Granulocyte-macrophage Colony-stimulating Factor Signal Transduction Research Articles

MODL-09. Exploring the role of Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) inhibition as a potential therapeutic intervention in medulloblastoma

Abstract BACKGROUND: Brain tumors are the second most common type of pediatric cancer and the leading cause of all cancer-related deaths in children. Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor and has a five-year overall survival ranging from 40-75%, depending on the patient’s age and other prognostic features. There are various anti-cancer therapies against medulloblastoma, but the treatment of recurrent and refractory disease remains a challenge. As a result, the need for new and novel therapies remain a top priority. One area of interest in CNS tumors are targets within mitochondria. Magmas overexpression has been reported in multiple types of metabolically active tissue and cancer cells, including prostate cancer, pituitary adenoma, and glioma. Some new data suggest that specific subgroups of medulloblastoma may also overexpress Magmas. This ongoing study aims to examine whether Magmas inhibition by compound “BT9” could be beneficial in the treatment of medulloblastoma. METHODS: We continue to study the ability of a Magmas inhibitor (BT#9) as a therapeutic agent in stable medulloblastoma cell lines and patient-derived primary cultures by performing MTT assays, tunnel assays, flow cytometry, migration assays, and invasion assays. RESULTS: Similar to the adult GBM studies, Magmas inhibition by BT#9 had significant cytotoxic effects, causing both decreased cell proliferation, increased apoptosis, and blocked cell migration in medulloblastoma cell lines DAOY, D283, and D425. IC50s determined for each during different time points demonstrated an average range 2-5μM compared to the average range seen in adult glioblastoma cell cultures which could range up to 10 μM. These findings suggest that the inhibition of Magmas could potentially optimize clinical outcomes in recurrent/refractory medulloblastoma and warrants further investigation. Our future studies will include the determination of IC50s for primary cell cultures and in vitro testing with patient-derived xenograft models.

DDRE-31. MITOCHONDRIAL TRAFFICKING AS A TARGET FOR GBM THERAPY

Abstract Glioblastoma (WHO Grade IV glioma) is the most aggressive brain cancer. The current standard of care treatment includes surgery, radiation, and chemotherapy. Tumor recurrence is almost inevitable as less than 50% of patients survive more than two years. The low survival rate poses a dire need to develop an effective therapy for GBM patients. GBM cells are resistant to treatment, as they activate their DNA damage response mechanisms to overcome the effects of radiation and temozolomide (TMZ) treatments. Recurrent tumors can arise from slow cycling and self-renewing stem/tumor-initiating cells resistant to radiation and TMZ. No second-line therapy was proven to prolong survival after TMZ failure. Magmas (Mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) is a subunit of the TIM23 complex regulating precursor protein trafficking into the mitochondrial matrix. Magmas is encoded by pam16, known to be upregulated in human pituitary adenomas, prostate cancer and GBM. Previous studies have demonstrated that Magmas negatively regulates the stimulatory activity of Pam18, which in turn stimulates the ATPase activity of mitochondrial heat shock protein 70 (mtHsp70). No small molecules targeting Magmas are in clinical use. We developed a novel small molecule inhibitor (BT9) that has been specifically designed to inhibit Magmas binding to Pam18. BT9 induces apoptosis through cleavage of caspase-3, reduced mitochondrial respiration and glycolysis. Our recent findings also demonstrate that BT9 treatment reduced protein trafficking of Lon protease into the mitochondrial matrix. Pretreatment of glioma cells with BT9 sensitizes cells to radiation treatment and enhances the TMZ activity. BT9 can cross the blood-brain-barrier and improve survival in intracranial glioma PDX models. BT9 has potential therapeutic value by directly dysregulating mitochondrial function in GBM, enhancing radiation and chemotherapy response, and improving survival in a relevant animal model.

DDRE-38. MAGMAS INHIBITION IN MEDULLOBLASTOMA CELL CULTURES AND PATIENT-DERIVED XENOGRAFT MODELS: POTENTIAL THERAPEUTIC IMPLICATIONS

Abstract BACKGROUND Brain tumors are the second most common type of pediatric cancer and are the leading cause of all cancer-related deaths in children. Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor and has a five-year overall survival ranging from 40-75%, depending on the patient’s age and other prognostic features. There are current anti-cancer therapies against medulloblastoma, but the treatment of recurrent disease remains a challenge. Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) overexpression has been reported in multiple types of metabolically active tissue and cancer cells, including prostate cancer, pituitary adenoma, and glioma. Limited data suggest that specific subgroups of medulloblastoma may also overexpress Magmas. This study aims to examine whether Magmas inhibition by compound “BT#9” could be beneficial for the treatment of medulloblastoma. METHODS We studied the ability of a Magmas inhibitor (BT#9) as a therapeutic agent in stable medulloblastoma cell lines (DAOY and D283) and patient-derived primary cultures with MTT assays, migration assays, and invasion assays. RESULTS Similar to the adult GBM studies, Magmas inhibition by BT9 had significant cytotoxic effects, causing both decreased cell proliferation and blocked cell migration in medulloblastoma cell lines DAOY and D283. IC50s determined for each during different time points demonstrated an average range of less than 3μM compared to the average range seen in adult glioblastoma cell cultures (< 10 μM). These findings suggest that the inhibition of Magmas warrants further investigation as a potential therapeutic target to optimize clinical outcomes in medulloblastoma. Our future studies will include the determination of IC50s for primary cell cultures and in vitro testing with patient-derived xenograft models.

EXTH-19. EVALUATING THE ANTI-TUMOR EFFECT OF A NOVEL THERAPEUTIC AGENT, MAGMAS INHIBITOR, IN MALIGNANT GLIOMA

Abstract BACKGROUNDS Glioblastoma (GBM) is an aggressive infiltrative brain tumor, and has an extremely poor prognosis despite the use of multiple treatment modalities, including surgery, radiation, and chemotherapy. Meanwhile, mitochondrial changes represent a significant part of cancer cell biology since cancer cells must survive and adapt to challenging microenvironments, specifically in conditions where tumor growth makes oxygen and glucose scarce. As GBM is characterized by extensive hypoxia-induced phenotypic changes such as abnormal vascular proliferation and necrosis, regulation of mitochondrial function could be a novel approach for treating GBM that currently lacks effective therapies. Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) is a nuclear gene that encodes for the mitochondrial import inner membrane translocase subunit Tim16. We previously demonstrated that a novel Magmas inhibitor, BT#9, significantly exerted anti-tumor effect in glioma in vitro, and may cross the blood brain barrier in vivo, indicating that Magmas inhibitor may be a new chemotherapeutic agent for the treatment of GBM. METHODS In this study, the antitumor effect of Magmas inhibitor BT#9 was tested in an orthotopic xenograft model of human GBM. The molecular mechanism of BT#9 was investigated using glioma cell lines. RESULTS The mice were tolerated to BT#9, and there was no statistical difference in the weight of animals between the control and MTD (Maximum-tolerated Dose, 50mg/kg) groups. The immunocompromised mice, intracranially implanted with human D-54 GBM xenografts, survived significantly longer than the controls (P< 0.5) when treated with BT#9 at MTD. In vitro study showed that the MAP kinase pathways are involved in BT#9-induced tumor suppression. DISCUSSION This is the first study on the role of Magmas in glioma in vivo. Our findings suggested that Magmas plays a key role in glioma survival and targeting Magmas by Magmas inhibitor has the potential to become a therapeutic strategy in glioma patients.

Magmas inhibition as a potential treatment strategy in malignant glioma.

Magmas (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction) is a nuclear gene that encodes the mitochondrial import inner membrane translocase subunit Tim16. Magmas is highly conserved, ubiquitously expressed in mammalian cells, and is essential for cell viability. Magmas expression levels are increased in prostate cancers and pituitary adenomas. Moreover, silencing Magmas by RNAi sensitizes pituitary adenoma cells to pro-apoptotic stimuli and induces a G0/G1 accumulation. The aim of this study was to examine whether inhibition of Magmas by small molecule inhibitors could be beneficial for the treatment of malignant gliomas. We evaluated the expression of Magmas in patient-derived glioblastoma tissue samples and xenograft models. We studied the feasibility of a small molecule Magmas inhibitor (BT#9) as a therapeutic agent in stable human glioma cell lines and high-grade patient derived glioma stem-like cells. Magmas was overexpressed in tissue sections from glioma patients and xenografts. In vivo studies revealed that BT#9 could cross the blood-brain barrier in the animal model. Magmas inhibition by BT#9 in glioma cell lines significantly decreased cell proliferation, induced apoptosis along with vacuole formation, and blocked migration and invasion. In addition, BT#9 treatment decreased the respiratory function of glioma cells, supporting the role that Magmas serves as a reactive oxygen species regulator. This is the first study on the role of Magmas in glioma. Our findings suggest that Magmas plays a key role in glioma cell survival and targeting Magmas by small molecule inhibitors may be a therapeutic strategy in gliomas.

Clinical and Genetic Characterization Of Patients With C-CBL Mutated Juvenile Myelomonocytic Leukemia By Whole-Exome/Deep Sequencing

IntroductionJuvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in a mutually exclusive manner in >80% affected children. Patients and MethodsWe examined 108 children (71 boys and 37 girls) diagnosed with JMML in institutions throughout Japan. Written informed consent for sample collection was obtained from the parents of the patients. Molecular analysis, including whole-exome sequencing, was approved by the Ethics Committees of Nagoya University Graduate School of Medicine and Graduate School of Medicine, the University of Tokyo, in accordance with the Declaration of Helsinki. JMML diagnoses were based on internationally accepted criteria.Using exome sequencing and target deep sequencing, we identified 17 (7 boys and 10 girls) patients with c-CBLmutations. The median age at diagnosis was 11 months (range, 1-67 months). Seven of these 17 (41%) patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). ResultsComparison of the clinical characteristics between patients with c-CBL mutations (n = 17) and those without c-CBL mutations (n = 91) revealed a statistically significant male predominance restricted to the patients without c-CBL mutations [7/17 (41%) vs. 64/91 (70%); p = 0.02]. The genetic alterations of c-CBLobserved in the 17 patients were as follows: 12 point mutations, 1 splice site mutation, and 4 deletions [c.1105del66: p.Glu369_Asp390del (n = 1); c.1217del22: p.Thr406fs (n = 1); c.1096-110del643: p.Glu366_Phe468del (n = 1); and c.1177_1227+86del: p.Ile393_Gln409del (n = 1)]. We could confirm heterozygous germline mutations in all the 5 patients (100%) whose germline sample was available [buccal smear and nail (n = 2), buccal smear (n = 1), and CD3+T cell (n = 2)].Deep sequencing using a next generation sequencing platform enabled precise estimation of the mutated allele frequencies of each mutation, and we categorized the patients with c-CBL mutations into 3 distinctive groups: (A) homozygous (allele frequencies >86%; n = 10), (B) heterozygous (35%–42%; n = 5), and (C)small clone mutations (19% and 7%; n = 2).While the majority of point mutations and splice site mutations are homozygous [11/13 (85%)], which is consistent with previous reports, all 4 deletion mutations showed heterozygosity (p = 0.0037). Surprisingly, both patients with small clone mutations harbored other RAS pathway mutations simultaneously with higher mutated allele frequencies (%) [PTPN11 (c.181G>T, p.Asp61Tyr; 46%; n = 1) and KRAS (c.38G>A, p.Gly13Asp; 42%; n = 1)], suggesting that c-CBL mutations occurred as secondary genetic events in these patients. Any patient with JMML with a c-CBL mutation does not have SETBP1 and JAK3 mutations, which we recently identified as secondary mutations in JMML (Sakaguchi et.al., Nature Genetics2013).Unexpectedly, central nervous system complications were observed in 3 of the 17 (18%) patients (Moyamoya disease, n = 2, and acute disseminated encephalomyelitis, n = 1). Although 4 patients survived without post HSCT event, late graft rejection (n = 1), relapse as acute myeloid leukemia (n = 1), and death due to unknown reasons (n = 1) was observed among the 7 patients who underwent HSCT. Nine of 10 patients with JMML survived without HSCT. Consequently, the probability of 5-year transplantation-free survival [95% confidence interval (CI)] of the 17 patients with c-CBL mutations was significantly superior to that of the other 91 patients without c-CBL mutations [38% (9%–69%) vs. 14% (5%–26%), p < 0.001]. ConclusionJMML patients with c-CBL mutations have genetic and clinical heterogeneity. However, this subgroup of JMML showed better survival outcome compared with c-CBL wild-type JMML, although with several characteristic clinical events precluding patients' quality of life. Further clinical research is warranted to elucidate determining factors for clinical heterogeneity of c-CBL mutated JMML patients. Disclosures:Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Clinical and Genetic Characterization Of Patients With C-CBL Mutated Juvenile Myelomonocytic Leukemia By Whole-Exome/Deep Sequencing

IntroductionJuvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in a mutually exclusive manner in >80% affected children. Patients and MethodsWe examined 108 children (71 boys and 37 girls) diagnosed with JMML in institutions throughout Japan. Written informed consent for sample collection was obtained from the parents of the patients. Molecular analysis, including whole-exome sequencing, was approved by the Ethics Committees of Nagoya University Graduate School of Medicine and Graduate School of Medicine, the University of Tokyo, in accordance with the Declaration of Helsinki. JMML diagnoses were based on internationally accepted criteria.Using exome sequencing and target deep sequencing, we identified 17 (7 boys and 10 girls) patients with c-CBLmutations. The median age at diagnosis was 11 months (range, 1-67 months). Seven of these 17 (41%) patients underwent allogeneic hematopoietic stem cell transplantation (HSCT). ResultsComparison of the clinical characteristics between patients with c-CBL mutations (n = 17) and those without c-CBL mutations (n = 91) revealed a statistically significant male predominance restricted to the patients without c-CBL mutations [7/17 (41%) vs. 64/91 (70%); p = 0.02]. The genetic alterations of c-CBLobserved in the 17 patients were as follows: 12 point mutations, 1 splice site mutation, and 4 deletions [c.1105del66: p.Glu369_Asp390del (n = 1); c.1217del22: p.Thr406fs (n = 1); c.1096-110del643: p.Glu366_Phe468del (n = 1); and c.1177_1227+86del: p.Ile393_Gln409del (n = 1)]. We could confirm heterozygous germline mutations in all the 5 patients (100%) whose germline sample was available [buccal smear and nail (n = 2), buccal smear (n = 1), and CD3+T cell (n = 2)].Deep sequencing using a next generation sequencing platform enabled precise estimation of the mutated allele frequencies of each mutation, and we categorized the patients with c-CBL mutations into 3 distinctive groups: (A) homozygous (allele frequencies >86%; n = 10), (B) heterozygous (35%–42%; n = 5), and (C)small clone mutations (19% and 7%; n = 2).While the majority of point mutations and splice site mutations are homozygous [11/13 (85%)], which is consistent with previous reports, all 4 deletion mutations showed heterozygosity (p = 0.0037). Surprisingly, both patients with small clone mutations harbored other RAS pathway mutations simultaneously with higher mutated allele frequencies (%) [PTPN11 (c.181G>T, p.Asp61Tyr; 46%; n = 1) and KRAS (c.38G>A, p.Gly13Asp; 42%; n = 1)], suggesting that c-CBL mutations occurred as secondary genetic events in these patients. Any patient with JMML with a c-CBL mutation does not have SETBP1 and JAK3 mutations, which we recently identified as secondary mutations in JMML (Sakaguchi et.al., Nature Genetics2013).Unexpectedly, central nervous system complications were observed in 3 of the 17 (18%) patients (Moyamoya disease, n = 2, and acute disseminated encephalomyelitis, n = 1). Although 4 patients survived without post HSCT event, late graft rejection (n = 1), relapse as acute myeloid leukemia (n = 1), and death due to unknown reasons (n = 1) was observed among the 7 patients who underwent HSCT. Nine of 10 patients with JMML survived without HSCT. Consequently, the probability of 5-year transplantation-free survival [95% confidence interval (CI)] of the 17 patients with c-CBL mutations was significantly superior to that of the other 91 patients without c-CBL mutations [38% (9%–69%) vs. 14% (5%–26%), p < 0.001]. ConclusionJMML patients with c-CBL mutations have genetic and clinical heterogeneity. However, this subgroup of JMML showed better survival outcome compared with c-CBL wild-type JMML, although with several characteristic clinical events precluding patients' quality of life. Further clinical research is warranted to elucidate determining factors for clinical heterogeneity of c-CBL mutated JMML patients. Disclosures:Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Whole Exome Analysis Reveals Spectrum of Gene Mutations in Juvenile Myelomonocytic Leukemia

AbstractAbstract 170 Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods:We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation.Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers’ protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results:For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases.Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion:Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

A Favorable Outcome In Children with Juvenile Myelomonocytic Leukemia (JMML) with RAS Mutations

AbstractAbstract 2909 Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that affects young children. It is characterized by a specific hypersensitivity of JMML cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. The pathogenesis of JMML involves disruption of GM-CSF signal transduction resulting from mutations of the components of the RAS signaling pathway, including NF1, PTPN11, NRAS, and KRAS. Somatic point mutations of the RAS genes at codons 12, 13, and 61 are found in approximately 20% of patients. Although most patients with JMML die due to progressive disease within 12 months unless treated with hematopoietic stem cell transplantation (HSCT), Matsuda et al reported that JMML patients with NRAS or KRAS glycine to serine substitution improved spontaneously. Other groups in Europe did not confirm this observation, and treatment for patients with JMML and RAS mutations is controversial. Therefore, in the present study, we analyze the association between the mutational status of RAS and prognoses of patients with JMML. Patients and Methods:Eighty children diagnosed with JMML between 1988 and 2010 were studied retrospectively. We performed a mutational analysis of NRAS, KRAS, PTPN11, and C-CBL genes. Results:Seventeen patients (21%) had RAS mutations [NRAS (n = 13) and KRAS (n = 4)], while PTPN11 and C-CBL mutations were found in 28 patients (35%) and 5 patients (6.3%), respectively (Four patients were included in the previous report; Matsuda et al, Blood, 2007). Five children had clinical evidence of NF1 mutations. Among NRAS mutations, G12D and G13D were the most common (n = 6 and n = 5, respectively). Only one patient carried a G12S substitution, which was reported as a favorable mutation. Three patients with KRAS mutations had G13D substitutions. Compared to patients with other mutations or without any aberrations, patients with RAS mutations were significantly younger at diagnosis (median age: 12 months vs. 24 months, p = 0.011), while other known predictive factors such as HbF level and platelet count were not significantly different at diagnosis (median HbF level: 9.1 % vs. 22.2 %, p = 0.295; median platelet count: 27.5 × 109/L vs. 49.0 × 109/L, p = 0.390). Monosomy 7 was observed in seven patients without RAS mutations, and all patients with RAS mutations had normal karyotypes. Among untransplanted patients with RAS mutations, three achieved long-term survival (20, 84, and 209 months after diagnosis). The probability of 5-year overall survival estimated by the Kaplan-Meier method was significantly higher for patients with RAS mutations than for those without (85.7% vs. 30.4%, p = 0.033). Conclusion:These results suggest that JMML patients with RAS mutations may be a distinct subgroup with favorable outcomes in spite of other than G12S.TablePatients characteristics and 5 year OSAll patientsPatients with RAS mutationPatients without RAS mutationp-valueNo. of Patients801763Gender (M/F)50/3013/437/26NSAge (Months), median (range)16 (0–75)12 (2–46)24 (0–75)0.011WBC (×109/L), median (range)31.9 (0.96–563.0)29.4 (0.96–79.3)32.2 (3.93–563.0)NSMono (%), median (range)15 (3–49)15 (3–49)15 (4–49)NSBlast (%), median (range)3 (0–35)1.8 (0–25)3.5 (0–35)NSHb (g/dL), median (range)9.3 (4.9–13.7)8.7 (6.1–11.3)9.4 (4.9–13.7)NSPlt (×109/L), median (range)48.0 (1.4–320)27.5 (3–154)49.0 (1.4–320)NSHbF (%), median (range)21.8 (0–62)9.1 (1.1–61)22.2 (0–62)NSMonosomy 77/80 (8.8%)07/63 (11.1%)NS5-year OS (%)39.985.730.40.033 Disclosures:No relevant conflicts of interest to declare.

Magmas, a Gene Newly Identified as Overexpressed in Human and Mouse ACTH-Secreting Pituitary Adenomas, Protects Pituitary Cells from Apoptotic Stimuli

Pituitary tumors are mostly benign, being locally invasive in 5-35% of cases. Deregulation of several genes has been suggested as a possible alteration underlying the development and progression of pituitary tumors. We here report the identification of a cDNA, corresponding to Magmas gene (mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction), which is highly expressed in two different ACTH-secreting mouse pituitary adenoma cell lines as compared with normal pituitary as well as in two thirds of 64 examined pituitary adenomas as compared with human normal pituitary. Tim 16, the mitochondrial protein encoded by Magmas, was indeed expressed in a mouse ACTH-secreting pituitary adenoma cell line, AtT-20 D16v-F2 cells, in a subcellular compartment likely corresponding to mitochondria. Magmas silencing determined a reduced rate of DNA synthesis, an accumulation in G1 phase, and a concomitant decrease in S phase in At-T20 D16v-F2 cells. Moreover, Magmas-silenced cells displayed basal caspase 3/7 activity and DNA fragmentation levels similar to control cells, which both increased under proapoptotic stimuli. Our data demonstrate that Magmas is overexpressed in mouse and human ACTH-secreting pituitary adenomas. Moreover, our results show that Magmas protects pituitary cells from apoptosis, suggesting its possible involvement in neoplastic transformation.

C-Cbl but Not TET2 Mutations Are Present in Patients with Juvenile Myelomonocytic Leukemia.

Abstract 420Juvenile myelomonocytic leukemia (JMML) is a distinct subtype of myelodysplastic syndrome/myeloproliferative disorder (MDS/MPD) which, in analogy to chronic myelomonocytic leukemia (CMML), is characterized by excessive proliferation of myelomonocytic cells, but unlike CMML it occurs in young children and shows characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Chromosomal defect are present in 22% of patients in particular involving del7/7q. Mutations of genes involved in GM-CSF signal transduction, including RAS and PTPN11, can be identified in a majority of children with JMML, constitutional mutations of NF1 can be found in another 10% of patients with JMML, but in significant proportion of patients no molecular lesions were identified. To further clarify the molecular pathogenesis of JMML we have applied a high density SNP array-based karyptyping and screened for associated mutations including established defected in RAS, PTPN11 and NF1 but also c-Cbl and TET genes, recently identified in patients with CMML and MDS/MPD.We studied 49 children with JMML diagnosed between 1988 and 2008 in 28 institutions throughout Japan. The median age at diagnosis was 28 months (range, 1-75 months). Karyotypic abnormalities were detected in 11 patients, including 7 patients with monosomy 7. Two children had clinical evidence of NF1 mutations. Out of 49 patients, 32 received hematopoietic stem cell transplantation (HSCT).We performed mutational analysis of the genes known to be affected by mutations in JMML. PTPN11 mutations were found in 26/49 (53%) while NRAS and KRAS mutations were found in 2/49 (4%) and 1/49 (2%), respectively. None of the patients screened showed the presence of TET2 mutations, previously shown to be present in a significant proportion of patients with MDS/MPD, including CMML.High-density Affymetrix 250K single nucleotide polymorphism array (SNP-A) were applied as a karyotyping platform to identify LOH and submicroscopic copy number changes. Signal intensity was analyzed and SNP calls determined using Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE). Copy number and areas of UPD were investigated using Hidden Markov Model and CNAG v3.0 software. Compared to the results of conventional metaphase cytogenetics (MC), SNP-A identified significantly more genetic abnormalities (25% vs 49%; p=.02). In 1 patient UPD17q was present ivolving NF1 locus. In 4 patients UPD11q involving c-cbl locus (11q23.1) was found.Sequencing of c-Cbl gene family revealed mutations of c-Cbl in 5/49 (10%), and no Cbl-b mutations. All but 1 mutations were homozygous and were located in the RFD (exon 8 and intron 8). C-Cbl mutations were mutually exclusive with PTPN11, NRAS, and KRAS mutations or had clinical diagnosis of NF1. Unlike in CMML, no UPD4q24 or homo- or heterozygous TET2 mutations were found. Histomorphologic analysis did not reveal any distinct c-Cbl mutation-associated features or differences in count between patients grouped based on the presence of specific mutations. Similarly, there were no differences in gender, or the presence of cytogenetic abnormalities and the probability of 2 year overall survival of c-Cbl mutant cases between patients grouped according to mutational status. All patients with c-Cbl mutations displayed GM-CSF hypersensitivity at initial presentation but did not differ in this feature from the remaining JMML patients. However, mutant c-Cbl cases showed earlier presentation (median age 12 months vs. 29 months, p = .037) and lower median hemoglobin F fraction (3.5 % vs. 25%, p=.02). In sum, c-Cbl mutations constitute a novel important pathogenic lesion in JMML. While their presence suggests functional similarity to CMML, absence of TET2 mutation in JMML and rarity of PTPN11 mutations in CMML constitute important distinctive features of both diseases. Disclosures:No relevant conflicts of interest to declare.

Involvement of suppressors of cytokine signaling in toll-like receptor–mediated block of dendritic cell differentiation

AbstractDendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage–colony-stimulating factor (GM-CSF)–mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

Correlation of Clinical Features with Mutational Status in Children with Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that affects young children. It is characterized by specific hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Mutations in RAS, NF1, or PTPN11 positioned in the GM-CSF signal transduction pathway, which contribute to myeloid proliferation, have been recognized in the pathogenesis of JMML. Recently a multi-step model for leukemogenesis has been proposed. In this model, the pathogenesis of leukemia requires at least two classes of mutations:primary mutations of genes implicated in cell differentiation such as AML1 and PU.1 andadditional mutations of genes contributing to myeloid proliferation such as FLT3, RAS, and PTPN11.We hypothesized that in patients with JMML, in addition to known mutations of genes in the GM-CSF pathway involved in myeloid proliferation, potential causative mutations of other classes might be acquired. AML1 encodes a transcription factor that is essential for definitive hematopoiesis, and its mutations have recently been found in adults with acute myeloid leukemia and myelodysplastic syndrome. However, no information is available on the profiles of mutations in these genes and the relationship between these mutations and clinical features of JMML in children. We analyzed mutations of N-RAS, K-RAS, and PTPN11 in 50, and of AML1 in 30 Japanese children with JMML by direct sequencing. Correlation between the mutational status and clinical and laboratory findings, including age at diagnosis, sex, fetal hemoglobin (HbF), platelets count, cytogenetic abnormalities, and hepatomegaly, all which are suggested prognostic factors for JMML, were also assessed. Of the 50 patients with JMML, cytogenetic abnormalities were detected in 14, including 8 with monosomy 7. PTPN11 and N-/K-RAS mutations were found in 24 (48%) and 7 (14%) patients, respectively, and a novel mutation in AML1 was identified in one patient. No simultaneous mutations in these genes were found. Age at diagnosis was older (median 36 vs 11 months, p=0.0005) and HbF level was higher (31.0% vs 5.1%, p=0.033) in patients with the PTPN11 mutation than those with the RAS mutation. No difference was observed between patients with PTPN11 and RAS mutations in sex ratio, white blood cell count, platelets count, and the incidence of cytogenetic abnormalities and hepatomegaly. Our results suggest that the clinical outcome of patients with PTPN11 might differ from those with RAS mutations because a higher level of HbF and older age have been reported to be poor prognostic factors. In one patient with JMML, we identified a novel mutation in the AML1 gene that belongs to the class of genes contributing to cell differentiation instead of the class of genes in the GM-CSF pathway involved in myeloid proliferation.

Identification and characterization of Magmas, a novel mitochondria-associated protein involved in granulocyte-macrophage colony-stimulating factor signal transduction.

The aim of this study was to identify granulocyte-macrophage colony-stimulating factor (GM-CSF) responsive genes. Potential GM-CSF responsive genes were identified by comparing the mRNA expression pattern of the murine myeloid cell line PGMD1 grown in either interleukin-3 (IL-3) or GM-CSF by differential display. Human and murine cDNA clones of one of the bands having increased expression in GM-CSF were isolated. mRNA expression of the gene was examined by Northern blot. Immunohistochemistry and studies with a green fluorescent fusion protein were used to determine its intracellular location. Growth factor-stimulated proliferation of PGMD1 cells transfected with constitutively expressed sense and anti-sense cDNA constructs of the gene was measured by 3H-thymidine incorporation. A gene, named Magmas (mitochondria-associated granulocyte macrophage CSF signaling molecule), was shown to be rapidly induced when cells were switched from IL-3 to GM-CSF. Analysis of the amino acid sequence of Magmas showed it contained a mitochondrial signal peptide, but not any other known functional domains. The human and murine clones encode nearly identical 13-kDa proteins that localized to the mitochondria. Magmas mRNA expression was observed in all tissues examined. PGMD1 cells that overexpressed Magmas proliferated similarly to untransfected cells when cultured in IL-3 or GM-CSF. In contrast, cells with reduced protein levels grew normally in IL-3, but had impaired proliferation in GM-CSF. Magmas is a mitochondrial protein involved in GM-CSF signal transduction.

Down-regulation of interleukin-3/granulocyte-macrophage colony-stimulating factor receptor β-chain in BCR-ABL+human leukemic cells: association with loss of cytokine-mediated Stat-5 activation and protection from apoptosis after BCR-ABL inhibition

AbstractSeveral signaling cascades are engaged by expression of the p210 bcr-abl tyrosine kinase, and evidence suggests that these signals drive leukemogenesis. In this report, signaling pathways were examined and compared between cells derived from leukemic patients and cells expressing a bcr-abl construct (MBA). The effects of acute inhibition of bcr-abl with STI-571 on these signals and the survival of bcr-abl–expressing cells were also evaluated. Expression of bcr-abl in interleukin-3 (IL-3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)–dependent Mo7e cells (MBA) resulted in growth factor independence, constitutive activation of Stat-5 phosphorylation, engagement of mitogen-activated protein (MAP) kinase signals, and increased expression of PTP1B and bcl-xL. STI-571 inhibited cell growth and induced apoptosis in bcr-abl–expressing cells (MBA, K562, BV-173, KBM5) but not in bcr-abl− tumor cells (Mo7e, KG-1, ME-180, Daudi). STI-571–mediated apoptosis correlated with the inhibition of Stat-5 and MAP kinase activation and a reduction in overexpressed bcl-xL but not in PTP1B. Inhibitor had no effect on IL-3/GM-CSF–dependent Mo7e cell signaling and did not prevent activation of the other Jak/Stat pathways (interferon α, IL-3/GM-CSF). However, neither IL-3 nor GM-CSF could reactivate Stat-5 after the STI-571–mediated inhibition of bcr-abl. Expression of the common β-chain of the IL-3/GM-CSF receptor was down-regulated in Stat-5–activated myeloid leukemic cells, suppressing IL-3/GM-CSF signal transduction and the ability of these cytokines to provide apoptotic protection. These studies suggest that bcr-abl activates cytokine-independent mechanisms of survival while inactivating intrinsic cytokine signaling cascades, making bcr-abl+myeloid cells vulnerable to apoptosis after bcr-abl inactivation.

GM-CSF Induces Phosphorylation of CREB Through Activation of pp90rsk in Myeloid Cells. • 664

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) regulates the proliferation and maturation of myeloid progenitors and enhances the function of differentiated effector cells. GM-CSF stimulation of myeloid cells results in the rapid and transient induction of the immediate early gene, egr-1, independent of protein synthesis. We previously demonstrated that the cAMP response element (CRE) contained within the -116 nucleotide egr-1 promoter region is necessary, but not sufficient, for transcriptional activation of egr-1 in GM-CSF-treated cells. We also found that the CRE-binding protein, CREB, constitutively associates with the CRE and is phosphorylated on serine 133 through a protein kinase A-independent pathway. Based on previous studies with Nerve Growth Factor-treated PC12 cells (Xing, et al. Science 273:959-963), we hypothesized that CREB is phosphorylated by pp90rsk. We identified pp90rsk in both the nucleus and cytoplasm of TF-1 cells by Western blot analysis. With GM-CSF stimulation, no increase in pp90rsk protein levels was observed. To determine whether pp90rsk phosphorylates CREB in response to GM-CSF treatment, we performed in vitro kinase assays with TF-1 cell extracts. Cells were serum- and factor-starved for 24 hours and treated with GM-CSF(1nM) for 1, 2, 5, 10, 20, 30, and 60 minutes. Extracts were incubated with pp90rsk antibody and immunoprecipitation was performed. CREB was phosphorylated within 2 to 5 minutes following GM-CSF stimulation and decreased by 30 minutes. Our results suggest that pp90rsk phosphorylates CREB during GM-CSF signal transduction. These studies provide insights into the mechanisms regulating myeloid cell proliferation and differentiation.

Granulocyte-Macrophage Colony-stimulating Factor Induces the Transcriptional Activation of egr-1 through a Protein Kinase A-independent Signaling Pathway

Granulocyte-macrophage colony-stimulating factor (GM-CSF) rapidly and transiently induces the transcriptional activation of the early growth response gene-1 (egr-1) in the human factor-dependent myeloid leukemic cell line, TF-1. We previously demonstrated that the cAMP response element (CRE) is required for GM-CSF-induced egr-1 expression and that phosphorylation of CREB on serine 133 plays a critical role during GM-CSF signal transduction. To determine whether GM-CSF activates signaling pathways through a protein kinase A-dependent or -independent pathway, we measured cAMP levels following GM-CSF or forskolin treatment of TF-1 cells. Forskolin but not GM-CSF stimulation resulted in an increase in cAMP levels. Transient transfection assays with TF-1 cells were also performed with a -116-nucleotide egr-1 promoter construct and the protein kinase inhibitor, PKI. Although PKI inhibited forskolin induction of the -116-nucleotide construct, it did not affect GM-CSF stimulation of this construct. In the present study, we demonstrated that GM-CSF induces egr-1 expression through a protein kinase A-independent pathway.

Phosphorylation of a Fes-related Protein in Response to Granulocyte-Macrophage Colony Stimulating Factor

Previous work has suggested that a 97-kDa protein (p97) is involved in the signal transduction pathway of granulocyte-macrophage colony stimulating factor (GM-CSF) as well as interleukin 3, erythropoietin, and interleukin 2. We have examined the relationship of p97 to the protein tyrosine kinase Fes in the GM-CSF signal transduction pathway in erythroid and myeloid cell lines. GM-CSF stimulation of three different cell lines induced tyrosine phosphorylation of p97 as well as a number of other phosphotyrosylproteins. Although each cell line expressed the proto-oncogene product Fes, antisera specific for Fes did not recognize p97 in immunoblotting experiments. Furthermore, immunodepletion of Fes did not reduce the amount of p97 in GM-CSF-treated cells. Two-dimensional gel electrophoresis demonstrated that p97 and Fes have similar charge to mass ratios, and limited proteolytic mapping of p97 and Fes suggested that these proteins may be related but are not identical. Our studies demonstrate that p97 is not Fes but is probably a Fes-related protein.

GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production.

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.

PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments.

We have used microphysiometry and antisense methodology to show that the epsilon isoenzyme of protein kinase C (PKC) is involved in the signal transduction pathway of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a human bone marrow cell line, TF-1. These cells require GM-CSF or a related cytokine for proliferation. When the cells are appropriately exposed to GM-CSF, they exhibit a burst of metabolic activity that can be detected on the time scale of minutes in the microphysiometer, a biosensor-based instrument that measures the rate at which cells excrete protons. These cells express PKC alpha and -epsilon, as determined by Western blot analysis. Treatment with isoenzyme-specific antisense oligonucleotides inhibits expression appropriately, but only inhibition of PKC epsilon appreciably diminishes the burst of metabolic activity induced by GM-CSF. Consistent with the involvement of PKC epsilon, GM-CSF appears to activate phospholipase D and does not cause a detectable increase in cytosolic [Ca2+].