SPRED1, a RAS MAPK Pathway Inhibitor That Causes Legius Syndrome, Is a Tumour Suppressor Downregulated in Paediatric Acute Myeloblastic Leukaemia
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Oncogene (2015) 34, 631–638 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE SPRED1, a RAS MAPK pathway inhibitor that causes Legius syndrome, is a tumour suppressor downregulated in paediatric acute myeloblastic leukaemia E Pasmant1,2, B Gilbert-Dussardier3, A Petit4,5,6, B de Laval7, A Luscan1,2, A Gruber1, H Lapillonne6,8, C Deswarte4,5,6, P Goussard2, I Laurendeau1, B Uzan9, F Pflumio9, F Brizard10, P Vabres11, I Naguibvena9, S Fasola4,5, F Millot12, F Porteu7,13, D Vidaud1,2,3,13, J Landman-Parker4,5,6,13 and P Ballerini8 Constitutional dominant loss-of-function mutations in the SPRED1 gene cause a rare phenotype referred as neurofibromatosis type 1 (NF1)-like syndrome or Legius syndrome, consisted of multiple cafe´-au-lait macules, axillary freckling, learning disabilities and macrocephaly. SPRED1 is a negative regulator of the RAS MAPK pathway and can interact with neurofibromin, the NF1 gene product. Individuals with NF1 have a higher risk of haematological malignancies. SPRED1 is highly expressed in haematopoietic cells and negatively regulates haematopoiesis. SPRED1 seemed to be a good candidate for leukaemia predisposition or transformation. We performed SPRED1 mutation screening and expression status in 230 paediatric lymphoblastic and acute myeloblastic leukaemias (AMLs). We found a loss-of-function frameshift SPRED1 mutation in a patient with Legius syndrome. In this patient, the leukaemia blasts karyotype showed a SPRED1 loss of heterozygosity, confirming SPRED1 as a tumour suppressor. Our observation confirmed that acute leukaemias are rare complications of the Legius syndrome. Moreover, SPRED1 was significantly decreased at RNA and protein levels in the majority of AMLs at diagnosis compared with normal or paired complete remission bone marrows. SPRED1 decreased expression correlated with genetic features of AML. Our study reveals a new mechanism which contributes to deregulate RAS MAPK pathway in the vast majority of paediatric AMLs. Oncogene (2015) 34, 631–638; doi:10.1038/onc.2013.587; published online 27 January 2014 Keywords: SPRED1; tumour suppressor gene; childhood leukaemia; Legius syndrome; cafe´-au-lait spots; neurofibromatosis type 1 INTRODUCTION share characteristic overlapping features, including predisposition In 2007, germline dominant loss-of-function mutations in the to develop multiple types of cancer. Individuals with NF1 and SPRED1 (sprouty-related, EVH1 domain containing 11; NM_152594) Noonan syndrome have a higher risk of haematological mali- gene were identified in patients fulfilling the NIH clinical gnancies, including acute leukaemia (AL) and the rare disorder diagnostic criteria for neurofibromatosis type 1 (NF1) with no juvenile myelomonocytic leukaemia. NF1 mutation found.1–4 Their phenotype referred as NF1-like We previously reported the observation of an 11-month-old boy syndrome or Legius syndrome (OMIM 611431), consisted of with a SPRED1 constitutional mutation, who developed a multiple cafe´-au-lait macules, axillary freckling, mild neuroco- paediatric acute myeloblastic leukaemia (AML).6 Although rare, gnitive impairment and macrocephaly but without features inherited predispositions to myeloid leukaemia have uncovered a common in NF1 such as neurofibromas and iris Lisch nodules. critical role of hyperactive RAS MAPKs signalling in normal Current knowledge of natural history of Legius syndrome is based myeloid growth and leukemogenesis.7 SPRED1 and SPRED2 are on clinical manifestations of fewer than 150 individuals with a members of the evolutionarily conserved SPROUTY/SPRED family molecularly confirmed diagnosis.1–4 of membrane-associated negative regulators of growth factor- NF1 and Legius syndrome belong to the neuro-cardio-facio- induced ERK (extracellular-regulated kinase) activation.8 In mice, cutaneous syndromes that are caused by deregulating constitu- SPRED1 inhibits ERK activation by suppressing RAF1 kinase tional mutations of the RAS MAPKs (mitogen-activated protein phosphorylation. SPRED1 is highly expressed in haematopoietic kinases) signalling pathway.5 These RASopathies that include cells and negatively regulates haematopoiesis by suppressing Noonan syndrome, LEOPARD syndrome, cardio-facio-cutaneous stem cell factor and interleukin-3 (IL-3)-induced ERK activation.9 syndrome, Costello syndrome, NF1 and the Legius syndrome, Bone marrow (BM)-derived mast cells and eosinophils from 1UMR_S745 INSERM, Faculte´ des Sciences Pharmaceutiques et Biologiques, Universite´ Paris Descartes Sorbonne Paris Cite´, Paris, France; 2Service de Biochimie et Ge´ne´tique Mole´culaire, Hoˆpital Cochin, Assistance Publique-Hoˆpitaux de Paris, Paris, France; 3Service de Ge´ne´tique, C.H.U. de Poitiers, Poitiers, France; 4Service d’He´matologie-Oncologie, Hoˆpital A Trousseau, Assistance Publique-Hoˆpitaux de Paris, Paris, France; 5Universite´ Pierre et Marie Curie Paris 6, Paris, France; 6UMR938, Universite´ Paris 6, Pierre et Marie Curie, Paris, France; 7INSERM 1016, Institut Cochin, CNRS UMR8104, Universite´ Paris Descartes, Paris, France; 8Service d’He´matologie Biologique, Hoˆpital Trousseau, Assistance Publique- Hoˆpitaux de Paris, Paris, France; 9UMR967, CEA, Universite´ Paris 7, Fontenay aux Roses, France; 10Laboratoire d’He´matologie Biologique, C.H.U. de Poitiers, Poitiers, France; 11Service de Dermatologie, C.H.U. de Dijon et EA 4271, Universite´ de Bourgogne, France and 12Service de Pe´diatrie, C.H.U de Poitiers, Poitiers, France. Correspondence: Dr E Pasmant, UMR_S745 INSERM, Faculte´ des Sciences Pharmaceutiques et Biologiques, Universite´ Paris Descartes Sorbonne Paris Cite´, 75006, Paris, France or Dr P Ballerini, Service d’He´matologie Biologique, Hoˆ pital Trousseau, Assistance Publique-Hoˆ pitaux de Paris, 75012, Paris, France. E-mail: [email protected] or [email protected] 13These authors contributed equally to this work. Received 30 April 2013; revised 11 November 2013; accepted 3 December 2013; published online 27 January 2014 SPRED1 as a tumour suppressor downregulated in paediatric AML E Pasmant et al 632 Table 1. Clinical characteristics of the four patients with SPRED1 constitutional variants Patient Age Gender LS clinical Leukaemia BM cells karyotype SPRED1 Associated molecular Follow-up (years) diagnosis type variation lesions AML/3 9.9 F À M4 with 46, XX, t(6;9) c.124G4A DEK-CAN fusion FLT3- Alive, 4, 8 yearsa basophiles (p23;q34)[23]/ 46, XX [2] p.Val42Ile ITD excess B-ALL/1 10.8 M À B-ALL 53–54, XY, þ X[9], þ 5[7], c.1089A4G FLT3 hyperexpression Alive, 48 years þ 8[7], þ 12[3], þ 17[8], p.Ile363Met þ 18[8], þ 21[3], þ 21 Â 2[6] B-ALL/2 3.8 M À B-ALL 46, XY c.674C4T TEL-AML1 fusion Alive, 4 7 years B-ALL/3 11.2 F þ B-ALL with 46,XX[30]/45,XX, c.395dupA – Alive, 4 8 years osteolytic der(5),del(15) p.Asn133fs lesions (q11q25), À 20[3] Abbreviations: LS, Legius syndrome; M, male; F, female. Individuals AML/3, B-ALL/1 and B-ALL/2 had no typical cutaneous symptoms of Legius syndrome (including cafe´ -au-lait macules and axillary freckling) and no NF1 characteristic complications. Patient B-ALL/3 showed typical cafe´ -au-lait spots, and axillary freckling, and developed later learning disabilities. aThis child received allogenic BM treatment. Spred1–/– mice were more sensitive to IL-3 and IL-5, respectively, domains.4 Moreover, the two missense mutations both affect two than those from wild-type mice.9,10 Moreover, recent evidences highly conserved amino acids (Supplementary Figure 1). indicate that SPRED1 regulates neurofibromin RAS-GTPase activity The four heterozygous nucleotide variations were also found at by interacting with the NF1 gene product.11 With regards to these the time of remission. This finding demonstrated that these observations, SPRED1 seemed to be a good candidate gene for variations were constitutional rather than somatic. Recently the leukaemia predisposition or transformation. p.Val42Ile missense variation was described in two unrelated In this study, we performed SPRED1 mutation screening and individuals affected by Legius syndrome.4 However, whether the expression status in a large series of 230 paediatric acute two constitutional missense variations are disease causing lymphoblastic and myeloblastic leukaemias. We found four new mutations, or only rare neutral variants, remain to be established SPRED1 variations that were constitutional events. Among them, and their precise consequences should be determined at the one patient had Legius syndrome clinical features, which were not functional protein level. Patient B-ALL/3 with the frameshift SPRED1 diagnosed at the time of leukaemia onset. In this patient, the loss-of-function mutation (c.395dupA; p.Asn133fs), showed typical leukaemia blasts karyotype showed a loss of heterozygosity (LOH) Legius syndrome clinical features: cafe´-au-lait spots and axillary of the SPRED1 locus, confirming SPRED1 as a tumour suppressor. freckling. Clinical information of the four patients with SPRED1 Our observation confirmed that AL are complication of the Legius constitutional variations is summarized in Table 1. syndrome stressing the risk of developing leukaemia in constitu- tional carriers of SPRED1 mutations. Moreover, SPRED1 was significantly decreased at RNA and protein levels in the majority SPRED1 somatic LOH was found in leukaemia blasts in one patient of AML at diagnosis compared with normal BM or paired complete with a constitutional SPRED1 mutation remission (CR) BM samples. Interestingly, SPRED1 decreased At the time of diagnosis,