Letters to the Editor
c-Src/ mice, which are severely osteopetrotic due to an absence of osteoclast activity rather than a reduction in osteoclast number.8–10 This is thought to be primarily due to the defective osteoclast actin ring and sealing zone formation following inhibition of signalling by integrins such as avb3.8,9 The effect of dasatinib on c-Src phosphorylation in osteoclast precursors was examined by western blot. Dasatinib treatment decreased basal levels of Src phosphorylation at doses of X20 nM in both huCD14 þ and mBM (IC50 ¼ 15.5 and 17.15, respectively; Figures 2c–f). These results suggest that, under the culture conditions used in the experiments reported here, dasatinib treatment inhibits c-Src phosphorylation at concentrations higher than those that inhibit osteoclast activity in mBM and huCD14 þ . In summary, our results suggest that dasatinib can inhibit osteoclast formation and activity in vitro, at least in part through an inhibition of c-fms signal transduction. Inhibition of osteoclast activity, if not corrected by a decrease in osteoblast activity, may lead to an overall increase in bone volume. To this end, current evidence suggests that patients undergoing treatment with imatinib mesylate exhibit dysregulated bone remodelling, resulting in an overall increase in trabecular bone volume.11–13 This is thought to result from an inhibition of osteoclast activity and a concomitant activation of osteoblast activity by imatinib, through inhibition of c-fms and PDGFR, respectively.5,11 Although it is unknown whether dasatinib alters the mineralization activity of osteoblasts, it could be predicted to promote osteogenesis as it is a potent inhibitor of PDGFR. If this is the case, a decrease in osteoclast activity and a concurrent increase in osteoblast activity by dasatinib may result in decoupling of the bone remodelling process. This hypothesis awaits further investigation in suitable animal models of normal bone remodelling. These studies also suggest the need to monitor skeletal remodelling in patients receiving dasatinib, as has been prescribed for imatinib mesylate.11 Furthermore, it highlights the potential application of dasatinib therapy in cases of pathologies characterized by increased osteoclast activity, such as in osteoporosis, osteoarthritis, bone metastatic breast cancer and prostate cancer, and multiple myeloma.
Acknowledgements We wish to thank Lee Anne Griffiths, Francis Lee and Richard Smykla from Bristol-Myers Squibb for the provision of dasatinib drug and helpful discussions.
K Vandyke1,2, AL Dewar1, AN Farrugia1, S Fitter1, L Bik To3, TP Hughes3 and ACW Zannettino1,2 1 Myeloma Research Program, Bone and Cancer Research Laboratories, Division of Haematology, Hanson Institute,
Institute of Medical and Veterinary Science, Adelaide, Australia; 2 School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, Australia; 3 Division of Haematology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, Australia E-mail:
[email protected]
997
References 1 Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 2004; 305: 399–401. 2 Melnick JS, Janes J, Kim S, Chang JY, Sipes DG, Gunderson D et al. An efficient rapid system for profiling the cellular activities of molecular libraries. Proc Natl Acad Sci USA 2006; 103: 3153–3158. 3 Hochhaus A, Kantarjian HM, Baccarani M, Lipton JH, Apperley JF, Druker BJ et al. Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood 2007; 109: 2303–2309. 4 Dewar AL, Cambareri AC, Zannettino AC, Miller BL, Doherty KV, Hughes TP et al. Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib. Blood 2005; 105: 3127–3132. 5 Dewar AL, Farrugia AN, Condina MR, Bik To L, Hughes TP, Vernon-Roberts B et al. Imatinib as a potential antiresorptive therapy for bone disease. Blood 2006; 107: 4334–4337. 6 Dai XM, Ryan GR, Hapel AJ, Dominguez MG, Russell RG, Kapp S et al. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood 2002; 99: 111–120. 7 Wang X, Hochhaus A, Kantarjian HM, Agrawal S, Roy A, Pfister M et al. Dasatinib pharmacokinetics and exposure-response (E-R): relationship to safety and efficacy in patients (pts) with chronic myeloid leukemia (CML). J Clin Oncol 2008; 26 (Suppl): 3590. 8 Xing L, Venegas AM, Chen A, Garrett-Beal L, Boyce BF, Varmus HE et al. Genetic evidence for a role for Src family kinases in TNF family receptor signaling and cell survival. Genes Dev 2001; 15: 241–253. 9 Lowe C, Yoneda T, Boyce BF, Chen H, Mundy GR, Soriano P. Osteopetrosis in Src-deficient mice is due to an autonomous defect of osteoclasts. Proc Natl Acad Sci USA 1993; 90: 4485–4489. 10 Boyce BF, Yoneda T, Lowe C, Soriano P, Mundy GR. Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J Clin Invest 1992; 90: 1622–1627. 11 Fitter S, Dewar AL, Kostakis P, To LB, Hughes TP, Roberts MM et al. Long-term imatinib therapy promotes bone formation in CML patients. Blood 2008; 111: 2538–2547. 12 Berman E, Nicolaides M, Maki RG, Fleisher M, Chanel S, Scheu K et al. Altered bone and mineral metabolism in patients receiving imatinib mesylate. N Engl J Med 2006; 354: 2006–2013. 13 Grey A, O’Sullivan S, Reid IR, Browett P. Imatinib mesylate, increased bone formation, and secondary hyperparathyroidism. N Engl J Med 2006; 355: 2494–2495.
Molecular profile of CD34 þ stem/progenitor cells according to JAK2V617F mutation status in essential thrombocythemia
Leukemia (2009) 23, 997–1000; doi:10.1038/leu.2008.357; published online 8 January 2009
Essential thrombocythemia (ET) is mainly characterized by the abnormal proliferation of a malignant megakaryocytic clone and by persistent thrombocytosis. Recently, a JAK2 mutation
(JAK2V617F) has been reported in ET and other myeloproliferative neoplasms. In particular, 40–60% of ET patients harbor this mutation and a substantial proportion of them are heterozygous with allele burden below 50%.1 JAK2V617F mutation distinguishes the disease into two biologically distinct subtypes with the V617F-positive subgroup exhibiting many laboratory and clinical similarities to polycythemia vera. Furthermore, 50 Leukemia
Letters to the Editor
998 patients with V617F-negative ET remained V617F-negative for more than 6 years, indicating that this disorder is distinct from, and not an early ‘pre-JAK2’ phase of V617F-positive ET.2 Despite this finding, the molecular pathology of ET according to the presence or the absence of JAK2V617F mutation is still unknown. Gene expression profiling of granulocytes from ET patients showed that patients lacking the mutation do not display constitutively active JAK–STAT signaling and show significantly lower expression of the target genes Pim1 and SOCS2.3 More recently, we read with interest the study by Puigdecanet et al.,4 which compared the gene expression profile of two groups of ET, selected for presence or absence of JAK2V617F mutation in granulocytes. A supervised hierarchical clustering analysis on differentially expressed genes showed a classification of two main groups of patients, which is independent from JAK2V617F mutation status. However, the authors confirmed that some genes involved in the JAK–STAT signaling pathway (CISH, SOCS2, SOCS3 and PIM1) present lower expression levels in JAK2V617F-negative patients. It can be noted that, in all the above studies, the source used for microarray analysis is granulocytes and no data are reported about the CD34 þ stem/progenitor cells molecular profile. As ET is a stem cell disease, the molecular analysis of the CD34 þ population would be of particular interest. Therefore, the aim of this study was to evaluate whether the JAK2V617F mutation causes altered gene expression in the CD34 þ stem cell compartment of ET. We firstly showed that bone marrow CD34 þ stem/progenitor cells of a subset of patients with ET carry the JAK2V617F mutation. Table 1 summarizes the principal laboratory and clinical data of eight JAK2V617Fpositive and eight JAK2V617F-negative patients, diagnosed according to WHO criteria. All patients/controls provided written informed consent. Bone marrow CD34 þ cells were isolated using immunomagnetic cell sorting as described earlier.5 The JAK2V617F mutation was identified in the CD34 þ stem cells by RT-PCR (reverse transcription-polymerase chain reaction) followed by enzymatic digestion with BsaXI restriction enzyme. Afterwards, JAK2V617F-positive CD34 þ stem cell gene expression profile was compared with that of the negative counterpart to identify differentially expressed genes. Total RNA from CD34 þ cells was extracted using RNeasy Micro Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s recommendations, and two-cycle target labeling assays, as well as the Affymetrix HG-U133A GeneChip array hybridization, staining and scanning, were performed, using
Affymetrix standard protocols (Affymetrix, Santa Clara, CA, USA).5 All the data have been deposited in the Gene Expression Omnibus MIAME compliant public database, at http://www.ncbi.nlm.nih.gov/geo (Supplementary Table S1). RMA (robust multi-array average) procedure was applied to the entire set of raw signals (that is, CEL files) to adjust the background and normalize microarray intensities and to generate gene expression values. To explore the relationship among samples as natively defined by gene expression profiles, we performed an unsupervised hierarchical cluster analysis using the ‘condition tree’ option implemented in GeneSpring GX 7.3 package and applying the Pearson’s correlation equation as described.5 As shown in Figure 1, the unsupervised analysis of gene expression in CD34 þ cells from all ET patients revealed that the presence of the JAK2V617F mutation is not sufficient to determine the separation of the two classes, in agreement with results obtained by Puigdecanet et al.4 Then, to deeply analyze whether or not differences exist between the JAK2V617F-positive and negative classes, we selected only those genes that showed a fold change of more than 2 or less than 0.5 between the signal mean value of patients carrying or not the JAK2V617F mutation. Successively, two additional analyses were performed on the list of genes deriving from the fold change filter. The first was a t-test with the Benjamini and Hochberg correction of false discovery rate as already described.5 Moreover, the data was also analyzed using SAM (significance analysis of microarray) method. Consistent with the results of clustering analysis, no genes were differentially expressed according to these analyses. To confirm that there are no significant differences in the gene expression between JAK2V617F-positive and negative patients, the expression levels of the following target genes of the JAK2– STAT pathway (BCL2L1, MYC, PIM1, SOCS1, SOCS2) was quantitated by RTQ-PCR (real-time quantitative reverse transcription-polymerase chain reaction). These results were validated in an independent test cohort consisting of 12 patients with JAK2V617F mutation and 10 with wild-type JAK2.
Table 1 Clinical and laboratory characteristics, at the time of sample collection, of ET study patients Patient characteristics Males/Females Age (Years) Median (range) Platelet number, 109/l Median (range) Hemoglobin, g/100 ml Median (range) Diagnosis/off cytotoxic treatment Thrombosis Hemorrhage Homozygous/heterozygous JAK2V617F mutation Cytogenetic aberrations
Leukemia
JAK2V617Fpositive ET
JAK2V617Fnegative ET
3/5
2/6
46 (26–63)
49 (30–68)
912 (556–1230) 834 (580–1088) 15.2 (12.1–15.9) 14.5 (13–15.1) 4/4 5/3 1 0 0 1 0/8 / 0
0
Figure 1 The unsupervised clustering of gene expression profiles of BM CD34 þ stem/progenitor cells. The columns marked in green, blue and red refer respectively to the results obtained from healthy individuals (four cases), JAK2V617F-positive ET patients (eight cases) and JAK2V617F-negative (eight cases) ET patients.
Letters to the Editor
999 RTQ-PCR was carried out with TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA) (Supplementary Table S2) using the ABI Prism 7900 HT sequence detection system (Applied Biosystems) as described earlier.5 Once again, none of the selected genes showed significant differences in the level of expression among the two subgroups (Figure 2). In turn, the expression of these genes was not significantly different between the JAK2V617F-positive/negative patients and the normal counterpart (Figure 2). To the best of our knowledge this is the first time that the gene expression profile of CD34 þ malignant stem/progenitor cell of ET is investigated. We provide here evidence that JAK2V617F mutation does not influence the gene expression profile of malignant CD34 þ stem/progenitor cells of ET. Whether our findings are due to the low expression level of the mutant in ET, which may not be able to modify the gene expression profile of CD34 cells in our system, remains a matter of speculation. In polycythemia vera, where the vast majority of patients carry the mutation with high mutant allele burden, in vitro experiments suggest that the JAK2V617F mutation is present in hematopoietic stem cells (that is, CD34 þ /CD38-/Lin-/CD90 þ long-term culture initiating cells). However, once again, no data are reported about hematopoietic stem cell molecular profile. Similar to ET, altered gene expression profile has been reported
in granulocytes from polycythemia vera patients in comparison to the normal counterparts.6 Consistent with our findings, recent data by Moliterno et al.7 suggest that the mutant allele burden measured in CD34 þ cells, rather than that of granulocytes, should be considered for clinical correlates because the burden measured in peripheral blood granulocytes was 1.56 and 1.14-fold higher, respectively, in polycythemia vera and ET than in the CD34 þ cells. As it has been shown that expression of a homodimeric type I cytokine receptors is required for JAK2V617F-mediated transformation, the JAK2 pathway activation could depend on cytokine receptor expression levels that are strongly increased in granulocytes as compared with CD34 þ cells. In addition, we cannot exclude that alterations of different pathways regulating JAK2 activity (deregulation of phosphatases and suppressors of cytokine signaling proteins, polymorphism or mutations in cytokine receptors) may contribute to the pathogenesis of the disease. Consistently, indirect evidence, based on studies of mutation prevalence within clonal granulocytes as well as familial myeloproliferative disorder studies, suggests that the JAK2V617F mutation may be acquired as a secondary genetic change, especially in ET and idiopathic myelofibrosis.8 Additional mutations of JAK2 in exon 12 and of the thrombopoietin receptor gene (MPL) were recently found in
MYC
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Figure 2 RTQ-PCR of selected genes. The data were expressed as RQ log10. Patients were categorized according to the absence (V617F;10 cases) or the presence of JAK2V617F mutation (V617F þ ;12 cases). Boxes represent the interquartile range that contains 50% of the subjects, the horizontal line in the box marks the median, and bars show the range of values. Abbreviations: CTR, control; RQ, relative quantitation. Leukemia
Letters to the Editor
1000 a subset of ET patients. Moreover, a large proportion of ET patients, who are JAK2V617F-negative, have evidence of clonal hematopoiesis. Taken together, these data suggest that there are patients with ET in whom a different genetic event contributes to disease pathogenesis. Therefore, our findings provide a strong molecular support to the emerging view that unknown additional or alternative molecular lesions contribute to disease pathogenesis in ET.
Acknowledgements This work was supported by Fondi ex 60% of the University of Bologna 2006 (LC), PRIN 2006 (AMV), PRIN 2006 (RM), Programma di ricerca oncologica 2006- Regione Emilia-Romagna (RML), Programma di ricerca Regione-Universita` 2007–2009 (RML), and by BolognaAIL (Italian association against Leukemia, Bologna section). This paper is dedicated to the memory of Stefano Ferrari, Professor of Biochemistry at University of Modena and Reggio Emilia.
L Catani1, R Zini2, D Sollazzo1, E Ottaviani1, AM Vannucchi3, S Ferrari2, M Baccarani1, N Vianelli1, RM Lemoli1 and R Manfredini2 1 Department of Hematology and Oncological Science ‘L& A Sera`gnoli’, University of Bologna, Bologna, Italy; 2 Department of Biomedical Science, Section of Biological Chemistry, University of Modena and Reggio Emilia, Modena, Italy and 3 Department of Hematology, University of Florence, Florence, Italy E-mail:
[email protected]
References 1 Passamonti F, Rumi E, Pietra D, Della Porta MG, Boveri E, Pascutto C et al. Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders. Blood 2006; 107: 3676–3682. 2 Campbell PJ, Baxter EJ, Beer PA, Scott LM, Bench AJ, Huntly BJ et al. Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation. Blood 2006; 108: 3548–3555. 3 Schwemmers S, Will B, Waller CF, Abdulkarim K, Johansson P, Andreasson B et al. JAK2V617F-negative ET patients do not display constitutively active JAK/STAT signalling. Exp Hematol 2007; 35: 1695–1703. 4 Puigdecanet E, Espinet B, Lozano JJ, Sumoy L, Bellosillo B, Arenillas L et al. Gene expression profiling distiguishes JAK2V617F-negative from JAK2V617F-positive patients in essential thrombocythemia. Leukemia 2008; 22: 1368–1376. 5 Manfredini R, Zini R, Salati S, Siena M, Tenedini E, Tagliafico E et al. The kinetic status of hematopoietic stem cell (HSC) subpopulations underlies a differential expression of genes involved in self-renewal, commitment and engraftment. Stem Cells 2005; 23: 496–506. 6 Kovacs R, Teo S, Buser AS, Brutsche M, Tiedt R, Tichelli A et al. Altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2. Blood 2005; 106: 3374–3376. 7 Moliterno AR, Williams DM, Rogers O, Isaacs MA, Spivak JL. Phenotypic variability within the JAK2 V617F-positive MPD: roles of progenitor cell and neutrophil allele burdens. Exp Hematol 2008; 36: 1486–1492. 8 Rumi E, Passamonti F, Pietra D, Della Porta MG, Arcaini L, Boggi S et al. JAK2 (V617F) as an acquired somatic mutation and a secondary genetic event associated with disease progression in familial myeloproliferative disorders. Cancer 2006; 107: 2206–2211.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Exclusion of ABCB8 and ABCB10 as cancer candidate genes in acute myeloid leukemia
Leukemia (2009) 23, 1000–1002; doi:10.1038/leu.2008.358; published online 8 January 2009
Acute myeloid leukemia (AML) exhibits great heterogeneity in manifestation, sensitivity to therapy and genetic basis of pathology. Numerous studies have implied that AML arises
from the sequential accumulation of mutations at the level of the hematopoietic stem/progenitor cell, resulting in a disturbed balance of proliferation, differentiation and apoptosis of immature myeloid progenitors.1 To gain more insight into somatic mutations that affect biological pathways in tumorigenesis, a systematic mutation analysis of virtually all annotated human protein-coding genes in eleven breast and eleven
Figure 1 Location of nucleotide changes observed in ABCB8 and ABCB10 in 94 AML patients compared with GenBank sequences. The conserved domains (transmembrane and adenosine triphosphate (ATP) binding domains) and segments of low compositional complexity (empty blocks) are depicted in the schematic representation of the two transporters. The upper panels of the two schematic diagrams show the variants identified in our patient cohort; the lower panels show the earlier described SNPs not found in our patient cohort. Newly discovered genetic variants and single nucleotide polymorphisms (SNPs) are indicated by their positions at the cDNA level. The filled and empty circles represent new and known SNPs, respectively. The frequency of each variant in our cohort is indicated by the number of patients who carried that variant. (a) Variants observed in the ABCB8 coding region. Three new variants were also present in healthy controls: c.567 C4T (V158V, n ¼ 1, heterozygous), c.2202 A4C (P703P, n ¼ 5, heterozygous) and c.2222 G4T (G710V, n ¼ 8, one homozygous and seven heterozygous). Two new variants were found in remission samples: c.1076 G4A (R328H, n ¼ 1, homozygous) and c.463 C4G (L124V, n ¼ 2, heterozygous, present in the remission sample of one patient and in the T cells cultured from the other patient’s cryopreserved peripheral blood sample). Another two new variants were detected in cultured T cells: c.1572 C4T (G493G, n ¼ 1, heterozygous) and c.223 C4T (R44W, n ¼ 1, heterozygous). The remaining new variant, c.915 G4A (S274S, n ¼ 1, heterozygous), was not determined to be somatic or not due to lack of associated normal material. The nonsynonymous SNPs detected in our patient cohort include: c.496 G4A (V135I, n ¼ 5, heterozygous), c.1003 C4T (R304C, n ¼ 9, seven heterozygous and two homozygous) and c.1717 G4A (V542I, n ¼ 5, heterozygous). The SNP c.1003 C4T had a different frequency (0.123) in our patient cohort compared with that described in GenBank (0.339). One synonymous SNP was found in our panel of patients: c.1077 T4A (R328R, n ¼ 30, 25 heterozygous and five homozygous). Seven SNPs present in the GenBank database were not present in our patients. (b) Variants identified in ABCB10. The new variant, c.923 C4T (L254T, n ¼ 1, heterozygous), located in the transmembrane domain was present in the remission material of the same patient. Two known SNPs were detected in our patient panel: SNP c.946 C4T (S301S, n ¼ 28, heterozygous) and SNP c.1676 G4A (N545D, n ¼ 2, heterozygous). Six SNPs described in GenBank in the ABCB10 coding region were not present in our patient cohort. Leukemia
