Evidence for PTEN Deficiency in Juvenile Myelomonocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3428-3428
Author(s):  
Y. Lucy Liu ◽  
Likang Xu ◽  
Robert P. Castleberry ◽  
Peter Dean Emanuel
Keyword(s):  
Bone Marrow ◽  
Negative Regulator ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Mrna Levels ◽  
Pten Protein ◽  
Gm Csf ◽  
The Status ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative disorder (MDS/MPD) of young children. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), low percentage of myeloblasts in bone marrow, and absence of the Philadelphia chromosome. 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 vitro dose-response assays. PTEN, a major negative regulator of the PI3-kinase pathway by virtue of its PIP3 phosphatase activity, was initially isolated as a tumor suppressor in a variety of malignancies. In order to evaluate the role of PTEN in the pathogenesis of JMML, we examined the status of PTEN in JMML patient samples. Peripheral blood or bone marrow was collected from 40 patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a concentration of 107/ml. Total RNA was extracted from MNCs of patients and 17 normal individuals. Protein and mRNA levels of PTEN were evaluated by Western-blot and relative-quantitative real-time RT-PCR, respectively. We found that PTEN protein was decreased in 18 of 30 (60%) JMML patients, and the patients had significantly lower RNA expression of PTEN than normal controls (p=0.015). With the available samples we also evaluated AKT activity and MAP kinase (MAPK) levels. We found that MAPK levels were correlated well with the status of the PTEN in 12 of 27(44%), and AKT activity in 13 of 25 patients (52%). Our data indicates that PTEN is significantly deficient in JMML patients, and the low PTEN protein level is related to its low transcription of RNA in JMML patients. The role of PTEN in regulation of MAPK and AKT activities in JMML is under further evaluation by studying the upstream status of the RAS pathway prior to PTEN. This is the first investigation of PTEN deficiency in JMML patients, and additional investigations may help to further understand the pathogenetic mechanisms in JMML, as well as to guide the development of targeted therapeutics for JMML.

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.

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.

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.

Genetic Disruption of Rac1 but Not Rac2 Is Sufficient To Ameliorate the Development of Juvenile Myeloproliferative Myeloid Leukemia in Nf1−/− Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3612-3612
Author(s):  
Fengchun Yang ◽  
Jayme Allen ◽  
Shi Chen ◽  
Yan Li ◽  
Jin Yuan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Genetically Engineered ◽  
Juvenile Myelomonocytic Leukemia ◽  
Spleen Weight ◽  
Myeloproliferative Disease ◽  
Gm Csf ◽  
Cell Counts ◽  
Genetically Engineered Mice ◽  
Myelomonocytic Leukemia ◽  
Myeloid Progenitors

Abstract Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML) associated with loss of the normal NF1 allele. Genetically engineered mice containing nullizygous loss of Nf1 in the hematopoietic system develop a leukemia with complete penetrance that is similar to JMML, including hepatosplenomegaly, elevated peripheral blood cell counts, and elevated numbers of myeloid progenitors that are hypersensitive to multiple cytokines, particularly GM-CSF. Though we and others have found that p21ras is hyperactivated in Nf1−/− myeloid progenitors, inhibiting Ras specifically as a therapeutic target has been challenging. Therefore, identification of alterations in distinct p21ras effector pathways that control leukemia progression in Nf1-deficient cells is critical for understanding disease pathogenesis and identifying therapeutic targets. Here we intercrossed MxCre; Nf1flox/flox mice with mice that are deficient in the small Rho GTPases Rac1 or Rac2 to generate syngeneic progeny that were MxCre; Nf1flox/flox, MxCre; Nf1flox/flox;Rac1flox flox or MxCre; Nf1flox/flox; Rac2 −/−. Consistent with previous studies (Le, Blood 2004) MxCre; Nf1flox/flox mice develop a progressive myeloproliferative disease with 100% penetrance 6 months following interferon inducible induction of the MxCre transgene to disrupt the Nf1flox alleles. Eighty percent of MxCre; Nf1flox/flox die by 9 months after inactivation. Genetic disruption of Rac2 was not sufficient to diminish the onset or severity of the characteristic myeloproliferative disease of MxCre; Nf1flox/flox mice. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice followed for 11 months all survived and had normal bone marrow cellularity, spleen weight and splenic architecture. MxCre; Nf1flox/flox mice have elevated numbers of both HPP-CFC and LPP-CFC in the bone marrow and spleen. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice had myeloid progenitor numbers that were comparable to wildtype, age-matched controls. Further, though myeloid progenitors from MxCre; Nf1flox/flox mice are hypersensitive to GM-CSF, myeloid progenitors from MxCre; Nf1flox/flox; Rac1flox/flox have a sensitivity to GM-CSF comparable to wildtype controls. The correction in the myeloproliferative phenotype isolated from MxCre; Nf1flox/flox; Rac1flox/flox mice was associated with the reduction in Rac-GTP and prolonged Erk phosphorylation, a MAPK effector that is characteristically elevated in Nf1−/− myeloid progenitors. Collectively, these genetic data identify Ras-Rac1 signaling pathway as a key axis in the genesis of juvenile myelomonocytic leukemia and provide evidence that Rac1 is a therapeutic molecular target for this myeloproliferative disease that currently has no effective therapies.

Phosphatidylinositol-3-Kinase p110d Uniquely Promotes Gain-Of-Function PTPN11-Induced GM-CSF Hypersensitivity In a Model Of Juvenile Myelomonocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3678-3678
Author(s):  
Charles B Goodwin ◽  
Raghuveer Mali ◽  
Gordon Chan ◽  
Benjamin Neel ◽  
Brian Lannutti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Conditional Knockout ◽  
Juvenile Myelomonocytic Leukemia ◽  
Erk Pathway ◽  
Spleen Size ◽  
Gain Of Function ◽  
Mek Inhibition ◽  
Gm Csf ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a fatal leukemia affecting children under the age of 4 years and is characterized by myelomonocytic cell overproduction and hypersensitivity to GM-CSF. The only curative therapy is allogeneic stem cell transplantation; however, half of children relapse after this aggressive therapy. Approximately 85% of JMML patients bear loss-of-function (LOF) mutations in NF1 or CBL or gain-of-function (GOF) mutations in KRAS, NRAS, or PTPN11. Typically, these mutations are non-overlapping, with the net effect being Ras hyperactivation. Children bearing somatic GOF mutations within PTPN11, which encodes the protein tyrosine phosphatase, Shp2, exhibit the poorest prognosis. GOF Shp2 (Shp2D61Y and Shp2E76K) induces hyperactivation of both the Ras-MEK- Erk and PI3K-Akt pathways. While the Ras-MEK-Erk pathway clearly contributes to the pathogenesis of JMML, we hypothesize that the PI3K-Akt pathway cooperates with the Ras-MEK-Erk pathway to promote JMML. Recently published work indicates that genetic disruption of the PI3K regulatory subunit, p85a, reduces GOF Shp2-induced hypersensitivity to GM-CSF. However, as PI3K regulatory subunits cannot be easily inhibited pharmacologically, we examined the contribution of class IA PI3K catalytic subunits in GOF Shp2-induced JMML. Shp2 D61Y/+ ;Mx1Cre+ mice were crossed with mice bearing conditional knockout of p110a (Pik3caflox/flox) or bearing a kinase dead mutant of p110d (Pik3cdD910A/D910A). Shp2D61Y/+;Mx1Cre-, Shp2D61Y/+;Mx1Cre+, Shp2D61Y/+;Mx1Cre+; Pik3caflox/flox, and Shp2D61Y/+;Mx1Cre+; Pik3cdD910A/D910A mice were treated with polyI;polyC, and 8 weeks post-treatment, animals were euthanized followed by evaluation of spleen size, hypersensitivity of bone marrow low density mononuclear cells (LDMNCs) to GM-CSF, frequency of bone marrow phenotypically-defined common myeloid, granulocyte-monocyte, and megakaryocyte-erythroid progenitors (CMPs, GMPs, and MEPs), and GM-CSF-stimulated Erk and Akt activation. Genetic disruption of p110a failed to normalize GOF Shp2-induced splenomegaly, GM-CSF hypersensitivity in proliferation assays and methylcellulose-based progenitor assays, or hyperphosphorylation of Erk or Akt. In contrast, genetic ablation of p110d kinase activity significantly reduced spleen size, normalized progenitor hypersensitivity to GM-CSF, and reduced both Akt and Erk hyperactivation. Additionally, genetic inhibition of p110d normalized the skewed hematopoietic progenitor distribution reported in the Shp2D61Y/+;Mx1Cre+ mice, while genetic disruption of p110a failed to do so. This unique function of p110d in the context of GOF Shp2-expressing mice is significant, as p110d expression is restricted to hematopoietic cells and p110d bears transforming properties independent of Ras. While previously published work indicates that the PI3K p110a and p110d inhibitor, GDC-0941, inhibits proliferation of GOF Shp2-expressing cells, we tested if the potent p110d-specific inhibitor, GS-9820, is similarly effective. GOF Shp2-expressing bone marrow LDMNCs treated with GS-9820 demonstrated significantly reduced proliferation in a dose-dependent fashion, while GS-9820 failed to inhibit the proliferation of WT Shp2-expressing cells. GS-9820 treatment decreased Akt phosphorylation (S473 and T308) as well as reduced Erk phosphorylation, indicating that p110d inhibition also reduces signaling within the Ras-MEK-Erk pathway. While PI3K activates the canonical Akt-mTORC1 pathway, it also positively feeds back to the Ras-MEK-Erk pathway via activation of Rac-Pak-MEK; therefore, we evaluated if p110d inhibition adds to or is redundant with MEK inhibition. Treatment of GOF Shp2-expressing hematopoietic cells with the MEK inhibitor, PD0325901, effectively reduced proliferation, and addition of GS-9820 further significantly reduced proliferation, indicating that p110d works cooperatively with MEK to promote GOF Shp2-induced disease. Collectively, our findings suggest that PI3K catalytic subunit p110d functions in a Ras-MEK-Erk pathway-independent manner to promote GOF Shp2-induced hypersensitivity to GM-CSF, and suggest that PI3K p110d inhibition in combination with MEK inhibition may be a novel, optimal approach for the treatment of JMML. Disclosures: No relevant conflicts of interest to declare.

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.

5-Azacytidine Reduces Leukemic Burden in a Xenograft Model of Juvenile Myelomonocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1655-1655
Author(s):  
Christopher Felix Krombholz ◽  
Angelina Meier ◽  
Konrad Aumann ◽  
Silvia Fluhr ◽  
Matthias Kollek ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Dna Methyltransferase ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Patient Specific ◽  
Juvenile Myelomonocytic Leukemia ◽  
Methylation Patterns ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood with often fatal outcome. Despite many attempts to develop alternative treatment options allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative modality. In the past our group has linked the prognosis of JMML to differential DNA methylation patterns (Olk-Batz, Blood 2011;117:4871-80 and Poetsch, Epigenetics 2014;9:1252-60), suggesting a key role of epigenetic modifications in JMML pathophysiology. To overcome the lack of suitable preclinical JMML research models we have developed an ex vivo JMML xenotransplantation system using neonatal Rag2-/- gamma-c-/- mice. Transplantation of 1x106 primary JMML cells resulted in stable xenologous engraftment and reproduced a characteristic JMML phenotype including myelomonocytic expansion; infiltration of spleen, liver and, notably, lung; splenomegaly; and reduced survival (median 26 weeks). Persistent human engraftment and leukemic organ infiltration was confirmed by both flow cytometry and immunohistology. Ras pathway mutations present in xenotransplanted patient samples were invariably confirmed in engrafted tissues. In addition, the model sustained serial transplantations and can therefore be used to amplify scarce patient material. We first tested if DNA methylation patterns in JMML cells were stable even after xenologous engraftment because such stability would be a prerequisite if the model were to be used for preclinical investigation of DNA methyltransferase inhibitors. JMML cells before xenotransplantation and those retrieved from the bone marrow of engrafted mice were profiled for global CpG methylation using Illumina 450K arrays. DNA methylation patterns in JMML were patient-specific and surprisingly robust in functional regions over several months of engraftment time (on average, 0.29% of 30877 promoters and 0.25 % of 30725 intragenic regions were called as "differentially methylated" between source and xenograft; 0.2 β-value change cutoff). These findings confirm the suitability of the xenograft model to investigate JMML epigenetics and, more importantly, indicate that patient-specific epigenetic profiles originate in leukemia-initiating stem cells, reinforcing a fundamental role of these alterations in JMML biology. Our group recently published a retrospective case series demonstrating unprecedented clinical efficacy of the DNA methyltransferase inhibitor 5-azacytidine (5AC) to induce partial or complete remissions in JMML before allogeneic HSCT (Cseh, Blood 2015;125:2311-3). To further investigate the drug on the preclinical level we administered 5AC to Rag2-/- gamma-c-/- mice xenografted with primary JMML cells. After a leukemia establishment phase the mice were divided into treatment or mock groups and treated with 5AC (3mg/kg body weight i.p., N=6) or saline (N=6) for 2 cycles (1 dose daily for 5 days; 9 days of recovery). This regimen was tolerated well by the animals. We found that 5AC reduced JMML infiltration in all organs analyzed, with most pronounced effects in spleen (human CD45+ fraction of all CD45+ cells, 0.24% +/- 0.04% vs 39.78% +/- 10.72%; p<0.01) and lung (0.41% +/-0.18% vs 42.88% +/-8.42%; p<0.01). The proportion of early progenitor cells (CD34+) within the human leukemia population in murine bone marrow was dramatically reduced after 5AC treatment (7.89% +/-0.74% vs 32.65% +/-3.76%; p<0.01) while the amount of granulocytes increased simultaneously (44.90% +/-1.74% vs 9.35% +/-1.95%; p<0.01). These findings suggest a loss of JMML cells induced by forced differentiation of more immature cells into mature myelomonocytic cells with reduced proliferation potential. Bisulfite pyrosequencing of the human BMP4 promoter CpG island, a locus frequently hypermethylated in JMML, showed significantly reduced DNA methylation in JMML cells retrieved from 5AC-treated mice (31.32% +/-2.66% vs 52.46% +/-1.39%; p<0.001). In summary we created an ex vivo JMML xenograft model in immunodeficient mice that reflects many important aspects of this disorder and proved its usefulness for preclinical research of DNA methyltransferase inhibition because of extraordinary stability of leukemic DNA methylation patterns. 5AC showed clear preclinical efficacy in this model, supporting its further development in clinical treatment strategies for JMML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Prevents Dissemination and Induces Remission of Juvenile Myelomonocytic Leukemia in Engrafted Immunodeficient Mice

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4910-4917 ◽  
Author(s):  
Per O. Iversen ◽  
Ian D. Lewis ◽  
Suzanne Turczynowicz ◽  
Henrik Hasle ◽  
Charlotte Niemeyer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Juvenile Myelomonocytic Leukemia ◽  
Mouse Bone Marrow ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Myelomonocytic Leukemia ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor α (TNFα) have been implicated in the pathogenesis of the fatal childhood disease termed juvenile myelomonocytic leukemia (JMML). We used a severe combined immunodeficient/nonobese diabetic (SCID/NOD) mouse model of JMML and examined the effect of inhibiting these cytokines in vivo with the human GM-CSF antagonist and apoptotic agent E21R and the anti-TNFα monoclonal antibody (MoAb) cA2 on JMML cell growth and dissemination in vivo. We show here that JMML cells repopulated to high levels in the absence of exogeneous growth factors. Administration of E21R at the time of transplantation or 4 weeks after profoundly reduced JMML cell load in the mouse bone marrow. In contrast, MoAb cA2 had no effect on its own, but synergized with E21R in virtually eliminating JMML cells from the mouse bone marrow. In the spleen and peripheral blood, E21R eliminated JMML cells, while MoAb cA2 had no effect. Importantly, studies of mice engrafted simultaneously with cells from both normal donors and from JMML patients showed that E21R preferentially eliminated leukemic cells. This is the first time a specific GM-CSF inhibitor has been used in vivo, and the results suggest that GM-CSF plays a major role in the pathogenesis of JMML. E21R might offer a novel and specific approach for the treatment of this aggressive leukemia in man.

scholarly journals Inhibition of Granulocyte-Macrophage Colony-Stimulating Factor Prevents Dissemination and Induces Remission of Juvenile Myelomonocytic Leukemia in Engrafted Immunodeficient Mice

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4910-4917 ◽  
Author(s):  
Per O. Iversen ◽  
Ian D. Lewis ◽  
Suzanne Turczynowicz ◽  
Henrik Hasle ◽  
Charlotte Niemeyer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Juvenile Myelomonocytic Leukemia ◽  
Mouse Bone Marrow ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Myelomonocytic Leukemia ◽  
Stimulating Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF ) and tumor necrosis factor α (TNFα) have been implicated in the pathogenesis of the fatal childhood disease termed juvenile myelomonocytic leukemia (JMML). We used a severe combined immunodeficient/nonobese diabetic (SCID/NOD) mouse model of JMML and examined the effect of inhibiting these cytokines in vivo with the human GM-CSF antagonist and apoptotic agent E21R and the anti-TNFα monoclonal antibody (MoAb) cA2 on JMML cell growth and dissemination in vivo. We show here that JMML cells repopulated to high levels in the absence of exogeneous growth factors. Administration of E21R at the time of transplantation or 4 weeks after profoundly reduced JMML cell load in the mouse bone marrow. In contrast, MoAb cA2 had no effect on its own, but synergized with E21R in virtually eliminating JMML cells from the mouse bone marrow. In the spleen and peripheral blood, E21R eliminated JMML cells, while MoAb cA2 had no effect. Importantly, studies of mice engrafted simultaneously with cells from both normal donors and from JMML patients showed that E21R preferentially eliminated leukemic cells. This is the first time a specific GM-CSF inhibitor has been used in vivo, and the results suggest that GM-CSF plays a major role in the pathogenesis of JMML. E21R might offer a novel and specific approach for the treatment of this aggressive leukemia in man.

scholarly journals Targeting M2-Tumor Associated Macrophages By Arginase-1 and PD-L1 in Regulating Juvenile Myelomonocytic Leukemia (JMML) Development and Relapse

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1471-1471
Author(s):  
Santhosh Kumar Pasupuleti ◽  
Baskar Ramdas ◽  
Kai Yang ◽  
Chujing Zhang ◽  
Elliot Stieglitz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Cells ◽  
Juvenile Myelomonocytic Leukemia ◽  
M2 Macrophages ◽  
Arginase 1 ◽  
Donor Cells ◽  
Transplantation Experiment ◽  
Leukemia Relapse ◽  
Myelomonocytic Leukemia

Abstract Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. Macrophages are largely characterized as M1 or M2 types, and TAMs have been shown to express an M2-like phenotype. TAMs endorse tumor progression and contribute to resistance to chemotherapies. However, it is unclear what the composition of M2 macrophages is in patients with Juvenile myelomonocytic leukemia (JMML) and how do these cells mechanistically contribute to JMML and/or relapse after bone marrow transplantation. To study the role of M2- TAMs in JMML development, we first examined the bulk RNA-sequence data in 90 JMML patients. These data demonstrated a significant increase in the expression of arginase-1 (Arg-1) and programmed cell death-1 (PD-1). Furthermore, single cell RNA-sequencing analysis of monocytes/macrophages from 4 JMML patients revealed higher expression of M2- macrophage markers/genes such as IL-10, CD163, MRC1/CD206, TGF-β1 and IL-1R1 compared to M1 macrophage (CD80, CCR7, IL-6, CXCL10, CXCL11 and TNF) expression. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-macrophages express higher levels of arginase and PD-1, which may contribute to the local suppression of immune responses and damage the bone marrow microenvironment (BME) leading to poor engraftment of normal donor cells, resulting in relapse. To study how alterations in bone marrow (BM) macrophages (M1/M2) contribute to JMML development and relapse, we utilized a mouse model bearing Shp2 E76K mutation (Ptpn11 E76K/+) driven by lysosome-cre (Ptpn11 E76K/+; LysM-Cre+, indicated as Shp2* mice hereafter). This model is frequently used to study JMML as it manifests cardinal features of human JMML. In a competitive transplantation experiment using, Shp2* + Boy/J BM cells (1:1 ratio) transplanted into lethally irradiated Shp2* recipient mice, we show that Shp2* mutant cells out compete WT BoyJ cells and result in rapid growth of CD45.2+ Shp2* mutant mature myeloid cells, hematopoietic stem and progenitors (HSC/Ps) and M2- macrophages (F4/80+/CD206+) in the BM and spleen leading to leukemia relapse. To determine if modulating Arg-1 and PD-1/PD-L1 levels in the background of Shp2* mutant leukemic stem cells in Shp2* recipients would alter the overall engraftment and JMML development and relapse, we again performed a competitive transplantation experiment using, Shp2* + Boy/J (BM cells, 1:1 ratio) into Shp2* and WT recipient mice. After 8 weeks post transplantation, we investigated the role of Arg-1 and PD-L1 in Shp2* recipients using pharmacological inhibitors, CB-1158 (Arg-1 inhibitor; 100 mg/kg, orally) + anti-PD-L1 antibody (10 mg/kg, i.p) for 30 days. The Arg-1 + PD-L1 treatment significantly reduced the number of white blood cells, neutrophils, monocytes and improved RBC and platelet counts. The spleen and liver weights were significantly rescued as well. Interestingly, CD45.1 WT donor cells in the PB, BM, and spleen were significantly increased and a significant reduction of Shp2* mutant CD45.2+ mature myeloid cells in the PB, BM, and spleen was observed. Importantly, the frequency and absolute number of leukemic blasts, LSK (Lin-/Sca1+/c-KIT+) cells, short term hematopoietic cells (ST-HSCs), common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP) were significantly reduced. Furthermore, the M2- TAMs were significantly reduced in the BM and spleen of Arg-1 + PD-L1 drug treated group compared to vehicle treated mice. Notably the CD8+ T-cells (IFN-γ+ and TNF-α+) were significantly improved in the drug treated mice. These data suggest that the suppression of arginase-1 allows for the arginine levels to increase, which promotes the proliferation of T-cells. Increasing arginine levels also promotes an anti-tumor immune response resulting in the emergence of CD45.1 WT HSCs as opposed to mutant CD45.2 HSCs, suggesting that Arg-1 + PD-L1 treatment is a novel therapeutic approach to treat patients with JMML and for preventing leukemia relapse after BM transplantation. Disclosures No relevant conflicts of interest to declare.

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