Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 45-45 ◽  
Author(s):  
Hui Huang ◽  
Daniel E. Bauer ◽  
Mignon L. Loh ◽  
Govind Bhagat ◽  
Alan B. Cantor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Phosphorylation ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Brdu Incorporation ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

CREB Deficiency: A Potential Critical Factor for Selective GM-CSF Hypersensitivity in Juvenile Myelomonocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2604-2604
Author(s):  
Y. Lucy Liu ◽  
Priyangi A Malaviarachchi ◽  
Shelly Y. Lensing ◽  
Robert P. Castleberry ◽  
Peter Dean Emanuel
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Protein ◽  
Gm Csf ◽  
Myelomonocytic Leukemia ◽  
Normal Controls

Abstract Abstract 2604 Poster Board II-580 Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures: No relevant conflicts of interest to declare.

Human NUP98/Iqcg Fusion Gene Induced An Acute Myelomonocytic Leukemia In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3725-3725
Author(s):  
Qi-Yao Zhang ◽  
Ping Liu ◽  
Mengmeng Pan ◽  
Sai-Juan Chen
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Self Renewal ◽  
Acute Myelomonocytic Leukemia ◽  
Myelomonocytic Leukemia ◽  
Marrow Cells

Abstract NUP98 gene encoding a nucleoporin located at 11p15 has been reported to be fused with about 30 different partner genes by chromosomal translocations in hematological malignances. NUP98/IQCG was cloned in a patient of Myeloid/ T-lymphoid bi-phenotypic leukemia with 47, XX, t(3;11)(q29q13;p15)der(3)(q29), +21 karyotype. However, NUP98/IQCG’s ability for leukemogenesis has not been identified yet.In this study, we established a retrovirus-mediated murine bone marrow transduction and transplantation (BMT) model of NUP98/IQCG to investigate its oncogenicity. In our model, half of the NUP98/IQCG mice developed a penetrable and transplantable acute myelomonocytic leukemia ,which was similar to the phenotype of patient with t(3;11). It suggested that NUP98/IQCG could induce the disease development. To investigate how the fusion gene promoted leukemogenesis, we transduced NUP98/IQCG into primary bone marrow cells, and found that NUP98/IQCG-expressing cells retained the ability to generate colonies in serial replating, while seldom control cells did, which indicated the increasing self-renewal ability caused by NUP98/IQCG. Meanwhile, when induced by Macrophage-Colony Stimulating Factor, NUP98/IQCG -expression bone marrow cells showed enhanced proliferation in vitro. Further molecular mechanism studies revealed that NUP98/IQCG could be involved in both NFκB and CREB pathways during leukemia development. In summary, we showed that NUP98/IQCG promoted leukemogenesis in BMT mouse model through increasing the bone marrow cells’ proliferation and self-renewal capacity, which explained the fusion gene’s oncogenicity in patient with t(3;11). Our mouse model will be a powerful tool both to investigate the leukemogenic mechanism of NUP98-related fusion gene, and to find the drugs for treating the disease. Disclosures: No relevant conflicts of interest to declare.

Rapamycin -- a Potential Mechanistically Targeted Therapeutic for Juvenile Myelomonocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2378-2378
Author(s):  
Y. Lucy Liu ◽  
Robert P. Castleberry ◽  
Peter Dean Emanuel
Keyword(s):  
Philadelphia Chromosome ◽  
Disease Free Survival ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Sensitivity Testing ◽  
Blood Concentrations ◽  
Patient Sample ◽  
Gm Csf ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD) of infancy and early childhood. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. The pathogenesis of JMML has been clearly and definitively linked to dysregulated signal transduction through the RAS signaling pathway. A series of studies conducted over the last decade have shown that mutations or other abnormalities in RAS, NF1, and PTPN11, are potentially responsible for the pathogenesis of JMML in up to 75% of cases. Treatment has been very difficult. There is no effective therapy for JMML. Only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28–55%), with 5-year disease-free survival rates of 25-40%. Rapamycin is a macrolide antibiotic with established clinical applications in organ transplantation. Recent studies have proved that the Mammalian Target of Rapamycin (mTOR) plays an important role in cytokine receptor signaling and induction of apoptosis. Numerous studies have suggested that mTOR functions as a nutritional checkpoint and is connected to energy sensing through AMP-dependent kinase (AMPK) which senses the AMP: ATP ratio in cells. Its function is regulated by the RAS/PI3-kinase pathway. In searching for novel mechanistically-targeted reagents to treat JMML, we conducted an in vitro pilot study with JMML cells. The CFU-GM formation assay was used to test the therapeutic sensitivity of rapamycin to JMML cells. Mononuclear cells (MNCs) from peripheral blood of 9 JMML patients were collected and plated on 0.3% agar medium with rapamycin at a concentration of 1-8nM(0.91-7.28μg/L) and carrier (DMSO). Greater than 50% inhibition of spontaneous CFU-GM growth was observed in all cultures in a dose-dependent fashion, with the exception of one patient sample which had colonies resistant to rapamycin. The effective concentrations in our cultures are equivalent to the safe and tolerable whole blood concentrations achieved in organ transplant patients in clinical settings (5-30μg/L). Our data suggests that rapamycin may be considered as a potentially safe and effective reagent to treat JMML, but that in vitro sensitivity testing might be recommended since one patient sample demonstrated complete resistance to rapamycin in vitro. Further studies are ongoing to explore the mechanism of rapamycin in inhibiting hypersensitivity of JMML cells to GM-CSF.

Canonical NF-κB Signalling Is a Potential Target in FLT3/ITD AML.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2447-2447 ◽  
Author(s):  
Jing Zhang ◽  
Li Li ◽  
Alan D. Friedman ◽  
Donald Small ◽  
Ido Paz-Priel
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Myeloproliferative Neoplasm ◽  
Initial Response ◽  
Wild Type ◽  
Normal Numbers ◽  
Toxic Dose ◽  
Marrow Cells

Abstract Abstract 2447 Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor is common in acute myeloid leukemia (AML) and is associated with a dismal outcome. Despite initial response, FLT3/ITD AMLs often relapse early, suggesting a residual population of resistant leukemia stem cells (LSC). Clinically, FLT3 inhibitors asmonotherapy have yet to improve outcome significantly and therefore, targeting additional pro-survival pathways may be necessary for this group of AML patients. Mice genetically egineered to express a hemizygous FLT3/ITD mutation develop a progressive, fatal, myeloproliferative neoplasm. Lin−cells isolated from the bone marrow of FLT3/ITD or control mice were subjected to gel shift analysis using a radio-labeled NF-kB binding site. This analysis demonstrated high levels of nuclear activation of NF-kB in the FLT3/ITD-expressing cells, suggesting its activation downstream of mutant FLT3 signaling. MV4–11 is a human AML-derived cell line harboring a homozygous FLT3/ITD mutation. Cells expressing high levels of aldehyde dehydrogenase (ALDH) have been shown to be enriched for LSC in primary AML samples and cell lines. High ALDH expressing MV4–11 cells were isolated using FACS and analyzed for NF-kB activation. Western blot analysis demonstrated preferential phosphorylation of NF-kB p65 by activated IKK on Ser536 in this subpopulation, compared with cells with low ALDH activity. These findings indicate activation of NF-kB in MV4–11 LSCs. We wanted to next test the requirement for NF-kB signaling in transformation by FLT3/ITD mutations. NF-kB p65 null mice die in utero. We therefore established C57BL/6 p65(flox/flox);Mx1-Cre mice. Intra-peritoneal injection of pIpC every other day for 7 doses efficiently deletes the RelA/p65 gene, resulting in expression of <1% of the corresponding RNA or protein. Despite effective excision of p65, the mice survive. Bone marrow cells harvested from control or p65(del/del) mice were transduced with a FLT3/ITD-expressing lentivirus and seeded in methylcellulose without cytokines. Equal transduction rate was verified by measurement of GFP expression by flow cytometry. Reproducibly, p65(del/del) marrow transduced with FLT3/ITD was ineffective in forming cytokine independent colonies, in contrast to wild-type marrow (5 +/− 0.6 vs. 55 +/− 6 colonies per 1E5 cells, P<0.001), and the few p65(del/del) colonies that resulted were smaller than those from p65 expressing wild-type marrow cells. Cells transduced with a lentiviral vector expressing GFP but not FLT3/ITD did not form colonies without cytokines, and p65(del/del) marrow formed normal numbers of colonies of normal size and distribution in the presence of IL-3, IL-6, and SCF. Sorafenib inhibits FLT3 signaling and kills MV4–11 cells with an IC50 of approximately 10 nM. Reproducibly, a sub-toxic dose of sorafenib (5 nM) combined with sub-toxic levels of the IKKb inhibitor IMD-0354 (400 nM) resulted in synergistic cell killing as indicated by the calculated combination index of 0.55. Currently, clinical efforts in FLT3/ITD leukemia concentrate on FLT3 inhibition alone. Our data suggest that canonical NF-kB may be an important pathway in FLT3/ITD AML and that simultaneously targeting FLT3 and NF-kB in this disease may be an effective approach. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment of Stable Cell Lines Representing Juvenile Myelomonocytic Leukemia with SHP2 Mutations

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4353-4353
Author(s):  
Yuming Zhao ◽  
Yao Guo ◽  
Chunxiao He ◽  
Dengyang Zhang ◽  
Han Zhong Pei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cytokine Signaling ◽  
Ras Signaling ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Transformed Cell ◽  
Transformed Cell Lines ◽  
Myelomonocytic Leukemia

Abstract Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. Disclosures No relevant conflicts of interest to declare.

scholarly journals Secondary Mutations of JAK3 and SETBP1 in Juvenile Myelomonocytic Leukemia and Their Propagating Capacity; A Report from the AIEOP Study Group

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4625-4625
Author(s):  
Silvia Bresolin ◽  
Paola De Filippi ◽  
Francesca Vendemini ◽  
Riccardo Masetti ◽  
Franco Locatelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Hot Spot ◽  
Univariate Analysis ◽  
Driver Mutations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Nsg Mice ◽  
Myelomonocytic Leukemia

Abstract INTRODUCTION Juvenile myelomonocytic leukemia is a rare early childhood leukemia, characterized by excessive proliferation of granulocytic and monocytic cells. About 95% of JMML patients harbor driver mutations in the RAS signaling pathway. Recently, secondary hits in SETBP1 and JAK3 have been reported in a Japanese cohort of JMML patients showing an adverse clinical outcome of patients carrying these mutations. Here we report the mutational analysis of SETBP1 and JAK3 and clinical implications in a cohort of Italian JMML patients. METHODS Samples collected at diagnosis of 65 patients with JMML were analyzed by Sanger sequencing. Mutations were found in RAS (NRAS-KRAS) 31%, PTPN11 35%, CBL 5%, whereas in 29% of patients none of the above cited mutations was present. Mutation hot spot regions of SETBP1 (SKI domain) and of JAK3 (PTK domains) were sequenced. A xenografted murine model was used to assess the in vivo competitive repopulation advantage of clones carrying mutations of JAK3 and SETBP1. Mononuclear cells from a patient with JMML at diagnosis harboring PTPN11, SETBP1 and JAK3 mutations were transplanted in NSG mice and assessed for mutational status in the bone marrow and spleen after engraftment of JMML cells. RESULTS Screening for JAK3 and SETBP1 mutations in patients revealed 9 mutations in 8 out of 65 patients at diagnosis of JMML. All of the identified secondary mutations were associated with known driver mutations, more frequent with mutated PTPN11 and RAS (p=0.036 and p= 0.01 respectively) than with CBL or in cases without known driver mutations. Seventy-five percent of secondary mutations were found in SETBP1 and only 1 patient harbored a mutation in JAK3. Remarkably one patient carried mutations in JAK3 (L857P and L857Q, both predicted to damaging protein function), PTPN11 (G503A) and SETBP1 (D868N). All variants were identified as heterozygous mutations, confirmed bi-allelic expression at the transcriptome level. The only patient carrying JAK3 as secondary mutation at E958K showed wild-type expression of JAK3 pointing to absence of a functional role at the protein level. Univariate analysis revealed association between the presence of secondary mutations and patient’s age at diagnosis, with older patients carrying JAK3 and SETBP1 mutations (p=0.0067); no other clinical and biological characteristics (i.e. WBC count, percentage of monocyte, HbF level and platelet count) being significantly associated with the presence of secondary hits in bone marrow of JMML cases. Patients with secondary mutations showed a trend to shorter survival compared to those without secondary events in JAK3 and SETBP1 (5-years OS= 0% vs 54.01%, SE=8.1; p=0.41, respectively). Interestingly, the in vivo assay using xenografted mice revealed a different propagating capacity of JAK3 clones of patients carrying JAK3 (2 different clones), SETBP1 and PTPN11 mutations. Indeed, for JAK3 only the clone with the L857Q mutation engrafted in BM and spleen of the mouse, together with SETBP1 and PTPN11 mutations. Moreover, a second mouse engrafted with mononuclear cells of the same patients showed that only cells carrying the PTPN11 mutation had engrafted. CONCLUSIONS In conclusion we identified secondary mutations in JAK3 and SETBP1 in 12% of patients of a representative cohort of Italian JMML patients, showing a trend of adverse outcome for patients carrying these mutations. These secondary events in JMML patients showed to have distinct propagating capacities upon engraftment in NSG mice pointing to a different functional impact of these mutations. Disclosures No relevant conflicts of interest to declare.

JAK2 Inhibition with TG101348 Inhibits Monosomy 7 Myelodysplastic Syndromes (MDS) Bone Marrow Cells In Vitro: a Potential Targeted Therapy for Monosomy 7 MDS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 973-973 ◽  
Author(s):  
Matthew J. Olnes ◽  
Andrea Poon ◽  
Zachary Tucker ◽  
Neal S. Young ◽  
Elaine M Sloand
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Dependent Manner ◽  
Monosomy 7

Abstract Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML. Disclosures: No relevant conflicts of interest to declare.

Mir-183 Over-Expression: A Potential Biomarker for Juvenile Myelomonocytic Leukemia (JMML).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2558-2558
Author(s):  
Y. Lucy Liu ◽  
Yan Yan ◽  
Shelly Y. Lensing ◽  
Todd Cooper ◽  
Peter D. Emanuel
Keyword(s):  
Peripheral Blood ◽  
Robust Regression ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Potential Biomarker ◽  
Median Level ◽  
Myelomonocytic Leukemia

Abstract Abstract 2558 Juvenile myelomonocytic leukemia (JMML) is a rare disease of early childhood with a predilection for the monocyte/macrophage lineage. The pathogenesis of JMML is linked to dysregulated signal transduction through the NF1/RAS signaling pathway that is partially caused by genetic mutation of Ras, PTPN11, and c-CBL, or loss-of heterozygosity of Nf1. The hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro. We previously reported that protein deficiencies of PTEN, CREB, and Egr-1 were frequently observed in JMML (67–87%). Recent research indicated that CREB was regulated by miR-34b, and Egr-1 was targeted by miR-183. We hypothesized that microRNAs may play an important role in contributing to the deficiency of these proteins. Using relative-quantitative real-time PCR, we evaluated the expression levels of miR-34b and miR-183 in mononuclear cells from 47 JMML patients. We found that the median level of miR-183 was significantly higher in JMML in comparison to normal controls (median=13.8 vs 4.2, p<0.001); but the median level of miR-34b was only slightly higher in JMML subjects, and not significantly so, compared to normal individuals (median=1.4 vs 1.0, p>0.05). This suggests that miR-34b does not play a significant role in JMML. Since extreme monocyte accumulation is one of the critical characteristics of JMML, we analyzed the correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood. Strikingly, there was a significant correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood from 34 patients who had available data (p<0.05). Based on a robust regression analysis, for every unit increase in the square root of RQ miR-183, the monocyte percentage significantly increased by 0.73% (SE=0.32%, p=0.023). This is the first evidence suggesting that microRNAs may contribute to the pathogenesis of JMML. miR-183 may also serve as an important biomarker that can be directly and quantitatively linked to significant clinical parameters in JMML. It also may ultimately provide a target for JMML therapy. Disclosures: No relevant conflicts of interest to declare.

Autophagy Regulation and Dysfunction in Bone Marrow Malignancies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4623-4623
Author(s):  
Fernando V Pericole ◽  
Mariana Lazarini ◽  
Adriana S. S. Duarte ◽  
João Machado-Neto ◽  
Sara T. Olalla Saad
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Combination Treatments ◽  
Molecular Machinery ◽  
Total Bone Marrow

Abstract Abstract 4623 Introduction: Autophagy is a catabolic pathway by which cytoplasmic materials are degraded into the lysosome and it is also a quality control system for proteins and organelles. Autophagy plays an important role in cell adaptation to starvation, hypoxia, cell survival and cancer. Its core molecular machinery is tightly linked to metabolic pathways, such as LKB1/AMPK and mTORC1. Autophagy has been shown to play several important roles in cancer. Indeed, multiple autophagy genes have been characterized as tumor suppressor genes. In hematopoietic system, autophagy is required during myeloid and lymphoid differentiation, terminal erythroid mitochondrial clearance, production of proplatelets and also differentiation of monocytes into macrophages. Interestingly, autophagy seems disturbed in most bone marrow malignancies. Evidence in mice suggests that autophagy suppression (ATG7 or ATG5 knockdown models) in hematopoietic stem cells may be implicated in Acute Myeloid Leukemia (AML) pathogenesis. In Multiple Myeloma (MM), in vitro studies using cell lines showed autophagy activation and lysosome inhibitors (such as chloroquine) are currently been used in various combination treatments in clinical trials. Aim: The aim was to characterize the expression of autophagy machinery key genes (BECN1, MAP1LC3A, SQSTM1), as well as hypoxia master regulator (HIF1A) in total bone marrow cells from bone marrow malignancies: myelodysplasia (MDS), MM and AML patients, excluding acute promyelocytic leukemia. Methods: BECN1, MAP1LC3A, SQSTM1 and HIF1A levels were verified, by q-PCR, in diagnostic (or without any treatment) BM aspirates from 22 normal donors, 30 MDS (17 low-risk and 13 high-risk, according 2008 WHO classification), 43 AML and 11 MM patients. Results: BECN1 gene expression was increased in MM, compared with control group. All other groups did not differ from the control group. Comparing diseases amongst each other, AML had a lower BECN1 expression, compared with low-risk MDS and with MM (Figure 1A). Disclosures: No relevant conflicts of interest to declare.

Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 157-157 ◽  
Author(s):  
Caroline Marty ◽  
Nivarthi Harini ◽  
Christian Pecquet ◽  
Ilyas Chachoua ◽  
Vitalina Gryshkova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
In Vivo Model ◽  
Common Mechanism ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Platelet Counts

Abstract Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail. To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling. For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year. CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin. Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN. Disclosures No relevant conflicts of interest to declare.

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