukemic proliferation through NFkB activation. Disclosures: No relevant conflicts of interest to declare.

A Novel Inhibitor of Mutant IDH1 Induces Differentiation in Vivo and Prolongs Survival in a Mouse Model of Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3598-3598 ◽  
Author(s):  
Anuhar Chaturvedi ◽  
Michelle Maria Araujo Cruz ◽  
Ramya Goparaju ◽  
Nidhi Jyotsana ◽  
Heike Baehre ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Medical School ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Median Latency ◽  
Mutant Idh1

Abstract Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in patients with glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, cholangiocellular carcinoma and chondrosarcoma. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. We recently established an in vivo mouse model and investigated the function of mutant IDH1. By computational drug screening, we identified an inhibitor of mutant IDH1 (HMS-101), which inhibits mutant IDH1 cell proliferation, decreases R2HG levels in vitro, and efficiently blocks colony formation of AML cells from IDH1 mutated patients but not of normal CD34+ bone marrow cells. In the present study we investigated the effect of the inhibitor in our IDH1/HoxA9-induced mouse model of leukemia in vivo. To identify the maximally tolerated dose of HMS-101, we treated normal C57BL/6 mice with variable doses of HMS-101 for 9 days and measured the serum concentration. Mice receiving 0.5 mg and 1mg intraperitoneally once a day tolerated the drug well with mean plasma concentrations of 0.1 to 0.3 µM. To evaluate the effect of HMS-101 in the IDH1 mouse model, we transduced IDH1 R132C in HoxA9-immortalized murine bone marrow cells. Sorted transgene positive cells were then transplanted into lethally irradiated mice. After 5 days of transplantation, mice were treated with HMS-101 intraperitoneally for 5 days/week. The R/S-2HG ratio in serum was reduced 3-fold in HMS-101 treated mice after 8 weeks of treatment compared to control treated mice. HMS-101 or PBS treated mice had similar levels of transduced leukemic cells in peripheral blood at 2 and 6 weeks after transplantation. However, from week 6 to week 15 leukemic cells in peripheral blood decreased from 76% to 58, 63% to 29%, 67% to 7%, and 74% to 38% in 4/6 mice treated with HMS-101. In one mouse the percentage of leukemic cells was constant, and in one mouse it increased from week 6 to week 15 after transplantation. Leukemic cells increased constantly in peripheral blood until death in control treated mice. While the control cohort developed severe leukocytosis, anemia and thrombocytopenia around 8 to 10 weeks post transplantation, mice treated with HMS-101 still had normal WBC, RBC and platelet counts at 15 weeks after transplantation. Moreover, the HMS-101 treated mice had significantly more differentiated Gr1+CD11b+ cells in peripheral blood than control mice at 6 weeks and 15 weeks after transplantation and at death (P=.01). Morphologic evaluation of blood cells at 15 weeks or death from HMS-101 treated mice revealed a high proportion of mature neutrophils that were GFP positive and thus derived from IDH1 transduced cells, whereas control treated mice had monocytic morphology with a high proportion of immature cells. Importantly, HMS-101 treated mice survived significantly longer with a median latency of 87 days (range 80-118), whereas PBS-treated mice died with a median latency of 66 days (range 64-69) after transplantation (P<.001). Of note, HMS-101 was found to be specific for mutant IDH1, as mutant IDH2 cells were not preferentially inhibited over IDH2 wildtype cells in vitro. This data demonstrates that HMS-101 specifically inhibits R2HG-production of mutant IDH1 in vivo, inhibits proliferation, induces differentiation in leukemic cells, and thus prolongs survival of IDH1mutant leukemic mice. Therefore, HMS-101 - a novel inhibitor of mutant IDH1 - shows promising activity in vivo and warrants further development towards clinical use in IDH1 mutated patients. Disclosures Chaturvedi: Hannover Medical School: Patents & Royalties. Preller:Hannover Medical School: Patents & Royalties. Heuser:Hannover Medical School: Patents & Royalties.

scholarly journals HSP27: A Therapeutic Target in Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1963-1963 ◽  
Author(s):  
Margaux Sevin ◽  
Nicolas Pernet ◽  
Franck Vitte ◽  
Selim Ramla ◽  
Paul Sagot ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Bone Marrow Failure ◽  
Specific Inhibitor ◽  
White Pulp ◽  
Beneficial Effects

Abstract Myelofibrosis (MF) is the most aggressive myeloproliferative neoplasms (MPN) with the highest degree of morbidity and mortality, including progressive bone marrow fibrosis resulting into bone marrow failure. JAK2 kinase inhibitors have been successfully used for a few years in MPN and more particularly for MF treatment. Despite their beneficial effects on spleen size and symptoms, JAK2 inhibitors induce low molecular and survival responses underscoring the urgent need for other therapeutic approaches. Recently, heat shock protein 90 (HSP90) - known to stabilize JAK2 - has been reported as a promising therapeutic target in MPN. However HSP90 inhibitors show toxicity and induce the expression of stress-inducible proteins like HSP70 and, most likely HSP27 as previously shown in other cancers. In addition, we and others have shown that HSP27, was strongly expressed in patients with idiopathic pulmonary, lung or kidney tubulointerstitial fibrosis, underlying a relevant role of HSP27 in fibrotic processes. Taking into account both the beneficial effects of HSP inhibitors in leukemia and in MPN, and the possible implication of HSP27 in fibrosis, we have evaluated in this work, the status of HSP27 in MF patient's samples and assess the effectiveness of an HSP27 oligonucleotide inhibitor called OGX-427. In this study, we first assessed the extracellular and intracellular level of HSPs from MF patients by ELISA, flow cytometry and by immunohistochemistry. We observed for the first time a specific increase in both intracellular and extracellular HSP27 in CD34+ circulating hematopoietic progenitor cells (n=9-16; P=0.0097) and in the sera of patients (n=24-27; P<0.0001) with MF compared with healthy donors, respectively. Moreover, we identified the presence of HSP27 in the bone marrow's MF patients. We then investigated the in vivo impact of OGX-427, a specific inhibitor of HSP27, or an oligonucleotide control in a murine TPO-induced MF mouse model. The use of OGX-427 limited the progression of the disease in our MF mouse model (n=9). In particular, OGX-427 was associated with a marked reduction in both the spleen weight and size. Also, we noted a decrease of megakaryocyte hyperplasia in the bone marrow accompanied by a visible restoration of spleen structure and lymphoid white pulp territories with OGX-427. Taking altogether, our results support a key role of HSP27 in the pathophysiology in MF and highlight the potential therapeutic benefit of HSP27 inhibitors in this disorder. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Therapeutic Protein Asparaginase Is Efficiently Cleared By Bone Barrow and Spleen Resident Macrophages

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3630-3630 ◽  
Author(s):  
Laurens T van der Meer ◽  
Samantha YA Terry ◽  
Dorette S van IngenSchenau ◽  
Kiki C Andree ◽  
Peter M Hoogerbrugge ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Treatment Protocol ◽  
Leukemic Cells ◽  
Post Injection ◽  
Ct Image ◽  
Phagocytic Cells ◽  
Treatment Protocols

Abstract Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Recent studies monitoring ASNase kinetics in patients provide evidence of a large inter-patient variability of serum ASNase concentrations and call attention to the negative effects of ASNase underexposure on treatment response and relapse risk. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in patients. We used in vivo imaging to study the distribution and pharmacodynamics of ASNase in a mouse model. We injected mice with 3,000 International Units (I.U.)/kg ASNase, which was labeled with 20-25 MBq Indium-111 (In-111) and acquired micro-SPECT/CT images up 18 hours post injection. At this timepoint, serum ASNase activity has dropped to levels close to the detection limits. In addition to the expected uptake in the liver, SPECT/CT imaging revealed a rapid, strong and specific accumulation of radiolabeled ASNase in the bone marrow and spleen (Figure 1). Accumulation in these organs was confirmed by quantitative measurement of radiolabeled ASNase in the dissected organs (Figure 2). We hypothesized that macrophages which are present in high numbers in these organs, efficiently phagocytose the ASNase, thereby rapidly clearing the active enzyme from the blood. Autoradiography of spleen sections indeed showed high uptake of radiolabeled ASNase in the macrophage-rich red pulp of the spleen. Immunohistochemical stainings confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To provide additional evidence for the potential role of macrophages in the turnover of ASNase, we pretreated mice with a single injection of clodronate liposomes, which almost completely depletes the relevant organs from phagocytic cells. This pretreatment diminished the accumulation of ASNase in the liver, spleen and the bone marrow (Figure 2). Consistent with this notion, we found that clodronate pretreatment more than doubles the circulatory half-life of serum ASNase activity. We conclude from these experiments that ASNase is rapidly cleared from the serum by phagocytic cells. In particular, the efficient uptake of ASNase by spleen and bone marrow resident macrophages may protect leukemic cells from the nutrient depriving action of this drug and could thereby compromise therapeutic efficacy. Figure 1: SPECT/CT image of Asparaginase uptake Figure 1:. SPECT/CT image of Asparaginase uptake Lateral (A) and ventral (B) 3-dimensional volume projections of fused SPECT/CT scans of mice injected with 111Indium-labeled asparaginase (pseudocolor images with red being least intense and yellow most intense), 18 hours post injection. Numbers indicate relevant organs: 1 sternum, 2 liver, 3 spleen, 4 spine, 5, pelvis, 6 femur, 7 tibia. Figure 2: Biodistribution of Asparaginase in control and clodronate pretreated mice. Figure 2:. Biodistribution of Asparaginase in control and clodronate pretreated mice. Asparaginase uptake is depicted as percentage of the injected dose per gram of tissue (%ID/g) at 19 hours after injection in control (empty liposomes) and clodronate pretreated mice. Results are mean + standard deviation (n=5 for each group). 2-tailed t-test was used to test for significance: * p<0.05, ** p<0.01, *** p<0.001 Disclosures No relevant conflicts of interest to declare.

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