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.

Decitabine, a Potential Targeted Therapeutic for Juvenile Myelomonocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2516-2516 ◽  
Author(s):  
Y. Lucy Liu ◽  
Peter Dean Emanuel ◽  
Robert P. Castleberry
Keyword(s):  
Mononuclear Cells ◽  
Plasma Concentrations ◽  
Philadelphia Chromosome ◽  
Disease Free Survival ◽  
Adult Patients ◽  
Myelogenous Leukemia ◽  
Juvenile Myelomonocytic Leukemia ◽  
Pharmacokinetic Studies ◽  
Gm Csf ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. 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. Mutations or other abnormalities in RAS, NF1, and PTPN11, have been linked to be responsible for the pathogenesis of JMML in up to 75% of cases. Treatment has been very difficult 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%. Decitabine, as one of the second generation of hypomethylating agents, has been demonstrated to produce encouraging responses in adult patients with chronic myelogenous leukemia or other hematopoietic disorders. Our recent studies have demonstrated that PTEN deficiency is detected in 67% of JMML patients. We hypothesize that hypermethylation of the PTEN promoter is one of the causes that lead to PTEN deficiency in JMML, and that a hypomethylating agent may improve PTEN expression in JMML cells, and thus inhibit hypersensitivity to GM-CSF. In order to test our hypothesis, we conducted an in vitro pilot study with JMML cells. Hypermethylation of the PTEN promoter was detected in 23/30 (77%) of JMML patients using Methylation-specific PCR. Sequencing confirmed that the CpG islands of the PTEN promoter were hypermethylated. A CFU-GM formation assay was used to evaluate the therapeutic sensitivity of Decitabine to JMML cells. Frozen mononuclear cells from peripheral blood samples of 5 JMML patients were plated in 0.3% agar medium with Decitabine ranging in concentration from 1nM to 1000nM. Significant inhibition of spontaneous CFU-GM growth was observed in all cultures in a dose-dependent fashion. The effective Decitabine concentrations in the cultures were lower or equivalent to the safe and tolerable plasma concentrations achievable in adult patients in clinical settings (30 mg/m2/day). Our data suggests that hypermethylation of the PTEN promoter is a common event in JMML, and Decitabine may be a potentially safe and effective reagent to treat JMML. Further pharmacokinetic studies should be conducted in the clinic to clarify the plasma concentration in pediatric patients, and the mechanism of Decitabine in inhibiting hypersensitivity of JMML cells to GM-CSF should be further explored since multiple genes are hypermethylated in cancers.

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.

Transient hematologic and clinical effect of E21R in a child with end-stage juvenile myelomonocytic leukemia

Blood ◽  
2002 ◽  
Vol 99 (7) ◽  
pp. 2615-2616 ◽  
Author(s):  
Frédéric Bernard ◽  
Caroline Thomas ◽  
Jean François Emile ◽  
Timothy Hercus ◽  
Bruno Cassinat ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Juvenile Myelomonocytic Leukemia ◽  
Gm Csf ◽  
Spontaneous Proliferation ◽  
Bone Marrow Cultures ◽  
Macrophage Colony ◽  
End Stage ◽  
Myelomonocytic Leukemia

E21R is a modified granulocyte macrophage–colony-stimulating factor (GM-CSF) protein which results in antagonism of GM-CSF function via selective binding to the GM-CSF receptor complex. Juvenile chronic myelomonocytic leukemia (JMML) is a rare leukemia where spontaneous proliferation of myeloid and monocytic precursors in patients' bone marrow cultures is dependent on GM-CSF. For patients who progress after systemic chemotherapy, there are no effective therapies. In vitro and in vivo studies in an animal model demonstrating that E21R exerts an antileukemic action prompted us to consider its potential utility in a child with end-stage JMML. E21R was well-tolerated during the 3 courses of subcutaneous treatment. A clear in vivo efficacy was observed after 2 courses of E21R but the disease appeared completely refractory during the third course. This novel therapeutic approach clearly deserves further evaluation in JMML.

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.

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.

Inhibition of juvenile myelomonocytic leukemia cell growth in vitro by farnesyltransferase inhibitors

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 639-645 ◽  
Author(s):  
Peter D. Emanuel ◽  
Richard C. Snyder ◽  
Tonya Wiley ◽  
Balaganesh Gopurala ◽  
Robert P. Castleberry
Keyword(s):  
Signal Transduction ◽  
Colony Growth ◽  
Juvenile Myelomonocytic Leukemia ◽  
Farnesyltransferase Inhibitors ◽  
Inhibitory Effects ◽  
Gm Csf ◽  
Macrophage Colony ◽  
Myelomonocytic Leukemia ◽  
Dose Dependent

Juvenile myelomonocytic leukemia (JMML) is an early childhood disease for which there is no effective therapy. Therapy with 13-cis retinoic acid or low-dose chemotherapy can induce some responses, but neither mode is curative. Stem cell transplantation can produce lasting remissions but is hampered by high rates of relapse. The pathogenesis of JMML involves deregulated cytokine signal transduction through the Ras signaling pathway, with resultant selective hypersensitivity of JMML cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). A JMML mouse model, achieved through homozygous deletion of the neurofibromatosis gene, confirmed the involvement of deregulated Ras in JMML pathogenesis. With this pathogenetic knowledge, mechanism-based treatments are now being developed and tested. Ras is critically dependent on a prenylation reaction for its signal transduction abilities. Farnesyltransferase inhibitors are compounds that were developed specifically to block the prenylation of Ras. Two of these compounds, L-739,749 and L-744,832, were tested for their ability to inhibit spontaneous JMML granulocyte-macrophage colony growth. Within a dose range of 1 to 10 μmol/L, each compound demonstrated dose-dependent inhibition of JMML colony growth. An age-matched patient with a different disease and GM-CSF–stimulated normal adult marrow cells also demonstrated dose-dependent inhibitory effects on colony growth, but they were far less sensitive to these compounds than JMML hematopoietic progenitors. Even if the addition of L-739,749 were delayed for 5 days, significant inhibitory effects would still show in JMML cultures. These results demonstrate that a putative Ras-blocking compound can have significant growth inhibitory effects in vitro, perhaps indicating a potential treatment for JMML.

scholarly journals Correlation of Clinical Features With the Mutational Status of GM-CSF Signaling Pathway-Related Genes in Juvenile Myelomonocytic Leukemia

2009 ◽  
Vol 65 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Nao Yoshida ◽  
Hiroshi Yagasaki ◽  
Yinyan Xu ◽  
Kazuyuki Matsuda ◽  
Ayami Yoshimi ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Clinical Features ◽  
Juvenile Myelomonocytic Leukemia ◽  
Gm Csf ◽  
Mutational Status ◽  
Myelomonocytic Leukemia

Mutations of the NF1 Gene in Children With Juvenile Myelomonocytic Leukemia Without Clinical Evidence of Neurofibromatosis, Type 1

Blood ◽  
1998 ◽  
Vol 92 (1) ◽  
pp. 267-272 ◽  
Author(s):  
Lucy E. Side ◽  
Peter D. Emanuel ◽  
Brigit Taylor ◽  
Janet Franklin ◽  
Patricia Thompson ◽  
...  
Keyword(s):  
Clinical Diagnosis ◽  
Neurofibromatosis Type 1 ◽  
Neurofibromatosis Type ◽  
Juvenile Myelomonocytic Leukemia ◽  
Translation System ◽  
Polymorphism Analysis ◽  
Single Stranded Conformational Polymorphism ◽  
Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a pediatric myelodysplastic syndrome that is associated with neurofibromatosis, type 1 (NF1). The NF1 tumor suppressor gene encodes neurofibromin, which regulates the growth of immature myeloid cells by accelerating guanosine triphosphate hydrolysis on Ras proteins. The purpose of this study was to determine if the NF1gene was involved in the pathogenesis of JMML in children without a clinical diagnosis of NF1. An in vitro transcription and translation system was used to screen JMML marrows from 20 children for NF1mutations that resulted in a truncated protein. Single-stranded conformational polymorphism analysis was used to detect RASpoint mutations in these samples. We confirmed mutations of NF1in three leukemias, one of which also showed loss of the normalNF1 allele. An NF1 mutation was detected in normal tissue from the only patient tested and this suggests that JMML may be the presenting feature of NF1 in some children. Activating RASmutations were found in four patients; as expected, none of these samples harbored NF1 mutations. Because 10% to 14% of children with JMML have a clinical diagnosis of NF1, these data are consistent with the existence of NF1 mutations in approximately 30% of JMML cases.

γ-Globin, a Tumor-Associated Antigen for Juvenile Myelomonocytic Leukemia (JMML): A Cell-Based Approach To Identify Tumor Antigenic Epitopes That Are Naturally Processed and Presented.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3418-3418
Author(s):  
Naoto Hirano ◽  
Marcus O. Butler ◽  
Zhinan Xia ◽  
Seiji Kojima ◽  
Lee M. Nadler
Keyword(s):  
Mass Spectrometry ◽  
Tumor Cells ◽  
Fusion Gene ◽  
Peptide Sequencing ◽  
Juvenile Myelomonocytic Leukemia ◽  
Peptide Epitope ◽  
Peptide Epitopes ◽  
Tumor Associated Antigen ◽  
Myelomonocytic Leukemia

Abstract Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of early childhood. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high, and innovative approaches are needed. Since donor lymphocyte infusion in JMML is efficacious, T cell mediated immunotherapy may be effective, and appropriate antigenic targets must be identified. One candidate tumor-associated antigen for the immunotherapy of JMML is γ-globin, which is expressed at high levels in most JMML patients. Most clonogenic JMML cells constitutively express this onco-fetal protein, which is not necessary for the normal erythropoesis of children and adults. To determine whether γ-globin can serve as a target for immunotherapy in JMML, we sought to determine whether γ-globin is naturally processed and presented by the HLA complex. Using conventional bioinformatic techniques and the T2 binding assay to predict candidate epitopes, we identified 4 γ-globin derived peptides (g031, g071, g105, and g106) that were predicted to bind to the HLA-A2 molecule in vitro. Since this strategy provides no evidence for which predicted epitopes are processed and presented by tumor cells in vivo, we employed a biochemical strategy to determine which peptides are naturally processed and presented. This step is critical in certifying that a candidate peptide epitope is an appropriate target for immunotherapy treatments. Using our K562-derived artificial APC (aAPC), an APC that expresses A2 and no other HLA allele, we introduced the EGFP-γ-globin fusion gene. We then acid stripped peptides directly from the surface of one billion aAPC/EGFP-γ-globin cells without subjecting the cells to detergent mediated lysis. Peptides less than 5 kDa in size were fractionated by reverse phased HPLC analysis and analyzed by mass spectrometry. We identified two mass spectrometry peaks which corresponded to γ-globin derived peptides, g031 and g105. Of these, the identity of one peak, g105, was successfully confirmed by peptide sequencing, providing strong evidence that g105 is naturally processed and presented by aAPC/EGFP-γ-globin cells. Next, to confirm that g105 is processed and presented by primary JMML cells, we generated γ-globin specific CD8+ cytotoxic T cells (CTL) from A2 positive healthy donors using synthetic g105 peptide. γ-Globin specific CTL were able to specifically cytolyze A2+ γ-globin+ JMML cells but not A2+ γ-globin- JMML cells. Specific cytotoxicity was blocked by anti-A2 mAb but not isotype control. These results show for the first time that the γ-globin derived peptide, g105, can serve as a target epitope for the CTL directed immunotherapy of JMML. Furthermore, these results illustrate an innovative aAPC based strategy that can identify the antigenic peptide epitopes of putative tumor associated antigens that are naturally processed by tumor cells, presented via HLA class I, and can serve as targets for effective anti-cancer immunotherapy.

Phase II Window Study of the Farnesyltransferase Inhibitor R115777 (Zarnestra®) in Untreated Juvenile Myelomonocytic Leukemia (JMML): A Children’s Oncology Group Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2587-2587 ◽  
Author(s):  
Robert P. Castleberry ◽  
Mignon L. Loh ◽  
Nalini Jayaprakash ◽  
April Peterson ◽  
Vicky Casey ◽  
...  
Keyword(s):  
Phase Ii ◽  
Initial Dosage ◽  
Colony Growth ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cell Transplant ◽  
Monocyte Count ◽  
Similar Frequency ◽  
Gm Csf ◽  
The Impact

Abstract JMML is a rare and often fatal leukemia of young children exhibiting unique clinical, hematopoietic and genetic features including GM-CSF hypersensitivity, and mutations of NF1, RAS, and PTPN11. Ras proteins control a number of cell signaling events becoming activated in part by the addition of a farnesyl moiety via farnesyl protein transferase (FTPase). Given that hyperactive Ras is central to JMML pathogenesis, it is intuitive that an FTPase is an appropriate therapeutic target in JMML. One FTPase inhibitor, L739,749, has previously been shown to abrogate spontaneous in vitro colony growth in 9 JMML samples (Blood 95:639, 2000). R115777 is a potent in vitro and in vivo inhibitor of FTPase, abrogating the growth of H-ras, K-ras and N-ras transformed tumors. In humans, it is well tolerated with the dose-limiting toxicities being myelosuppression and diarrhea. To assess the efficacy and toxicity of R115777 in JMML, a phase II window study was conducted as a part of COG study AAML0122 in newly diagnosed patients who were given the option of receiving this agent prior to cytosine arabinoside, fludarabine and 13-cis retinoic acid followed by stem cell transplant. R115777 was administered PO BID for 21 days with a 7 day rest for two courses in the absence of disease progression or excessive toxicity. The starting dosage in the first 11 patients was 200mg/m2 with escalation in subsequent patients to 300mg/m2 if the initial dosage was tolerated. Overall response was based upon changes in WBC and organomegaly. The impact of R115777 upon in vitro spontaneous colony growth, GM-CSF hypersensitivity and farnesylation was monitored. A total of 47 patients were accrued: M:F=30:17, median (med) age 15 mos. (1–76); med WBC 30X109/L (4–151); med monocyte count 18X109/L (1–55); med platelet count 58X109/L (2–587); elevated fetal hemoglobin 30 (65%). RAS and PTPN11 mutations were tested in 42 cases and inhibition of prenylation in 33. R115777 was well tolerated at both dosages with the most common grade 3/4 toxicities being thrombocytopenia (40%), anemia (40%), neutropenia (15%), and diarrhea (6%). There were no deaths during the trial. The table details the responses in patients receiving one course (N=47) and 2 courses (N=38) of R115777. The 9 patients not receiving two courses were removed from study due to lack of response or progressive disease. WBC ONLY 0VERALL (WBC & organomegaly) COURSE #1 CR CR PR MR SD PD Total     200mg/m2 6 0 4 4 2 1 11     300mg/m2 18 1 17 9 4 5 36 COURSE #2     200mg/m2 6 0 6 1 2 1 10     300mg/m2 17 2 14 7 2 3 28 FTPase activity was inhibited in 13/15 cases (med 71%; range 38–91%) with similar frequency and degree of inhibition at both dosages of R115777. There was no relationship between FTPase inhibition or response and the presence of RAS/PTPN11 mutations or inhibition of prenylation in an HJ2 assay. In conclusion, R115777 provides an overall CR/PR rate of 58% with no significant differences between the two dosages (p=0.7). This agent should be considered in the future management of JMML.

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