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Vav3 Collaborates with P190-BCR-ABL in Lymphoid Progenitor Leukemogenesis, Proliferation, and Survival

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Vav3 Collaborates with P190-BCR-ABL in Lymphoid Progenitor Leukemogenesis, Proliferation, and Survival View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Dadun, University of Navarra LYMPHOID NEOPLASIA Vav3 collaborates with p190-BCR-ABL in lymphoid progenitor leukemogenesis, proliferation, and survival Kyung Hee Chang,1,2 Abel Sanchez-Aguilera,3 Shuhong Shen,4 Amitava Sengupta,1 Malav N. Madhu,1 Ashley M. Ficker,1 Susan K. Dunn,2 Ashley M. Kuenzi,1 Jorden L. Arnett,1 Rebecca A. Santho,1 Xabier Agirre,5 John P. Perentesis,6 Michael W. Deininger,7 Yi Zheng,1 Xose R. Bustelo,8 David A. Williams,3 and Jose A. Cancelas1,2 1Division of Experimental Hematology, Cincinnati Children’s Research Foundation, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Hoxworth Blood Center, University of Cincinnati College of Medicine, Cincinnati, OH; 3Division of Pediatric Hematology/Oncology, Children’s Hospital Boston, Dana-Farber Cancer Institute, and Harvard Stem Cell Institute, Harvard Medical School, Boston, MA; 4Department of Hematology/Oncology, Pediatric Translational Medicine Institute, Shanghai Children’s Medical Center, Shanghai Jiaotong University Medical School, Shanghai, China; 5Hematology Department and Area of Cell Therapy, Foundation for Applied Medical Research, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain; 6Division of Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 7Division of Hematology and Hematologic Malignancies, University of Utah Huntsman Cancer Institute, Salt Lake City, UT; and 8Centro de Investigacio´n del Ca´ncer, University of Salamanca, Campus Unamuno, Salamanca, Spain Despite the introduction of tyrosine ki- BCR-ABL and phenocopies the effect of and up-regulation of Bax, Bak, and Bik. nase inhibitor therapy, the prognosis for Rac2 deficiency, a downstream effector Finally, Vav3 activation only partly de- -p190-BCR-ABL؉ acute lymphoblastic leu- of Vav3. Compensatory up-regulation of pends on ABL TK activity, and Vav3 defi kemia remains poor. In the present study, expression and activation of Vav3 in Vav1/ ciency collaborates with tyrosine kinase we present the cellular and molecular Vav2–deficient B-cell progenitors in- inhibitors to inhibit CrkL activation and roles of the Rho GTPase guanine nucleo- creases the transformation ability of p190- impair leukemogenesis in vitro and tide exchange factor Vav in lymphoid BCR-ABL. Vav3 deficiency induces in vivo. We conclude that Vav3 represents leukemogenesis and explore the roles of apoptosis of murine and human leukemic a novel specific molecular leukemic effec- Vav proteins in BCR-ABL–dependent sig- lymphoid progenitors, decreases the acti- tor for multitarget therapy in p190-BCR- naling. We show that genetic deficiency vation of Rho GTPase family members ABL–expressng acute lymphoblastic leu- of the guanine nucleotide exchange fac- and p21-activated kinase, and is associ- kemia. (Blood. 2012;120(4):800-811) tor Vav3 delays leukemogenesis by p190- ated with increased Bad phosphorylation Introduction Philadelphia chromosome–positive (Phϩ) hematologic malignan- accumulate additional genetic mutations that result in a prolifera- cies arise from the t(9,22) (q34;q11.2) mutation, which encodes the tive advantage and differentiation arrest.6 Understanding the down- constitutively active tyrosine kinase oncofusion protein BCR-ABL. stream signaling cascades activated by BCR-ABL may lead to the BCR-ABL is both necessary and sufficient to induce leukemia.1 development of more effective therapeutic strategies that aim to Two types of BCR-ABL fusion proteins, associated with different prevent the development and/or selection of TKI-resistant clones. break points in the BCR gene, have been identified in patients Expression of p210-BCR-ABL activates the Rho-family with Phϩ B-cell acute lymphoblastic leukemia (Phϩ B-ALL). A GTPases Rac, RhoA, and Cdc42,7 possibly through the double 190-kDa fusion protein, referred to as p190-BCR-ABL, is present homology (DH) domain of guanine exchange factors (GEFs).8 We in 60%-80% of Phϩ B-ALL cases. Leukemia induced by this demonstrated previously that the absence of Rac proteins, specifi- BCR-ABL fusion protein arises from a transformed B-cell progeni- cally Rac2 or the combination of Rac1 and Rac2, impairs myeloid tor.2 A BCR-ABL isoform of 210 kDa, known as p210-BCR-ABL, leukemogenesis induced by p210-BCR-ABL expression in the is commonly expressed in patients with chronic myelogenous hematopoietic stem and progenitor cell compartment.9,10 Activation leukemia (CML) and in a minority of patients with Phϩ B-ALL. of Rac GTPases, particularly Rac2, has been shown to regulate The transforming effect of BCR-ABL is dependent on the tyrosine reactive oxygen species production by NADPH oxidase com- kinase (TK) activity of the fusion protein that leads to autophosphor- plexes11 and possibly to be responsible for DNA damage and ylation, recruitment of adaptor proteins, and subsequent activation genetic instability in BCR-ABL leukemias.12 Expression of p190- of downstream signaling. The TK inhibitor (TKI) imatinib and the BCR-ABL also activates Rac GTPases7 despite a lack of the DH second-generation TKIs dasatinib and nilotinib have been used as domain, suggesting that the activation of Rho-family GTPases by frontline treatment for CML and Phϩ B-ALL patients.3 However, p190-BCR-ABL must depend on the expression and activation of relapse is common in Phϩ B-ALL despite high rates of complete alternative GEFs. response with initial therapy,4,5 probably because of survival of Vav proteins are GEFs for Rho-family GTPase members.13 The leukemic progenitors. These BCR-ABLϩ progenitors appear to mammalian Vav family is made up of 3 members: Vav1, Vav2, and Submitted June 21, 2011; accepted May 29, 2012. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, June 12, 2012; DOI 10.1182/blood-2011-06-361709. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. The online version of this article contains a data supplement. © 2012 by The American Society of Hematology 800 BLOOD, 26 JULY 2012 ⅐ VOLUME 120, NUMBER 4 BLOOD, 26 JULY 2012 ⅐ VOLUME 120, NUMBER 4 Vav3 IN Phϩ LYMPHOBLASTIC LEUKEMIA 801 Vav3. Despite common functional domains and similar mechanism recombinant fragment of fibronectin, CH296 (Takara Bio) for 16 hours at of phosphorylation-dependent activation,14 the sequence homology 37°C. For the Vav3 rescue experiment, lentiviral particles encoding either between the 3 Vav isoforms is only approximately 65%. In WT Vav3 (full-length) cloned in a pCDH1-MCS1-EF1-copGFP vector addition, Vav1 expression is restricted to hematopoietic cells, (System Biosciences), or the empty vector, were used for stable expression in p190-BCR-ABLϩ B-cell lymphoid progenitors. Retroviral transduction whereas Vav2 and Vav3 are expressed broadly in multiple tissues.15 of human UCB CD34ϩ cells was performed under conditions similar to Vav Overexpression studies and various gene knockout mice have those used for murine cell transduction, but using recombinant human SCF revealed both unique and redundant roles of the 3 Vav family instead. Lentiviral transduction of UCB CD34ϩ cells was performed using 16 members in lymphoid cells. The phosphorylation of Vav proteins 2 rounds of infection with Vav3 shRNA-containing pLKO.1-puro-CMV- on specific tyrosine residues leads to conformational changes tGFP vectors (Sigma-Aldrich) at a multiplicity of infection of 20. required for binding to GTPase effectors.14 Vav1 has been shown to exist as a complex with both p190- and p210-BCR-ABL,17 with Transplantation of transduced leukemic cells uncertain significance. Whether other Vav proteins complex BCR- LDBM cells from WT or gene-targeted mice lacking Vav or Rac were ABL is not known. isolated and transduced with either a retroviral bicistronic vector encoding In the present study, we explored the upstream mechanism of p190-BCR-ABL with enhanced green fluorescent protein (EGFP) as a p190-BCR-ABL–dependent Rac activation through the Vav GEF reporter or an empty vector solely expressing the reporter protein (Mock). family members. We show that, although both Vav1 and Vav3 are Transduced LDBM cells (1-3 ϫ 106) were intravenously transplanted into hyperactivated in primary human and murine p190-BCR-ABLϩ lethally (7 ϩ 4.75 Gy) irradiated C57Bl/6 or C57Bl/10 mice. The multiplic- B-ALL, Vav3-deficient leukemogenesis induced by p190-BCR- ity of infection was kept low to allow 1 single copy of BCR-ABL per ABL is delayed. The proliferation and survival of B-cell progeni- transduced cell. Transduction efficiency of C57Bl/6 LDBM cells was not tors is impaired by genetic loss of Vav3 and can be reverted by its significantly different between groups (3.3% Ϯ 1.8%, 2.3% Ϯ 1.1%, and Ϯ Ϫ/Ϫ Ϫ/Ϫ reintroduction, and compensatory Vav3 up-regulation induced by 5.1% 2.3% for WT, Rac2 , and Rac3 BM cells, respectively). Transduction efficiencies of C57Bl/10 LDBM cells were not significantly genetic combined deficiency of Vav1 and Vav2 translates into different among the groups (10.66% Ϯ 2.12%, 10.86% Ϯ 1.63%, and increased survival and expansion ex vivo. Vav3 deficiency reduces 11.5% Ϯ 1.2% for WT, Vav3Ϫ/Ϫ, and Vav1Ϫ/Ϫ;Vav2Ϫ/Ϫ LDBM cells, p190-BCR-ABL–induced Rac GTPase activation and decreases respectively). Secondary recipients received the same irradiation dose and a ϩ proliferation and Vav3-deficient p190-BCR-ABL murine and cell dose of 4 ϫ 105 sorted EGFPϩ B220ϩcKitϩ BM cells along with human B-cell progenitors have enhanced apoptosis associated with 3 ϫ 106 normal, congenic C57Bl/10 BM cells. Peripheral blood was augmented expression of Bad and Bik and the downstream retro-orbitally collected on day 21 and on subsequent days after transplanta- effectors Bax and Bak. Our results demonstrate that Vav3 is a tion for complete blood counts and leukocyte differentials to check the critical GEF in p190-BCR-ABL–mediated activation of Rac engraftment of donor cells.
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