Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an IntroductionIn the adult bone marrow (BM), the hematopoietic function is supported by the proliferation and differentiation of a finite number of transplantable hematopoietic stem cells and progenitors (HSCs/Ps) that have self-renewal and multipotential differentiation capacity. Endosteal and neighboring vascular/perivascular niches have been proposed as anatomic sites that control HSC/P activity, providing signals that regulate the stem cell pool size, survival, and migration. [1][2][3][4] The definition of the cell components of the BM hematopoietic microenvironment (HM) is an area of debate. Endothelial cells, 2 mesenchymal stem cells (MSCs), 5 and osteoblasts and osteogenic progenitors (OB/Ps) 1,4,6 represent major cell components of the BM HM.A poorly studied mechanism of intercellular communication (IC) in the HM is mediated by gap junction (GJ) channels. GJ channels are formed by a large family of proteins called connexins (Cxs). Each neighboring cell contributes cell-to-cell channels with 1 hemichannel that dock head-to-head. GJ channels have the ability to transfer ions and low-molecular weight secondary messengers from one cell to another depending on concentration gradients and Cx-dependent selective permeability. 7 Connexin-43 (Cx43), one member of this protein family, is highly expressed by adult BM stromal cells, 8 osteoblasts, 9 endothelial cells, 2 and MSCs, 5 and is also expressed by HSCs. 10 The presence of a functional Cx43-dependence of GJIC between osteoblasts and stromal cells, as well as between stromal cells and HSCs, has been confirmed in vitro. 11 After HSC transplantation, HSCs must first home to the BM, and then localize and anchor in suitable microenvironments within the BM, a process known as lodgment. 12 This process involves the migration of HSC/Ps through different layers of cell components in the HM, including endothelial cells and mesenchymal-origin cells, which act as extrinsic HSC/P migration regulators, 13 and is directed by chemokine gradients. Myeloablation through irradiation has been postulated to modify the functional ability of the BM HM to allow HSC homing and retention through clearance of HSC niches. 14 The effect of irradiation on the BM HM is not well known, but diverse studies have shown that it is not innocuous 15 and includes the up-regulation of expression and secretion of Cxcl12 16 and the up-regulation of the expression of Cx43 in the BM. 17 It has been proposed that Cx43 hemichannels are important in reinforcing cell-to-cell migration and that Cx43-dependent GJ are needed in the process of neuronal migration during neocortex form...

Despite the introduction of tyrosine kinase inhibitor therapy, the prognosis for p190-BCR-ABL+ acute lymphoblastic leukemia remains poor. In the present study, we present the cellular and molecular roles of the Rho GTPase guanine nucleotide exchange factor Vav in lymphoid leukemogenesis and explore the roles of Vav proteins in BCR-ABL–dependent signaling. We show that genetic deficiency of the guanine nucleotide exchange factor Vav3 delays leukemogenesis by p190-BCR-ABL and phenocopies the effect of Rac2 deficiency, a downstream effector of Vav3. Compensatory up-regulation of expression and activation of Vav3 in Vav1/Vav2–deficient B-cell progenitors increases the transformation ability of p190-BCR-ABL. Vav3 deficiency induces apoptosis of murine and human leukemic lymphoid progenitors, decreases the activation of Rho GTPase family members and p21-activated kinase, and is associated with increased Bad phosphorylation and up-regulation of Bax, Bak, and Bik. Finally, Vav3 activation only partly depends on ABL TK activity, and Vav3 deficiency collaborates with tyrosine kinase inhibitors to inhibit CrkL activation and impair leukemogenesis in vitro and in vivo. We conclude that Vav3 represents a novel specific molecular leukemic effector for multitarget therapy in p190-BCR-ABL–expressng acute lymphoblastic leukemia.

Chronic myelogenous leukemia (CML) is

449 The Philadelphia chromosome-positive (Ph+) hematologic malignancies are characterized by the reciprocal translocation between the BCR serine/threonine kinase and the ABL tyrosine kinase genes. Fifty percent of adult B-cell Ph+ acute lymphoblastic leukemia (B-ALL) and most cases of pediatric Ph+ B-ALL express the p190 BCR-ABL isoform, which causes abnormal expansion of lymphoid progenitors and a poor prognosis despite the administration of tyrosine kinase inhibitors. A major signaling pathway activated by BCR-ABL is the Rac/Rho GTPase signaling pathway. We had previously demonstrated that p210-BCR-ABL activates Rac GTPases and the deficiency of Rac2, or even more, the deficiency of both Rac1 and Rac2, impairs myeloid leukemogenesis induced by p210 BCR-ABL in vitro and in vivo (Thomas EK et al., Cancer Cell 2007). The p190-BCR-ABL isoform also activates Rac GTPases and the deficiency of Rac2 induces resistance to develop proB-ALL (Sanchez-Aguilera et al., ASH 2008). Since p190-BCR-ABL is devoid of guanine nucleotide exchange factor (GEF) activity through the absence of the BCR Dbl-homology (DH) domain, it probably requires the recruitment of intermediate GEFs to activate Rac GTPases. The Vav family of GEF has been shown to bind the SH3/SH2 domains of ABL and become activated. Three isoforms of Vav (Vav1, Vav2 and Vav3) are expressed in hematopoietic cells. Several studies have indicated that Vav3 has a role distinct from Vav1 or Vav2. Here, we hypothesized that the hematopoietic specific Rho GTPase, Rac2, may mediate key signals in the transformation of B-cell progenitors upon Vav-mediated activation. We have found that both Vav1 and Vav3 are overexpressed and hyperphosphorylated in p190 BCR-ABL expressing human B-ALL cells as well as in BCR-ABL transduced murine proB cells. While the combined deficiency of Vav1 and Vav2 does not impair leukemogenesis in vivo, Vav3 deficiency reverses p190-BCR-ABL induced transformation phenotypes. Specifically, Vav3 deficiency in p190-BCR-ABL expressing proB-ALL cells associated with a significant reduction of Rho and Rac activation (75% and 56%), apoptosis induction (4-fold) and impaired ex-vivo expansion (33% reduction). In addition, p190-BCR-ABL-induced adhesion-to-fibronectin defect and hypermigratory response to CXCL12 were corrected by deletion of Vav3 (2.1-fold increase and 60% decrease, respectively), suggesting that Vav3 deficiency reverses the transformation-associated phenotype associated to BCR-ABL expression. Collectively, our work identifies a critical role of Rac2 and its activator Vav3 for p190 BCR-ABL induced B-cell progenitor leukemic transformation and may define a novel therapeutic target for this poor prognosis disease. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

3253 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and is characterized by myeloproliferation in the bone marrow (BM) and egress of leukemic stem cells and progenitors (LSC/P) to extramedullary sites. Persistence of BCR-ABL+ HSCs in patients under imatinib suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the LSC/P population. Rac GTPases represent integrative molecular switches for p210-BCR-ABL-induced HSC transformation and combined pharmacological and genetic attenuation of Rac GTPases significantly prolong survival in vivo, as reported in a retroviral transduction/transplantation model (Thomas EK & Cancelas JA et al, Cancer Cell 2008). Here, we analyzed the role of Rac2 GTPase in the leukemic maintenance and in the interaction of LSC/P with the leukemic microenvironment in vivo. We used a stem cell leukemia (Scl) promoter-driven, tet-off, Scl-tTA x TRE-BCR-ABL (Scl/p210-BCR-ABL) binary transgenic mouse model (Koschmieder S et al., Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment, allowing the study of the intrinsic molecular changes in LSC/P during leukemogenesis. In these mice, Scl-driven expression of BCR-ABL is active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+/Sca-1-; LK), and CML development is associated with the activation of downstream signaling effectors CrkL, p38-MAPK and JNK. Additionally, Scl/p210-BCR-ABL mice had increased cycling of LSK cells and expansion of circulating and splenic, but not BM, LSC/P, suggesting egress of LSC/Ps from the marrow. These mice share all the characteristics of HSC/P transformation in CML, including increased HSC/P proliferation and survival, severely reduced adhesion to fibronectin, increased migration towards CXCL12, increased cell surface expression of CD44 and decreased expression of L-selectin. Myeloproliferative disease (MPD) in these mice is transplantable into recipient mice, and CML splenocytes have a 10-fold increase in homing to the spleen than towards BM (P<0.05). Leukemic splenocytes are also enriched in endosteal lodging progenitors, compared to the BM-derived progenitors (1.9-fold, P≤0.05). In order to determine the contribution of Rac2 GTPase in the transformation phenotype of leukemic stem cells and progenitors, Scl/p210 mice were intercrossed with Rac2-/- mice. Interestingly loss of Rac2 GTPase alone significantly prolongs survival of the leukemic mice (P≤0.001). Prolonged survival, as observed in Scl/p210 x Rac2-/-, is associated with significantly reduced proliferation of leukemic LK (3-fold, P<0.05) and LSK (6-fold P<0.005) cells, both in BM as well as in spleen, in vivo. Scl/p210 x Rac2-/- mice are also characterized by increased apoptosis (1.7-fold) and lower frequency of LSK cells (2-fold) compared to the Scl/p210 mice in vivo. However, deletion of Rac2 does not significantly reverse the adhesion and migration transformation phenotype of LSC/P. In summary, Rac2 deficiency induces a significant survival of CML mice in a HSC-initiated model of disease through decrease proliferation and survival but does not reverse the transformation phenotype affecting adhesion and migration. This murine model may represent an adequate in vivo system to dissect out the specific signaling pathways involved in p210-BCR-ABL-induced stem cell transformation. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.