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Mutational Analysis of the DOK2 Haploinsufficient Tumor Suppressor

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Mutational Analysis of the DOK2 Haploinsufficient Tumor Suppressor Letters to the Editor 500 in MF. Second, the salutary effect of ruxolitinib therapy in alleviating 2 Tefferi A, Cervantes F, Mesa R, Passamonti F, Verstovsek S, Vannucchi AM et al. constitutional symptoms and reducing spleen size in MF is Revised response criteria for myelofibrosis: International Working Group- antagonized by treatment-related cytopenias and that of mome- Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and Eur- lotinib by treatment-emergent peripheral neuropathy. Furthermore, opean LeukemiaNet (ELN) consensus report. Blood 2013; 122: 1395–1398. fi 3 Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF et al. A double-blind, our long-term experience suggests that the bene t from both fi 366 drugs might not be long-lived. These observations highlight the placebo-controlled trial of ruxolitinib for myelo brosis. NEnglJMed2012; : 799–807. need for new drugs with alternative mechanism of action. 4 Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. ACKNOWLEDGEMENTS N Engl J Med 2012; 366:787–798. 5 Pardanani A, Laborde RR, Lasho TL, Finke C, Begna K, Al-Kali A et al. Safety and Drugs and support for the conduct of the clinical trials were provided by Incyte efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis. Leukemia 2013; 27: Corporation, Wilmington, DE, USA (ruxolitinib) and YM BioSciences, Inc., Mississauga, 1322–1327. Canada (momelotinib). 6 Pardanani A, Gotlib JR, Jamieson C, Cortes JE, Talpaz M, Stone RM et al. Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol 2011; 29:789–796. RA Abdelrahman, KH Begna, A Al-Kali, WJ Hogan, MR Litzow 7 Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J, Thomas DA and A Tefferi et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofi- Division of Hematology and Department of Medicine, Mayo Clinic, brosis. N Engl J Med 2010; 363: 1117–1127. Rochester, MN, USA 8 Tefferi A, Litzow MR, Pardanani A. Long-term outcome of treatment with rux- E-mail: [email protected] olitinib in myelofibrosis. N Engl J Med 2011; 365: 1455–1457. 9 Pardanani A, Gotlib J, Gupta V, Roberts AW, Wadleigh M, Sirhan S et al. Update on the long-term efficacy and safety of momelotinib, a JAK1 and JAK2 inhibitor, for REFERENCES the treatment of myelofibrosis. Blood 2013; 122: 108. 1 Tefferi A, Barosi G, Mesa RA, Cervantes F, Deeg HJ, Reilly JT et al. International 10 Gangat N, Caramazza D, Vaidya R, George G, Begna K, Schwager S et al. DIPSS Working Group (IWG) consensus criteria for treatment response in myelofibrosis plus: a refined Dynamic International Prognostic Scoring System for primary with myeloid metaplasia, for the IWG for Myelofibrosis Research and Treatment myelofibrosis that incorporates prognostic information from karyotype, platelet (IWG-MRT). Blood 2006; 108: 1497–1503. count, and transfusion status. J Clin Oncol 2011; 29:392–397. Mutational analysis of the DOK2 haploinsufficient tumor suppressor gene in chronic myelomonocytic leukemia (CMML) Leukemia (2015) 29, 500–502; doi:10.1038/leu.2014.288 Under informed consent, we analyzed the gene mutations in the bone marrow (BM) samples from 30 MD-CMML and 36 MP- CMML patients (Supplementary Table S1). Expectedly, mutations Downstream of tyrosine kinase (DOK) proteins are substrates of previously reported in CMML patients (such as NRAS, CBL, protein tyrosine kinases, acting as negative regulators of cell PTPN11, FLT3, JAK2 and NF1 genes)7 were present in our MP- signaling pathways.1 Loss of DOK2 gene expression has been CMML cohort. We analyzed somatic mutations in DOK1 and detected in human lung adenocarcinomas, and mice with Dok2 DOK2 genes. Genomic DNA from the BM cells was amplified with haploinsufficiency develop lung cancers.2 Mice lacking both six primer pairs covering the entire coding region (exons 1–5) of fi Dok1 and Dok2 genes (the two first described Dok gene family each DOK gene (Supplementary Material). We identi ed point members) present a myeloproliferative chronic myelogenous mutations in the two DOK genes. For DOK1, two variants were 3,4 found; L60Q in exon 1 coding for a functional protein–lipid leukemia-like syndrome. Moreover, genetic ablation of Dok 10 genes in a BCR/ABL transgenic background accelerates the interaction domain, a pleckstrin homology domain, and D263E in exon 5. For DOK2, four variants (2 × R201H, L238P and R215H) apparition of the blastic crisis and leukemia induced by the BCR- were found in 3/66 CMML and 1/2 unclassified myeloprolifera- ABL fusion oncoprotein,3,4 DOK1 and DOK2 adaptor proteins tive myelodysplastic neoplasm. These DOK2 point mutations are attenuate RAS/ERK- and PI3K/AKT-dependent signaling path- 3–5 locatedinexon4andthe5'endofexon5thatcodeforthe ways involved in myeloid cell proliferation. On the basis of 11 2–4 2,6 phosphotyrosine binding (PTB) domain of the DOK protein. these studies, animal models and data from solid tumors, Sorted CD3+ lymphocytes of the peripheral blood from DOK DOK genes are now considered as tumor suppressors. However, variant patients were only available for the MP-CMML patient the mutation status of DOK1 and DOK2 genes in patients with with DOK2 L238P mutation. The DOK2 L238P mutation was chronic myeloproliferative neoplasm (MPN) remains to be present in the myeloid cells but not in the lymphoid cells defined. 7 (Figure 1a). Mutations in cell signaling genes have been reported in MPNs. A three-dimensional structure model revealed that the L238P Chronic myelomonocytic leukemia (CMML) belongs to the MPN substitution would alter the structure of the DOK2 PTB domain, 8 class. Upon white blood cell count CMML has been subdivided in resulting in a loss of stable binding to phosphotyrosyl peptides myelodysplatic (MD-CMML) and myeloproliferative (MP-CMML) (Figure 1b). subtypes. These two subtypes are associated with different gene DOK2 binds via its PTB domain to tyrosine phosphorylated expression profiles.9 DOK1 protein.12 Two arginine residues in positions 200 and 201 Accepted article preview online 25 September 2014; advance online publication, 21 October 2014 Leukemia (2015) 491–514 © 2015 Macmillan Publishers Limited Letters to the Editor 501 DOK2 c.713 T>C: Leu238Pro Dok-2 (412aa) PH Y PTB Y Y Y YY Y * L238P Whole Bone Marrow L238P CD3+ T cells pDok2EGFP Dok2 Dok2 Dok2 Dok-1 IP Mock WT RR L238P Mock wt RR L238P --++-++-pV treatment 80kDa GFP WB 80kDa GFP WB 58kDa 58kDa Dok-1 WB β 46kDa -Tubulin WB 46kDa IgH 46kDa p-ERK1/2 WB WCL 80kDa GFP WB MEFs WT MEFs Dok DKO ) 50 pMIG 4 25 *** pMIG Dok2 WT pMIG Dok2 RR 40 20 *** pMIG Dok2 L238P *** 30 15 *** 20 10 10 5 *** *** * 0 Absolute cell number (x10 0 123456 123456 Days Days Figure 1. (a) Identification of an acquired and heterozygous DOK2 L238P point mutation in an MP-CMML patient. Sequencing profile of DOK2 MP-CMML patient (HD-1236). DNA samples were extracted from whole BM or sorted CD3+ T cells of the peripheral blood. The Human Genome Variation Society nomenclature of the identified DOK2 gene variation is c.713T4C (p.Leu238Pro) (Supplementary Table S2). (b) DOK2 L238P mutation alters the three-dimensional (3D) structure of the DOK2 PTB domain. Top: linear structure of the DOK2 molecule, containing an N-terminal pleckstrin homology (PH) domain, a central PTB domain and several phosphorylable tyrosine (Y) residues in the C-terminal part. The L238P point mutation (red asterisk) is located in the PTB functional domain. Bottom: 3D structural model of the DOK2 PTB domain (green ribbon) interacting with DOK1 EMLENLpY phospho-peptide (red) complex. The L238P single mutation (gray) would affect the 3D conformation of the protein, bending the helix α2 (represented by light green arrows that illustrate the consequences of such mutation) thus preventing the clamp between the C-terminal part of the protein and the phospho-peptide. (c) DOK2 L238P mutation induces a loss of DOK2 heterodimerization with the DOK1 protein. KG-1 myeloid cells were transfected by nucleofection with plasmids coding for GFP-tagged wild- type (WT) DOK2, a control DOK2 mutant with a loss-of-function in the PTB domain (RR200-201AA, RR), the L238P point mutant (L238P) or GFP alone (mock). KG-1 cells were treated with sodium pervanadate (pV) at 50 μM for 5 min at 37 °C. Cells were lysed and the lysates were immunoprecipitated using an anti-Dok1 antibody followed by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and anti-GFP western blot (WB) allowing the identification of DOK2 GFP fusion proteins with the endogenous DOK1 molecule. DOK1 immunoprecipitates were controlled by DOK1 WB. In parallel, whole-cell lysates (WCLs) were separated by SDS-PAGE and blotted for GFP expression. Molecular weight markers were reported in the left side of the blots. This panel shows representative blots of two independent experiments. (d) The L238P mutant DOK2 molecule is unable to reduce pervanadate-induced ERK-1/2 phosphorylation in KG-1 myeloid cells. KG-1 myeloid cells were transfected by nucleofection with plasmids encoding for GFP-tagged WT DOK2, a control DOK2 mutant with a loss-of- function in the PTB domain (RR200-201AA, RR), the L238P point mutant (L238P) or GFP alone (mock). KG-1 cells were treated or not with pV at 1 μM for 5 min at 37 °C. WCLs were separated by SDS-PAGE and subsequently immunoblotted for phospho-ERK-1/2 (p-ERK1/2 WB). The blots were reprobed for β-tubulin expression as a loading control and GFP expression to detect the presence of GFP-DOK2 fusion proteins. Molecular weight markers were reported in the left side of the blots. This panel shows representative blots of two independent experiments.
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