The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3–dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)–like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)–treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non– 5-FU–treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.

Human Ph + leukemias are caused by the BCR-ABL1 oncogene and include CML and B-ALL. CML has a triphasic clinical course, with chronic and accelerated phases followed by a terminal blast crisis phase resembling acute leukemia, in which myeloid or lymphoid blasts fail to differentiate. Together, lymphoid blast crisis of CML and Ph + B-ALL account for 20% of adult cases and 5% of pediatric cases of acute lymphoblastic leukemia. One-half of adult and 20% of pediatric BALLs express the p210 isoform of Bcr-Abl, and the remainder express the p190 isoform 1,2 . Whereas the Abl tyrosine kinase inhibitor imatinib mesylate induces a complete hematologic response in nearly all individuals with chronic phase CML 3 , imatinib is much less effective in treating CML blast crisis and Ph + B-ALL 2 owing to acquired resistance [4][5][6][7] . Drugs that target essential signaling molecules downstream of Bcr-Abl may help overcome or prevent imatinib resistance.Bcr-Abl activates multiple signaling pathways, including Ras, MAPK, STAT, JNK/SAPK, PI-3 kinase, NF-kB and c-Myc 8 . Furthermore, in myeloid cell lines Bcr-Abl activates the Src family kinases Lyn and Hck 9 . Multiple domains of Bcr-Abl interact with and activate Src kinases independently of Bcr-Abl kinase activity [10][11][12] , and studies with dominant-negative mutants and Src inhibitors suggest that Src kinases may contribute to the proliferation and survival of myeloid cell lines expressing Bcr-Abl in vitro [13][14][15] . Results in cell lines expressing Bcr-Abl often do not correlate with leukemogenesis in vivo 16 , emphasizing the need to evaluate signaling pathways in an animal model of leukemia. We developed accurate and quantitative mouse models of chronic phase CML 17 and Ph + B-ALL 17,18 . Here, we use these models to show that Src kinases are required for the induction of B-ALL by Bcr-Abl but are dispensable for induction of CMLlike myeloproliferative disease. RESULTS Bcr-Abl activates Lyn, Hck and Fgr in pre-B cellsBcr-Abl interacts with and activates the Src kinases Lyn and Hck in myeloid cells 9 . We investigated whether Bcr-Abl also activates Src kinases in pre-B lymphoid cells. We found Bcr-Abl protein and abundant tyrosine-phosphorylated cell proteins in BL-2 cells (isolated from a mouse with Bcr-Abl-induced B-ALL) and in ENU48515 cells (an N-ethyl-N-nitrosourea (ENU)-induced pre-B leukemia mouse line) transduced with BCR-ABL1 retrovirus, but not in vector-transduced cells (Fig. 1a). Of the eight Src family kinases expressed in hematopoetic cells (Blk, Fgr, Fyn, Hck, Lck, Lyn, c-Src and Yes), parental ENU48515 cells had moderate levels of constitutively activate Fyn and Lyn, and Bcr-Abl expression increased the activation of Lyn and also activated five other Src kinases (Blk, Fgr, Hck, Lck and c-Src), as indicated by increased tyrosine phosphorylation (Fig. 1b). Phosphorylation of Fyn was not increased with BcrAbl expression in ENU48515 cells, and Yes was not expressed. In primary leukemic cells isolated from peripheral blood of mice with

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.

It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibit its functions, leading to inactivation of its downstream signaling pathways and cure of the disease. Imatinib is highly effective at treating human Philadelphia chromosome-positive (Ph ؉ ) chronic myeloid leukemia (CML) in chronic phase but not Ph ؉ B cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABLactivated signaling pathways. This SRC pathway is essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Inhibition of both SRC and BCR-ABL kinase activities by dasatinib affords complete B-ALL remission. However, curing B-ALL and CML mice requires killing leukemic stem cells insensitive to both imatinib and dasatinib. Besides BCR-ABL and SRC kinases, stem cell pathways must be targeted for curative therapy of Ph ؉ leukemia.dasatinib ͉ imatinib ͉ SRC kinases

Targeting of cancer stem cells is believed to be essential for curative therapy of cancers, but supporting evidence is limited. Few selective target genes in cancer stem cells have been identified. Here we identify the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox5, BCR-ABL failed to induce CML in mice. This Alox5 deficiency caused impairment of the function of LSCs but not normal hematopoietic stem cells (HSCs) through affecting differentiation, cell division, and survival of long-term LSCs (LT-LSCs), consequently causing a depletion of LSCs and a failure of CML development. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly by affecting LT-LSCs, and prolonged survival. These results demonstrate that a specific target gene can be found in cancer stem cells and its inhibition can completely inhibit the function of these stem cells.

BackgroundA somatic activating mutation (V617F) in the JAK2 tyrosine kinase was recently discovered in the majority of patients with polycythemia vera (PV), and some with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis. However, the role of mutant JAK2 in disease pathogenesis is unclear.Methods and FindingsWe expressed murine JAK2 WT or V617F via retroviral bone marrow transduction/transplantation in the hematopoietic system of two different inbred mouse strains, Balb/c and C57Bl/6 (B6). In both strains, JAK2 V617F, but not JAK2 WT, induced non-fatal polycythemia characterized by increased hematocrit and hemoglobin, reticulocytosis, splenomegaly, low plasma erythropoietin (Epo), and Epo-independent erythroid colonies. JAK2 V617F also induced leukocytosis and neutrophilia that was much more prominent in Balb/c mice than in B6. Platelet counts were not affected in either strain despite expression of JAK2 V617F in megakaryocytes and markedly prolonged tail bleeding times. The polycythemia tended to resolve after several months, coincident with increased spleen and marrow fibrosis, but was resurrected by transplantation to secondary recipients. Using donor mice with mutations in Lyn, Hck, and Fgr, we demonstrated that the polycythemia was independent of Src kinases. Polycythemia and reticulocytosis responded to treatment with imatinib or a JAK2 inhibitor, but were unresponsive to the Src inhibitor dasatinib.ConclusionsThese findings demonstrate that JAK2 V617F induces Epo-independent expansion of the erythroid lineage in vivo. The fact that the central erythroid features of PV are recapitulated by expression of JAK2 V617F argues that it is the primary and direct cause of human PV. The lack of thrombocytosis suggests that additional events may be required for JAK2 V617F to cause ET, but qualitative platelet abnormalities induced by JAK2 V617F may contribute to the hemostatic complications of PV. Despite the role of Src kinases in Epo signaling, our studies predict that Src inhibitors will be ineffective for therapy of PV. However, we provide proof-of-principle that a JAK2 inhibitor should have therapeutic effects on the polycythemia, and perhaps myelofibrosis and hemostatic abnormalities, suffered by MPD patients carrying the JAK2 V617F mutation.

Philadelphia chromosome-positive (Ph þ ) chronic myeloid leukemia (CML) induced by the BCR-ABL oncogene is believed to be developed from leukemic stem cells (LSCs), and we have previously shown in mice that LSCs for CML express the same cell surface markers that are also expressed on normal hematopoietic stem cells (HSCs). Although the inhibition of BCR-ABL kinase activity by imatinib is highly effective in treating human Ph þ CML in chronic phase, it is difficult to achieve molecular remission of the disease, suggesting that LSCs remain in patients. In this study, we find that following imatinib treatment, LSCs not only remained but also accumulated increasingly in bone marrow of CML mice. This insensitivity of LSCs to imatinib was not because of the lack of BCR-ABL kinase inhibition by imatinib, and proliferating leukemic cells derived from LSCs were still sensitive to growth inhibition by imatinib. These results identify an LSC survival pathway that is not inhibited by imatinib. Furthermore, we show that b-catenin in the Wnt signaling pathway is essential for survival and self-renewal of LSCs, providing a new strategy for targeting these cells.

IntroductionThe human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. 1 The resulting chimeric BCR-ABL oncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL TKI, imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of chronicphase CML patients, 2 but is unable to completely eradicate BCR-ABL-expressing leukemic cells, 3,4 suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. [5][6][7][8][9][10][11] Resistance is predominantly due to emergence of kinase domain mutations. Three newly developed BCR-ABL kinase inhibitors-dasatinib, 12 AP23464, 13 and AMN107 14 -inhibit most of imatinib-resistant BCR-ABL mutants at biochemical and cellular levels, but are ineffective against the BCR-ABL-T315I mutant. 15,16 New approaches are needed to treat drug-resistant forms of CML as well as BCR-ABL-induced B-ALL, a leukemia that does not respond well to available TKIs. 15,16 Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins such as BCR-ABL, and affects the stability of these proteins. [17][18][19][20][21] When BCR-ABL contains resistance-conferring mutations, it becomes even more dependent on Hsp90 in vitro. 20 We therefore evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble inhibitor, 22 in drug-resistant animal models of leukemia induced by BCR-ABL-WT and T315I. Materials and methods Cell linesThe 32D myeloid cell line was grown in RPMI 1640 medium containing 10% FCS and 10% WEHI medium. The BaF/3 pre-B-cell line was grown in RPMI 1640 medium containing 10% FCS, 10% WEHI medium, and 50 M 2-mercaptoethanol. To generate the BCR-ABL-expressing 32D or BaF/3 line, the cells were transduced with the BCR-ABL-WT-or BCR-ABL-T315I-IRES-GFP-MSCV retrovirus, and the BCR-ABL-expressing cells were selected by GFP sorting by fluorescence-activated cell sorter (FACS). HistologyThe lungs from the placebo-or drug-treated mice were fixed in Bouin fixative (Fisher Scientific, Pittsburgh, PA) for 24 hours at room temperature, followed by an overnight rinse in water. Ten-m sections were stained with hematoxylin and eosin (H&E) and observed by a model DMRE compound microscope (Leica, Heidelberg, Germany). All sections were imaged with a 2.5 ϫ PH1 objective (NPLan, NA 0.25) and 10 ϫ PH1 objective (NPLan, NA 0.40). All images were imported into MetaMorph software (Molecular Devices, Downingtown, PA) as a series of tagged image files. All images were then constructed in Adobe Photoshop 6.0 (Adobe, San Jose, CA). Antibodies and Western blot analysisAntibodies against c-ABL, Hsp90, Hsp70, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein lysates were prepared by lysing cells...