Jun Okubo

IGF1 Receptor Signaling Regulates Adaptive Radioprotection in Glioma Stem Cells

Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular‐signal‐regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self‐renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection. STEM CELLS 2013;31:627–640

Aberrant activation of ALK kinase by a novel truncated form ALK protein in neuroblastoma

Anaplastic lymphoma kinase (ALK) was originally identified from a rare subtype of non-Hodgkins lymphomas carrying t(2;5)(p23;q35) translocation, where ALK was constitutively activated as a result of a fusion with nucleophosmin (NPM). Aberrant ALK fusion proteins were also generated in inflammatory fibrosarcoma and a subset of non-small-cell lung cancers, and these proteins are implicated in their pathogenesis. Recently, ALK has been demonstrated to be constitutively activated by gene mutations and/or amplifications in sporadic as well as familial cases of neuroblastoma. Here we describe another mechanism of aberrant ALK activation observed in a neuroblastoma-derived cell line (NB-1), in which a short-form ALK protein (ALKdel2-3) having a truncated extracellular domain is overexpressed because of amplification of an abnormal ALK gene that lacks exons 2 and 3. ALKdel2-3 was autophosphorylated in NB-1 cells as well as in ALKdel2-3-transduced cells and exhibited enhanced in vitro kinase activity compared with the wild-type kinase. ALKdel2-3-transduced NIH3T3 cells exhibited increased colony-forming capacity in soft agar and tumorigenicity in nude mice. RNAi-mediated ALK knockdown resulted in the growth suppression of ALKdel2-3-expressing cells, arguing for the oncogenic role of this mutant. Our findings provide a novel insight into the mechanism of deregulation of the ALK kinase and its roles in neuroblastoma pathogenesis.

Integrated analysis identifies different metabolic signatures for tumor-initiating cells in a murine glioblastoma model

BACKGROUND The metabolic preference of malignant glioma for glycolysis as an energy source is a potential therapeutic target. As a result of the cellular heterogeneity of these tumors, however, the relation between glycolytic preference, tumor formation, and tumor cell clonogenicity has remained unknown. To address this issue, we analyzed the metabolic profiles of isogenic glioma-initiating cells (GICs) in a mouse model. METHODS GICs were established by overexpression of H-Ras(V12) in Ink4a/Arf-null neural stem cells. Subpopulations of these cells were obtained by single-cell cloning, and clones differing in extracellular acidification potential were assessed for metabolic characteristics. Tumors formed after intracranial implantation of these clones in mice were examined for pathological features of glioma and expression of glycolytic enzymes. RESULTS Malignant transformation of neural stem cells resulted in a shift in metabolism characterized by an increase in lactic acid production. However, isogenic clonal populations of GICs manifested pronounced differences in glucose and oxygen consumption, lactate production, and nucleoside levels. These differences were paralleled by differential expression of glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2, with this differential expression also being evident in tumors formed by these clones in vivo. CONCLUSIONS The metabolic characteristics of glioma cells appear early during malignant transformation and persist until the late stages of tumor formation. Even isogenic clones may be heterogeneous in terms of metabolic features, however, suggesting that a more detailed understanding of the metabolic profile of glioma is imperative for effective therapeutic targeting.

IDH1 and IDH2 mutations are rare in pediatric myeloid malignancies

Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 1993; 73: 703–711. 4 Maciejewski JP, Mufti GJ. Whole genome scanning as a cytogenetic tool in hematologic malignancies. Blood 2008; 112: 965–974. 5 Gondek LP, Tiu R, Haddad AS, O’Keefe CL, Sekeres MA, Theil KS et al. Single nucleotide polymorphism arrays complement metaphase cytogenetics in detection of new chromosomal lesions in MDS. Leukemia 2007; 21: 2058–2061. 6 Bessler M, Mason P, Hillmen P, Luzzatto L. Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria. Lancet 1994; 343: 951–953. 7 Endo M, Ware RE, Vreeke TM, Singh SP, Howard TA, Tomita A et al. Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria. Blood 1996; 87: 2546–2557. 8 Young NS, Maciejewski JP. Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes ‘Why? Why? Why?’. J Clin Invest 2000; 106: 637–641.

Aberrations of NEGR1 on 1p31 and MYEOV on 11q13 in neuroblastoma.

MYEOV and NEGR1 are novel candidate gene targets in neuroblastoma that were identified by chromosomal gain in 11q13 and loss in 1p31, respectively, through single nucleotide polymorphism array analysis. In the present study, to assess the involvement of MYEOV and NEGR1 in the pathogenesis of neuroblastoma, we analyzed their mutation status and/or expression profiles in a panel of 55 neuroblastoma samples, including 25 cell lines, followed by additional functional studies. No tumor‐specific mutations of MYEOV or NEGR1 were identified in our case series. Expression of MYEOV was upregulated in 11 of 25 cell lines (44%) and in seven of 20 fresh tumors (35%). The siRNA‐mediated knockdown of MYEOV in NB‐19 cells, which exhibit high expression of MYEOV, resulted in a significant decrease in cell proliferation (P = 0.0027). Conversely, expression studies of NEGR1 revealed significantly lower expression of this gene in neuroblastomas at an advanced stage of the disease. Exogenous NEGR1 expression in neuroblastoma cells induced significant inhibition of cell growth (P = 0.019). The results of these studies provide supporting evidence for MYEOV and NEGR1 as gene targets of 11q13 gains and 1p31 deletions in a neuroblastoma subset. In addition, the findings suggest a possible prognostic value for NEGR1 in neuroblastoma. (Cancer Sci 2011; 102: 1645–1650)

Clinical features and prognostic impact of PRDM16 expression in adult acute myeloid leukemia

High PRDM16 (also known as MEL1) expression is a representative marker of acute myeloid leukemia (AML) with NUP98‐NSD1 and is a significant predictive marker for poor prognosis in pediatric AML. However, the clinical features of adult AML with PRDM16 expression remain unclear. PRDM16 is highly homologous to MDS1/EVI1, which is an alternatively spliced transcript of MECOM (also known as EVI1). We investigated PRDM16 expression in 151 AML patients, with 47 (31%) exhibiting high PRDM16 expression (PRDM16/ABL1 ratio ≥ 0.010). High PRDM16 expression significantly correlated with DNMT3A (43% vs. 15%, P < 0.001) and NPM1 (43% vs. 21%, P = 0.010) mutations and partial tandem duplication of KMT2A (22% vs. 1%, P < 0.001). Remarkably, high‐PRDM16‐expression patients were frequent in the noncomplete remission group (48% vs. 21%, P = 0.002). Overall survival (OS) was significantly worse in high‐PRDM16‐expression patients than in low‐PRDM16‐expression patients (5‐year OS, 18% vs. 34%; P = 0.002). This trend was observed more clearly among patients aged <65 years (5‐year OS, 21% vs. 50%; P = 0.001), particularly in FLT3‐ITD‐negative patients in the intermediate cytogenetic risk group (5‐year OS, 25% vs. 59%; P = 0.009). These results suggest that high PRDM16 expression is a significant predictive marker for poor prognosis in adult AML patients, similar to pediatric AML patients.

ASXL2 Mutations are Frequently Found in Pediatric AML Patients with t(8;21)/RUNX1-RUNX1T1 and Associated with a Better Prognosis.

ASXL2 is an epigenetic regulator involved in polycomb repressive complex regulation or recruitment. Clinical features of pediatric acute myeloid leukemia (AML) patients with ASXL2 mutations remain unclear. Thus, we investigated frequencies of ASXL1 and ASXL2 mutations, clinical features of patients with these mutations, correlations of these mutations with other genetic alterations including BCOR/BCORL1 and cohesin complex component genes, and prognostic impact of these mutations in 369 pediatric patients with de novo AML (0–17 years). We identified 9 (2.4%) ASXL1 and 17 (4.6%) ASXL2 mutations in 25 patients. These mutations were more common in patients with t(8;21)(q22;q22)/RUNX1‐RUNX1T1 (ASXL1, 6/9, 67%, P = 0.02; ASXL2, 10/17, 59%, P = 0.01). Among these 25 patients, 4 (27%) of 15 patients with t(8;21) and 6 (60%) of 10 patients without t(8;21) relapsed. However, most patients with relapse were rescued using stem cell transplantation irrespective of t(8;21). The overall survival (OS) and event‐free survival (EFS) rates showed no differences among pediatric AML patients with t(8;21) and ASXL1 or ASXL2 mutations and ASXL wild‐type (5‐year OS, 75% vs. 100% vs. 91% and 5‐year EFS, 67% vs. 80% vs. 67%). In 106 patients with t(8;21) AML, the coexistence of mutations in tyrosine kinase pathways and chromatin modifiers and/or cohesin complex component genes had no effect on prognosis. These results suggest that ASXL1 and ASXL2 mutations play key roles as cooperating mutations that induce leukemogenesis, particularly in pediatric AML patients with t(8;21), and these mutations might be associated with a better prognosis than that reported previously.

RUNX1mutations in pediatric acute myeloid leukemia are associated with distinct genetic features and an inferior prognosis

TO THE EDITOR: Acute myeloid leukemia (AML) is a complicated disease characterized by the uncontrolled proliferation of hematopoietic precursors and the loss of differentiation ability caused by various genetic alterations. Recent advances in massively parallel sequencing technologies have

Abstract 71: Mutational analysis for IDH1 and IDH2 in pediatric leukemia

Recently, mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that NADP+dependent isocitrate dehydrogenase (IDH)1/2-mutated AMLs display global DNA hypermethylation and a specific hypermethylation signature, suggesting a shared proleukemogenic effect. To explore the involvements of IDH1/2 in the pathogenesis in pediatric hematological malignancies. We analyzed mutations that involve the activation sites of IDH1/2 using polymerase chain reaction amplification/sequencing in a total of 244 samples of pediatric myeloid malignancies as well as infantile leukemia including 17 AML-derived cell lines, 115 primary cases of AML, 28 primary cases of MDS, 15 primary cases of juvenile myelomonocytic leukaemia (JMML), 6 chronic myeloid leukemia (CML)-derived cell line, 18 primary cases of CML and 45 infantile leukemia(6 AML and 39 acute lymphoblastic leukemia (ALL) patients). Moreover, to assess whetherIDH1/2 mutations overlap with known gene abnormalities, such as FLT3, c-KIT, and NPM1 mutations, mutational analyses of FLT3, c-KIT, and NPM1 were also performed. The common IDH2 R140Q mutation was detected in a single AML case, whereas no IDH1 mutation was detected in samples of myeloid malignancies. Although no IDH2 mutation was detected in infantile leukemias, novel P127S, H133I and I130V of IDH1 mutations were detected in 4 of 45 (8.9%) infantile ALL cases. No IDH1 and IDH2 mutations were detected in the JMML, MDS, or CML samples examined. Six AML samples including one cell line had c-KIT mutations (D816V, N822K, or D419fs), 12 AML cases had FLT3-ITD and 10 infantile leukemia cases had FLT3 mutations (D835E or I836). The NPM1 mutation was detected in 2 of 132 AML samples. The AML case harboring the IDH2 mutation, Case 39 was a 12-year-old boy diagnosed as AML-M2 according to the French-American-British cooperative group classification system having t(8;21)(q22;q22), showed no abnormalities of NPM1, c-KIT, and FLT3. Remarkably, among 4 infantile ALL cases with IDH1 mutations, 3 cases showed mixed lineage leukemia(MLL) rearrangements with t(4;11). The FLT3-D835 mutation was found in 1 of 4 patients with IDH1 mutations. These results suggest that although the involvements of IDH1/2 mutations in the pathogenesis of pediatric myeloid malignancies are extremely rare, closely positioned to near activation site, R132 IDH1, these IDH1 mutations are the candidate second genetic events in a subset of MLL-leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 71. doi:1538-7445.AM2012-71