Discovery of pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia.

Long-term survivors of glioblastoma multiforme (GBM) are at high risk of developing second primary neoplasms, including leukemia. For these patients, the use of classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate, is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. We aimed to develop agents useful for the treatment of patients with GBM and chronic myeloid leukemia (CML) using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization. Compounds 7 and 25, as planned, not only inhibited tubulin polymerization, but also inhibited the proliferation of both GMB and CML cells, including those expressing the T315I mutation, at nanomolar concentrations. In in vivo experiments in BALB/cnu/nu mice injected subcutaneously with U87MG cells, in vivo, 7 significantly inhibited GBM cancer cell proliferation, in vivo tumorigenesis, and tumor growth, tumorigenesis and angiogenesis. Compound 7 was found to block human topoisomerase II (hTopoII) selectively and completely, at a concentration of 100 µM.

3D-microenvironments initiate TCF4 expression rescuing nuclear ß-catenin activity in MCF-7 breast cancer cells.

Mechanical cues sensed by tumor cells in their microenvironment can influence important mechanisms including adhesion, invasion and proliferation. However, a common mechanosensitive protein and/or pathway can be regulated in different ways among diverse types of tumors. Of particular interest are human breast epithelial cancers, which markedly exhibit a heterogeneous pattern of nuclear ß-catenin localization, a protein known to be involved in both mechanotransduction and tumorigenesis. ß-catenin can be aberrantly accumulated in the nucleus wherein it binds to and activates lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors. At present, little is known about how mechanical cues are integrated into breast cancer cells harboring impaired mechanisms of ß-catenin's nuclear uptake and/or retention. This prompted us to investigate the influence of mechanical cues on MCF-7 human breast cancer cells which are known to fail in relocating ß-catenin into the nucleus due to very low baseline levels of LEF/TCFs. Exploiting three-dimensional (3D) microscaffolds realized by two-photon lithography, we show that surrounding MCF-7 cells have not only a nuclear pool of ß-catenin, but also rescue from their defective expression of TCF4 and boost invasiveness. Together with heightened amounts of vimentin, a ß-catenin/TCF-target gene regulator of proliferation and invasiveness, such 3D-elicited changes indicate an epithelial-to-mesenchymal phenotypic switch of MCF-7 cells. This is also consistent with an increased in situ MCF-7 cell proliferation that can be abrogated by blocking ß-catenin/TCF-transcription activity. Collectively, these data suggest that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of ß-catenin nuclear activity in MCF-7 cells. The employed methodology could, therefore, provide a mechanism-based rationale to dissect further aspects of mechanotranscription in breast cancerogenesis, somewhat independent of ß-catenin's nuclear accumulation. More importantly, by considering the heterogeneity of ß-catenin signaling pathway in breast cancer patients, these data may open alternative avenues for personalized disease management and prevention. STATEMENT OF SIGNIFICANCE: Mechanical cues play a critical role in cancer pathogenesis. Little is known about their influence in breast cancer cells harboring impaired mechanisms of ß-catenin's nuclear uptake and/or retention, involved in both mechanotransduction and tumorigenesis. We engineered 3D scaffold, by two-photon lithography, to study the influence of mechanical cues on MCF-7 cells which are known to fail in relocating ß-catenin into the nucleus. We found that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of ß-catenin nuclear activity that boost cell proliferation and invasiveness. Thus, let us suggest that our system could provide a mechanism-based rationale to further dissect key aspects of mechanotranscription in breast cancerogenesis and progression, somewhat independent of ß-catenin's nuclear accumulation.

Structure-activity relationship studies and in vitro and in vivo anticancer activity of novel 3-aroyl-1,4-diarylpyrroles against solid tumors and hematological malignancies.

Novel 3-aroyl-1,4-diarylpyrrole derivatives were synthesized to explore structure-activity relationships at the phenyls at positions 1 and 4 of the pyrrole. The presence of amino phenyl rings at positions 1 and 4 of the pyrrole ring were found to be a crucial requirement for potent antitumor activity. Several compounds strongly inhibited tubulin assembly through binding to the colchicine site. Compounds 42, 44, 48, 62 and 69 showed antitumor activity with low nanomolar IC50 values in several cancer cell lines. Compound 48 was generally more effective as an inhibitor of glioblastoma, colorectal and urinary bladder cancer cell lines; 69 consistently inhibited CML cell lines and demonstrated superiority in nilotinib and imatinib resistant LAMA84-R and KBM5-T315I cells. In animal models, compound 48 exhibited significant inhibition of the growth of T24 bladder carcinoma and ES-2 ovarian clear cell carcinoma tumors. Compounds 48 and 69 represent robust lead compounds for the design of new broad-spectrum anticancer agents active in different types of solid and hematological tumors.

Drug Design and Synthesis of First in Class PDZ1 Targeting NHERF1 Inhibitors as Anticancer Agents.

Targeted approaches aiming at modulating NHERF1 activity, rather than its overall expression, would be preferred to preserve the normal functions of this versatile protein. We focused our attention on the NHERF1/PDZ1 domain that governs its membrane recruitment/displacement through a transient phosphorylation switch. We herein report the design and synthesis of novel NHERF1 PDZ1 domain inhibitors. These compounds have potential therapeutic value when used in combination with antagonists of ß-catenin to augment apoptotic death of colorectal cancer cells refractory to currently available Wnt/ß-catenin-targeted agents.

ß-catenin knockdown promotes NHERF1-mediated survival of colorectal cancer cells: implications for a double-targeted therapy.

Nuclear activated ß-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/ß-catenin pathway mutations in APC/KRAS or ß-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic ß-catenin signaling negatively regulates the expression of NHERF1 (Na+/H+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear ß-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that ß-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon ß-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single ß-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/ß-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with ß-catenin knockdown alone. Collectively, our data unveil novel ß-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining ß-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/ß-catenin-targeted therapeutics.

3-Aroyl-1,4-diarylpyrroles Inhibit Chronic Myeloid Leukemia Cell Growth through an Interaction with Tubulin.

We designed 3-aroyl-1,4-diarylpyrrole (ARDAP) derivatives as potential anticancer agents having different substituents at the 1- or 4-phenyl ring. ARDAP compounds exhibited potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARDAP derivative 10 inhibited the proliferation of BCR/ABL-expressing KU812 and LAMA84 cells from chronic myeloid leukemia (CML) patients in blast crisis and of hematopoietic cells ectopically expressing the imatinib mesylate (IM)-sensitive KBM5-WT or its IM-resistant KBM5-T315I mutation. Compound 10 minimally affected the proliferation of normal blood cells, indicating that it may be a promising agent to overcome broad tyrosine kinase inhibitor resistance in relapsed/refractory CML patients. Compound 10 significantly decreased CML proliferation by inducing G2/M phase arrest and apoptosis via a mitochondria-dependent pathway. ARDAP 10 augmented the cytotoxic effects of IM in human CML cells. Compound 10 represents a robust lead compound to develop tubulin inhibitors with potential as novel treatments for CML.

Is Going for Cure in CML Targeting Aberrant Glycogen Synthase Kinase 3ß?

Chronic Myelogenous Leukemia (CML)-initiating cells (CICs) express the hybrid oncoprotein BCR-ABL at the highest levels compared to their differentiated progeny but fail to expand at the same rate as downstream leukemic myeloid cells. Moreover, the primitive stem cell clone that originates the indolent CML chronic phase (CP) remains almost invariant as the disease evolves to a fatal blast crisis (BC). Compared to their healthy counterpart, the most dormant BCR-ABL+ CICs show the tendency to remain in a somewhat unusual 'proliferative quiescence', i.e. a prolonged low-energy viable state that restrains the frequency of symmetrical (self-renewing) cell divisions while enforcing cell cycle entry and myeloid commitment under cytokine support. Over the past few years, we and others have proposed the nutrient-sensing protein serine/threonine kinase GSK3ß. (glycogen synthase kinase 3ß) as an attractive target to eradicate leukemia-initiating cells while sparing normal haematopoiesis. Beyond its natural negative effects on self-renewal, through the inhibitory phosphorylation of ß-Catenin (Wnt signalling) and c-MYC (Hedgehog signalling), hyperactive GSK3ß is reportedly crucial to link energy metabolism and nutrient availability to stem cell homeostasis processes. This review will integrate current evidence pertaining to the biological relevance of GSK3ß in normal and malignant haematopoiesis, with particular emphasis on its role(s) at the CML CP stage and BC transformation. Preclinical evidence earmarking the use of novel small-molecule inhibitors of GSK3ß as promsing anti-leukemia agents are also discussed.

Targeting of GSK3ß promotes imatinib-mediated apoptosis in quiescent CD34+ chronic myeloid leukemia progenitors, preserving normal stem cells.

The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of ß-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3ß (glycogen synthase kinase 3ß). Herein, we show that GSK3ß is constitutively Y(216) phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3ß activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3ß complex formation. Specifically, GSK3ß activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the ß-catenin, cyclinD1, C-EBPα, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib.

Mutation-independent anaplastic lymphoma kinase overexpression in poor prognosis neuroblastoma patients.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.

Gene expression profiling of bone marrow endothelial cells in patients with multiple myeloma.

PURPOSE: To determine a "gene/molecular fingerprint" of multiple myeloma endothelial cells and identify vascular mechanisms governing the malignant progression from quiescent monoclonal gammopathy of undetermined significance. EXPERIMENTAL DESIGN: Comparative gene expression profiling of multiple myeloma endothelial cells and monoclonal gammopathy of undetermined significance endothelial cells with the Affymetrix U133A Arrays was carried out in patients at diagnosis; expression and function of selective vascular markers was validated by real-time reverse transcriptase-PCR, Western blot, and small interfering RNA analyses. RESULTS: Twenty-two genes were found differentially expressed (14 down-regulated and eight up-regulated) at relatively high stringency in multiple myeloma endothelial cells compared with monoclonal gammopathy of undetermined significance endothelial cells. Functional annotation revealed a role of these genes in the regulation of extracellular matrix formation and bone remodeling, cell adhesion, chemotaxis, angiogenesis, resistance to apoptosis, and cell-cycle regulation. Validation was focused on six genes (DIRAS3, SERPINF1, SRPX, BNIP3, IER3, and SEPW1) not previously found to be functionally correlated to the overangiogenic phenotype of multiple myeloma endothelial cells in active disease. The small interfering RNA knockdown of BNIP3, IER3, and SEPW1 genes affected critical multiple myeloma endothelial cell functions correlated with the overangiogenic phenotype. CONCLUSIONS: The distinct endothelial cell gene expression profiles and vascular phenotypes detected in this study may influence remodeling of the bone marrow microenvironment in patients with active multiple myeloma. A better understanding of the linkage between plasma cells and endothelial cells in multiple myeloma could contribute to the molecular classification of the disease and thus pinpoint selective gene targets for more effective antiangiogenic treatments.

Epidermal growth factor receptor/beta-catenin/T-cell factor 4/matrix metalloproteinase 1: a new pathway for regulating keratinocyte invasiveness after UVA irradiation.

Previous studies have established that UV irradiation results in epidermal growth factor receptor (EGFR) activation in keratinocytes. However, the signaling pathways and cellular effects related to this process remain incompletely elucidated. Herein, we describe for the first time that UVA-mediated EGFR activation results in beta-catenin tyrosine phosphorylation at the Y654 residue responsible for the dissociation of E-cadherin/alpha-catenin/beta-catenin complexes. Moreover, UVA induces an EGFR-dependent, but Wnt-independent, beta-catenin relocalization from the membrane to the nucleus followed by its association with T-cell factor 4 (TCF4). This newly formed beta-catenin/TCF4 complex binds to a specific site on matrix metalloproteinase 1 (MMP1) promoter and governs MMP1 gene and protein expression, as well as cell migration in collagen and gelatin. Altogether, these results suggest that UVA stimulates keratinocyte invasiveness through two coordinated EGFR-dependent processes: loss of cell-to-cell contact due to beta-catenin/E-cadherin/alpha-catenin dissociation and increased cell migration through extracellular matrix component degradation due to beta-catenin/TCF4-dependent MMP1 regulation. These events may represent an important step in epidermis repair following UVA injury and their abnormal regulation could contribute to photoaging and photocarcinogenesis.

Validation of PDGFRbeta and c-Src tyrosine kinases as tumor/vessel targets in patients with multiple myeloma: preclinical efficacy of the novel, orally available inhibitor dasatinib.

Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/vessel growth associated with the disease progression. However, target specificity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFRbeta) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFRbeta kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangiogenic factors, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFRbeta/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFRbeta/c-Src TKs in MM, providing a framework for future clinical trials.

Zoledronic acid affects over-angiogenic phenotype of endothelial cells in patients with multiple myeloma.

Therapeutic doses of zoledronic acid markedly inhibit in vitro proliferation, chemotaxis, and capillarogenesis of bone marrow endothelial cells of patients with multiple myeloma. Zoledronic acid also induces a sizeable reduction of angiogenesis in the in vivo chorioallantoic membrane assay. These effects are partly sustained by gene and protein inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in an autocrine loop. Mevastatin, a specific inhibitor of the mevalonate pathway, reverts the zoledronic acid antiangiogenic effect, indicating that the drug halts this pathway. Our results provide evidence of a direct antiangiogenic activity of zoledronic acid on multiple myeloma patient-derived endothelial cells due to at least four different mechanisms identified either in vitro or in vivo. Tentatively, we suggest that the zoledronic acid antitumoral activity in multiple myeloma is also sustained by antiangiogenesis, which would partly account for its therapeutic efficacy in multiple myeloma.

Bcr-Abl stabilizes beta-catenin in chronic myeloid leukemia through its tyrosine phosphorylation.

Self-renewal of Bcr-Abl(+) chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated beta-catenin. Although beta-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of beta-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with beta-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate beta-catenin at Y86 and Y654 residues. This Y-phospho beta-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the beta-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated beta-catenin, which presents an increased binding affinity for the Axin/GSK3beta complex. Although Bcr-Abl does not affect GSK3beta autophosphorylation, it prevents, through its effect on beta-catenin Y phosphorylation, Axin/GSK3beta binding to beta-catenin and its subsequent S/T phosphorylation. Silencing of beta-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl(+) CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of beta-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+ neoplastic cells.

Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.

SKI-606 decreases growth and motility of colorectal cancer cells by preventing pp60(c-Src)-dependent tyrosine phosphorylation of beta-catenin and its nuclear signaling.

Inhibition of deregulated protein tyrosine kinases represents an attractive strategy for controlling cancer growth. However, target specificity is an essential aim of this strategy. In this report, pp60(c-Src) kinase and beta-catenin were found physically associated and constitutively activated on tyrosine residues in human colorectal cancer cells. The use of specific small-interfering RNAs (siRNA) validated pp60(c-Src) as the major kinase responsible for beta-catenin tyrosine phosphorylation in colorectal cancer. Src-dependent activation of beta-catenin was prevented by SKI-606, a novel Src family kinase inhibitor, which also abrogated beta-catenin nuclear function by impairing its binding to the TCF4 transcription factor and its trans-activating ability in colorectal cancer cells. These effects were seemingly specific, as cyclin D1, a crucial beta-catenin/TCF4 target gene, was also down-regulated by SKI-606 in a dose-dependent manner accounting, at least in part, for the reduced growth (IC50, 1.5-2.4 micromol/L) and clonogenic potential of colorectal cancer cells. Protein levels of beta-catenin remained substantially unchanged by SKI-606, which promoted instead a cytosolic/membranous retention of beta-catenin as judged by immunoblotting analysis of cytosolic/nuclear extracts and cell immunofluorescence staining. The SKI-606-mediated relocalization of beta-catenin increased its binding affinity to E-cadherin and adhesion of colorectal cancer cells, with ensuing reduced motility in a wound healing assay. Interestingly, the siRNA-driven knockdown of beta-catenin removed the effect of SKI-606 on cell-to-cell adhesion, which was associated with prolonged stability of E-cadherin protein in a pulse-chase experiment. Thus, our results show that SKI-606 operates a switch between the transcriptional and adhesive function of beta-catenin by inhibiting its pp60(c-Src)-dependent tyrosine phosphorylation; this could constitute a new therapeutic target in colorectal cancer.

Bcl-XL down-regulation suppresses the tumorigenic potential of NPM/ALK in vitro and in vivo.

Deregulated apoptosis is a common finding in tumorigenesis. The oncogenic tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) delivers a strong survival signal in anaplastic large cell lymphomas (ALCLs). Although NPM/ALK activates multiple antiapoptotic pathways, the biologic relevance and therapeutic potential of more downstream apoptotic effectors are mostly unknown. In this report, the NPM/ALK-mediated induction of Bcl-XL (but not of Bcl-2) was identified in human ALCL-derived cells. NPM/ALK kinase activity was required to promote Bcl-XL expression and its protective effect on mitochondrial homeostasis. Down-regulation of Bcl-XL significantly reduced the antiapoptotic potential of NPM/ALK in both transformed murine Ba/F3 pro-B cells and human ALCL-derived KARPAS-299 cells. To elucidate the role of Bcl-XL in vivo, Ba/F3-NPM/ALK+ cells expressing a doxycycline (Dox)-inducible Bcl-XL antisense transgene (pTet-ON) were injected into nude mice. Doxycycline administration prevented a fatal systemic disease in 15 of 15 intravenously injected mice and the appearance of subcutaneous tumor xenografts in 9 of 12 mice; in vivo down-regulation of Bcl-XL was also documented. Our results show a pivotal role for Bcl-XL in ALK-mediated oncogenicity; a single protein placed downstream of a known oncogene can be crucial for the survival of neoplastic cells both in vitro and in vivo. Bcl-XL deserves further investigation as a possible therapeutic target in ALK+ ALCLs.

Constitutive activation of Jak2 contributes to proliferation and resistance to apoptosis in NPM/ALK-transformed cells.

OBJECTIVE: The t(2;5) translocation results in a 80-kDa oncogenic fusion protein consisting of NPM and the kinase domain of the tyrosine kinase ALK and is present in over half the cases of anaplastic large cell lymphoma (ALCL). NPM/ALK exerts its transforming potential via activation of multiple signaling pathways promoting growth factor independence and protection from apoptosis. Jak/Stat signaling is aberrantly activated in several human hematopoietic malignancies. We investigated the role of Jak2 in the context of NPM/ALK-mediated oncogenesis. MATERIALS AND METHODS: Constitutive tyrosine phosphorylation of Jak2 was analyzed by Jak2 immunoprecipitation and subsequent anti-phosphotyrosine Western blotting. NPM/ALK-transformed cells were treated with the Jak2 inhibitor AG490 or transfected with wild-type or dominant-negative Jak2 expression constructs to measure 3[H]-thymidine incorporation. Apoptosis was assessed by flow cytometric analysis of annexin V-stained cells. The effect of Jak2 on Stat5-dependent transcriptional activity was measured by beta-casein promoter-dependent luciferase expression. RESULTS: Jak2 was found to be constitutively tyrosine phosphorylated in ALCL cells and in NPM/ALK-transformed hematopoietic cells. Also, NPM/ALK was present in immunoprecipitates of Jak2. Inhibition of Jak2 led to a reduction of NPM/ALK-mediated proliferation and induced apoptosis. Stat5-dependent transcriptional activity was inhibited by transfection of NPM/ALK-transformed cells with a dominant-negative Jak2 expression construct or treatment with AG490. CONCLUSION: Constitutive activation of Jak2 constitutes a pro-proliferative, anti-apoptotic signaling pathway in NPM/ALK-transformed hematopoietic cells.