KANK1, a candidate tumor suppressor gene, is fused to ... - Nature

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1052 the secondary malignancies and cardiovascular events, may not directly relate to SCT itself. Long-term follow-up should also consider issues of quality of life (QOL). This initial analysis shows that chronic graft-versushost disease rates and the duration of required immune suppression were lower after RIC. Chronic graft-versus-host disease is a dominant factor determining QOL, suggesting better long-term QOL with RIC; however, formal QOL assessment is required to determine this question. The major limitation of this study is the nonrandomized comparison and the selection biases in allocating patients to one of the regimens. However, because patients were selected for RIC based on high risk for non-relapse mortality, and because OS was similar among these patients given RIC and eligible patients given MAC, it is possible that results in standard risk patients may be similar. Randomized studies, for patients in CR (preferentially CR1) with long-term follow-up and assessment of late complication and QOL, will be needed to determine the best approach in this setting. MAC and possibly the new reduced toxicity myeloablative regimens are still the best approach in patients with active disease.

Conflict of interest The authors declare no conflict of interest.

A Shimoni, I Hardan, N Shem-Tov, R Yerushalmi and A Nagler The Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel E-mail: [email protected]

References 1 Blaise D, Vey N, Faucher C, Mohty M. Current status of reducedintensity-conditioning allogeneic stem cell transplantation for acute myeloid leukemia. Haematologica 2007; 92: 533–541.

2 Aoudjhane M, Labopin M, Gorin NC, Shimoni A, Ruutu T, Kolb HJ et al. Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic stem cell transplantation for patients older than 50 years of age with acute myeloblastic leukaemia: a retrospective survey from the Acute Leukemia Working Party (ALWP) of the European group for Blood and Marrow Transplantation (EBMT). Leukemia 2005; 19: 2304–2312. 3 Martino R, Iacobelli S, Brand R, Jansen T, van Biezen A, Finke J et al. Retrospective comparison of reduced-intensity conditioning and conventional high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLAidentical sibling donors in myelodysplastic syndromes. Blood 2006; 108: 836–846. 4 Ringdeń O, Labopin M, Ehninger G, Niederwieser D, Olsson R, Basara N et al. Reduced intensity conditioning compared with myeloablative conditioning using unrelated donor transplants in patients with acute myeloid leukemia. J Clin Oncol 2009; 27: 4570– 4577. 5 Shimoni A, Hardan I, Shem-Tov N, Yeshurun M, Yerushalmi R, Avigdor A et al. Allogeneic hematopoietic stem-cell transplantation in AML and MDS using myeloablative versus reduced-intensity conditioning: the role of dose intensity. Leukemia 2006; 20: 322– 328. 6 Socie´ G, Stone JV, Wingard JR, Weisdorf D, Henslee-Downey PJ, Bredeson C et al. Long-term survival and late deaths after allogeneic bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry. N Engl J Med 1999; 341: 14–21. 7 Mohty M, de Lavallade H, El-Cheikh J, Ladaique P, Faucher C, Fu¨rst S et al. Reduced intensity conditioning allogeneic stem cell transplantation for patients with acute myeloid leukemia: long term results of a ‘donor’ versus ‘no donor’ comparison. Leukemia 2009; 23: 194–196. 8 Valca´rcel D, Martino R, Caballero D, Martin J, Ferra C, Nieto JB et al. Sustained remissions of high-risk acute myeloid leukemia and myelodysplastic syndrome after reduced-intensity conditioning allogeneic hematopoietic transplantation: chronic graft-versus-host disease is the strongest factor improving survival. J Clin Oncol 2008; 26: 577–584. 9 Hegenbart U, Niederwieser D, Sandmaier BM, Maris MB, Shizuru JA, Greinix H et al. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 2006; 24: 444–453.

KANK1, a candidate tumor suppressor gene, is fused to PDGFRB in an imatinib-responsive myeloid neoplasm with severe thrombocythemia

Leukemia (2010) 24, 1052–1055; doi:10.1038/leu.2010.13 published online 18 February 2010

The majority of myeloproliferative neoplasms are associated with activating mutations in protein tyrosine kinase genes.1 About 20 different fusion products of the PDGFRB gene, which encodes platelet-derived growth factor receptor-b (PDGFRb), have been described in patients with myeloid neoplasms associated with eosinophilia.1,2 Myelodysplastic features and thrombocytopenia are often associated with PDGFRB translocations.3 Here, we describe a new acquired t(5;9) chromosomal translocation in a 67-year-old male patient who presented with thrombocythemia (platelets, 904 109/l) without prominent eosinophilia or neutrophilia. Bone marrow smears showed an increased number of polymorphic megakaryocytes without any blast cells or dysplastic features. Morphology was suggestive Leukemia

of myeloproliferative neoplasm, most likely essential thrombocythemia. There was no detectable BCR–ABL transcript or JAK2 V617F mutation, which is present in about 50% of patients with essential thrombocythemia. The karyotype performed on bone marrow cells showed 46,XY,t(5;9)(q31B32;p22B?24.3) in 16 of 18 metaphases (Figure 1a). Treatment with hydroxyurea was started, but elicited no response. After two-and-a-half months of treatment, the platelet count had increased to 1400 109/l and the neutrophil and eosinophil counts to 13 109/l and 0.9 109/l, respectively. The patient experienced a transient ischemic attack that was attributed to the thrombocythemia. Fluorescence in situ hybridization analysis established the disruption of the PDGFRB locus. As this tyrosine kinase receptor is highly sensitive to imatinib mesylate,2,3 the patient was treated with a low dose of this drug (100 mg/day). At 4 months after the initiation of this treatment, platelet and leukocyte counts were back to normal levels (Figure 1b). After 12 months of follow-up, the patient remains in complete hematological


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Figure 1 The t(5;9) translocation implicates KANK1 as a new fusion partner to PDGFRB in a patient with imatinib-sensitive thrombocythemia. (a) G-banded partial karyotype of the patient showing the t(5;9). (b) Evaluation of the patient’s platelet counts (continuous line) and white blood cell counts (dashed line) as a function of time. Arrows indicate when the treatments were initiated (HU, hydroxyurea). (c) The RNA and protein sequences of the KPb breakpoint are shown. (d) KPb expression was analyzed by nested RT-PCR in cDNA derived from bone marrow samples from the patient with the t(5;9)-positive myeloid neoplasm (lane 2), or from patients with chronic myelogenous leukemia (lanes 3 and 4) or with acute lymphoblastic leukemia (lane 5). (e) A schematic representation of KANK1, PDGFRb and KANK1–PDGFRb (KPb) proteins is shown. cc, coiled-coil domain; A, ankyrin domain; octagons in PDGFRb represent Ig-like domain; TM, transmembrane domain; and Kinase, kinase domain. The position of the breakpoint is indicated by arrowheads. Localization of the two primer sets used in nested PCR (d) is represented by arrows.

remission. The response to low-dose imatinib pointed to constitutive activation of PDGFRb as the cause of thrombocythemia in this patient. Using rapid amplification of cDNA ends PCR, we identified the KANK1 gene (also called ANKRD15) on chromosome 9 as the fusion partner of PDGFRB (Figure 1c). The fusion was confirmed by fluorescence in situ hybridization using specific KANK1 probes (Supplementary Figure S1) and by nested PCR (Figure 1d), as described in Supplementary Materials and methods. The translocation fuses exon 2 of KANK1 (ENST00000382293, Ensembl database, up to nucleotide 3020) to exon 9 of PDGFRB (ENST00000261799, from nucleotide 1714). The junction with exon 9 of PDGFRB is unusual and was mentioned only once regarding an ETV6–PDGFRB fusion (unpublished data in Curtis et al 4). In line with several previously reported PDGFRb hybrids, KANK1– PDGFRb (KPb) contains three coiled-coil domains of KANK1, which may mediate the oligomerization of the hybrid protein, as shown for wild-type KANK1.5 It also includes the fifth immunoglobulin (Ig)-like extracellular domain, the transmembrane domain and the kinase domain of PDGFRb (Figure 1e). The KANK1 gene was identified as a potential tumor suppressor gene at 9p24, whose expression was lost in renal cell carcinoma tissues and cell lines. The 9p chromosomal region, including KANK1, is also deleted in various cancers, such as in acute lymphocytic leukemia and in other diseases.5 The concomitant disruption of a tumor suppressor and activation of a receptor tyrosine kinase by translocation has been suggested in other cases, such as ETV6–PDGFRB. To assess the transforming activity of KPb, we generated lentiviral vectors expressing wild-type KANK1 or KPb and transduced the murine IL-3-dependent hematopoietic cell line Ba/F3. The KPb protein contains the first 741 residues of KANK1 fused to the last 692 residues of PDGFRb. In the absence of IL-3, only KPb-expressing cells proliferated, as shown in a [3H] thymidine incorporation assay (Figure 2a). Ba/F3–KPb cells

could be cultured for several weeks in the absence of IL-3 (data not shown), indicating that KPb is able to transform hematopoietic cells. To evaluate KPb sensitivity to imatinib mesylate, Ba/F3–KPb cells were exposed to increasing concentrations of the drug, which inhibited cell growth in the absence of IL-3 (Figure 2b). The estimated IC50 for KPb was around 7 nM, which corresponded to the reported values for ETV6–PDGFRb and FIP1L1–PDGFRa inhibition, and is about two orders of magnitude lower than the concentration required to inhibit BCR–ABL.2 Autophosphorylation of tyrosine residues in the intracellular part of receptor tyrosine kinases is a critical step to achieve their full activation.2 After immunoprecipitation of PDGFRbcontaining proteins, we showed that KPb was constitutively tyrosine phosphorylated in Ba/F3 cells (Figure 2c). As expected, this was not observed in the presence of imatinib, suggesting that KPb phosphorylated itself. STAT transcription factors are downstream targets of the PDGF receptors.2 Aberrant activation of STAT5 has an important role in hematopoietic cell transformation by many tyrosine kinase oncogenes, including ETV6–PDGFRB.2 By western blot with specific anti-phospho-site antibodies, we showed that STAT5, as well as the related factor STAT3, were phosphorylated in the presence of KPb in an imatinib-dependent manner (Figure 2d). The uncommon breakpoint within PDGFRB found in KPb leads to the addition of the fifth Ig-like domain of the PDGFRb extracellular part to the hybrid protein. In the wild-type receptor, this domain is not implicated in ligand binding, which is mediated by the first three Ig-like domains.2,6 Although the function of this domain has not been fully defined, it was suggested that in c-KIT, another member of the PDGFR family, homotypic interactions between the fourth and to a lesser extent between the fifth Ig-like domains allow the intracellular part to achieve the exact position needed for the receptor activation.6 Therefore, we investigated whether the fifth Ig-like domain could have a role in the activation of KPb. We generated a Leukemia


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Figure 2 KPb is an activated oncogene that stimulates cell proliferation. Ba/F3 cells were transduced with KPb, wild-type KANK1 or the empty vector control. (a, b) Cells were grown in absence of IL-3, and cell proliferation was measured by a [3H]thymidine incorporation assay as described in Material and methods. Cells proliferated to a similar extent in the presence of IL-3 (data not shown). (b) The effect of increasing imatinib concentrations was tested on proliferation of Ba/F3 cells stably expressing KPb. (c) Ba/F3 cells were treated for 4 h with imatinib (0.5 mM) as indicated. Cells were lysed and KPb was immunoprecipitated with an antibody against PDGFRb. Immunoprecipitates were separated by SDS–PAGE and blotted with an antibody against phosphorylated tyrosines (pTyr). As a control, the membrane was reprobed with an anti-PDGFR antibody. The bands shown are derived from the same blot and the same X-ray film. (d) Cells were left in cytokine-free medium for 4 h with imatinib (0.1 mM) as indicated. Cell lysates were immunoblotted with antibodies against phosphorylated or total STAT5 and STAT3. IL-3 was added for 10 min before lysis of Ba/F3.

mutant devoid of this domain (DIg5) in which the PDGFRb sequence starts with exon 11, as in most of the described hybrids (Supplementary Figure S2a). The DIg5 mutant stimulated Ba/F3 cell proliferation to a similar extent compared with KPb, suggesting no key function of this Ig-like domain in KPb activation (Supplementary Figure S2b). Altogether, this indicated that the breakpoint in PDGFRB can be located within intron 8 or intron 10 with little impact on the activation of the protein. The higher frequency of alterations in intron 10 may be related to the size of this intron (3142 base pairs versus 1316 for intron 8). We found no evidence of the recurrence of KANK1 rearrangement in myeloid neoplasms. In our database of B30 000 samples of patients with hematological malignancies, we found eight cases of myeloid malignancies with unexplained 9p terminal cytogenetic alterations and tested them with fluorescence in situ hybridization probes specific for the KANK1 locus. No additional case of KANK1 disruption was found. Future studies will have to search for other types of alterations in the KANK1 gene in such diseases, like deletions, mutations and epigenetic changes. Remarkably, Lerer et al.7 suggested that KANK1 is expressed only from one allele, and is even imprinted Leukemia

in familial cerebral palsy. It is therefore possible that the remaining wild-type KANK1 allele is not expressed in the patient cells. The patient presented thrombocythemia, but no prominent eosinophilia, in sharp contrast with the reported clinical features of myeloid neoplasms associated with PDGFRB rearrangements, which often include thrombocytopenia.1,3 Therefore, we speculate that KANK1 may also have a role in the KPb-associated thrombocythemia. This is in line with a previous report by Kralovics et al.,8 indicating that KANK1 expression was decreased in polycythemia vera and essential thrombocythemia. Furthermore, KANK1 was shown to inhibit cell migration and actin polymerization through the modulation of Rho GTPases.5 As Rho GTPases and the actin cytoskeleton have important functions during megakaryopoiesis, KANK1 might regulate thrombocyte production. In previous reports of treatment of PDGFRb-associated myeloid neoplasms with imatinib mesylate, a daily dose of 400 mg was administered to most patients (range, 200–800 mg), in line with common practice in BCR–ABL-positive chronic myelogenous leukemia.3 In FIP1L1–PDGFRa-positive


myeloproliferative neoplasm, a dose of 100 mg/day was shown to be effective.2 As PDGFRa and b have similar sensitivity to imatinib, a low dose is likely to be effective in the treatment of PDGFRBassociated myeloid neoplasms, as suggested by the present report, with potential benefits in terms of costs and side effects. In conclusion, we identified a new t(5;9) translocation product between PDGFRB and a novel partner, KANK1, in a patient with thrombocythemia who responded well to low-dose imatinib.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This work was supported by grants from the Salus Sanguinis foundation, the Bekales foundation and Action de Recherches Concerteés (Communaute´ Française de Belgique). SM is the recipient of a fellowship from the Maurange foundation (managed by the Roi Baudouin foundation), FD is a FRS-FNRS research fellow and FT is supported by a scholarship from Ope´ration Te´le´vie. We wish to thank Jean-Luc Vaerman for providing samples and Catherine Marbehant for excellent technical assistance. We are grateful to Thomas Michiels for generous donations of reagents and to Laurent Knoops and Stefan Constantinescu for very helpful discussions. This study was approved by the ethics committee of the medical faculty (ref # F/2005/02). Informed consent was obtained.

S Medves1,4, FP Duhoux2,4, A Ferrant3, F Toffalini1, G Ameye2, J-M Libouton2, HA Poirel2,4 and J-B Demoulin1,4 1 de Duve Institute, Universite´ catholique de Louvain, Brussels, Belgium;

2

Centre for Human Genetics, Cliniques universitaires SaintLuc, Universite´ catholique de Louvain, Brussels, Belgium and 3 Hematology Unit, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, Brussels, Belgium E-mail: [email protected] 4 These authors equally contributed to this work.

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References 1 Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 2008; 22: 14–22. 2 Jones AV, Cross NC. Oncogenic derivatives of platelet-derived growth factor receptors. Cell Mol Life Sci 2004; 61: 2912–2923. 3 David M, Cross NC, Burgstaller S, Chase A, Curtis C, Dang R et al. Durable responses to imatinib in patients with PDGFRB fusion gene-positive and BCR-ABL-negative chronic myeloproliferative disorders. Blood 2007; 109: 61–64. 4 Curtis CE, Grand FH, Waghorn K, Sahoo TP, George J, Cross NC. A novel ETV6-PDGFRB fusion transcript missed by standard screening in a patient with an imatinib responsive chronic myeloproliferative disease. Leukemia 2007; 21: 1839–1841. 5 Kakinuma N, Zhu Y, Wang Y, Roy BC, Kiyama R. Kank proteins: structure, functions and diseases. Cell Mol Life Sci 2009; 66: 2651–2659. 6 Yang Y, Yuzawa S, Schlessinger J. Contacts between membrane proximal regions of the PDGF receptor ectodomain are required for receptor activation but not for receptor dimerization. Proc Natl Acad Sci USA 2008; 105: 7681–7686. 7 Lerer I, Sagi M, Meiner V, Cohen T, Zlotogora J, Abeliovich D. Deletion of the ANKRD15 gene at 9p24.3 causes parent-of-origindependent inheritance of familial cerebral palsy. Hum Mol Genet 2005; 14: 3911–3920. 8 Kralovics R, Teo SS, 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.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Human leukocyte antigen (HLA) A1-B8-DR3 (8.1) haplotype, tumor necrosis factor (TNF) G-308A, and risk of non-Hodgkin lymphoma

Leukemia (2010) 24, 1055–1058; doi:10.1038/leu.2010.17; published online 11 February 2010

Large-scale consortial efforts now provide convincing evidence that the pro-inflammatory cytokine tumor necrosis factor (TNF) promoter polymorphism (TNF G-308A), which is thought to increase TNF-a protein expression resulting in inflammation, is associated with increased risk of non-Hodgkin lymphoma (NHL) and specifically with the NHL subtype, diffuse large B-cell lymphoma (DLBCL) among Caucasians.1 The largest effort to date from the International Lymphoma Epidemiology Consortium (InterLymph) comprising 7999 incidence NHL cases and 8452 controls report that TNF-308A carriers have a 1.25-fold (per allele) increased risk for DLBCL and a 1.35-fold increased risk for marginal zone lymphoma.2 A limitation to the published association studies, however, is the inability to delineate the association between TNF and NHL from human leukocyte antigen (HLA) alleles that are known to

be in linkage disequilibrium. TNF is located on chromosome 6p21.3 among the Class III genes of the major histocompatibility complex, 250 kb centromeric to the HLA-B locus and 850 kb telomeric to the Class II HLA-DR locus. It is well documented that Caucasian populations carry the 8.1 ancestral haplotype (AH) that includes the TNF-308A allele (HLA-A1-B8-TNF-308ADR3-DQ2).3 Notably, the 8.1 AH is implicated in the risk of numerous autoimmune conditions, including those associated with NHL (for example, systemic lupus erythematosus and Sjogren’s syndrome).3–5 It remains unknown, however, whether the association reported for TNF-308A is due to or independent from HLA alleles and/or haplotypes. Here, we present data from 555 controls and 610 cases from a US population-based case–control study of NHL where HLA Class I and Class II alleles were evaluated in the context of TNF with regard to their role in risk for NHL and NHL subtypes. As has been previously described,6 the multi-center National Cancer InstituteFSurveillance, Epidemiology and End Results (NCI-SEER) NHL case–control study population comprised 1321 Leukemia