Survivin silencing sensitizes imatinib-resistant CML cells to the cytotoxic effect of hydroxyurea

13119 Background: Imatinib Mesylate (IM), a semi-specific inhibitor of the BCR-ABL tyrosine kinase, is currently the treatment of choice for Chronic Myeloid Leukemia (CML). However, about one third of CML patients treated with IM develop resistance to the drug because of reactivation of BCR-ABL kinase activity. This phenomenon is usually ascribed to the amplification of the BCR-ABL gene or to the selection of leukemic clones harboring point mutations that abrogate IM binding. To identify novel anti-apoptotic signaling pathways employed by BCR-ABL and devise strategies capable of killing IM-resistant CML cells, we investigated the interplay between BCR-ABL and the Inhibitor of Apoptosis Protein Survivin. Methods: Murine hematopoietic cells (32D) transduced with p210 BCR-ABL and human cell lines either positive (K562, KCL22, KYO1 and LAMA84) or negative (HL60) for the BCR-ABL oncoprotein, were analyzed for Survivin expression by western blot before and after IM treatment. Three different pathways (MAPK, PI3K and JAK2/STA3) potentially involved in BCR-ABL-mediated induction of Survivin were studied using inhibitors specific for each signaling cascade. The effect of Survivin on the proliferation and viability of IM-sensitive and IM-resistant CML cells was investigated after silencing Survivin expression with small interfering RNAs. Results: BCR-ABL tyrosine kinase activity induced an over-expression of Survivin in both human and murine hematopoietic cell lines. This over-expression was both at the transcriptional and the translational level and required the JAK2/STAT3 pathway. Survivin silencing by siRNA increased IM cytotoxicity in IM-sensitive cells but failed to restore IM efficacy in IM-resistant cells. However, Survivin silencing sensitized CML cells to the cytotoxic effect of hydroxyurea and enhanced the efficacy of this compound on three different murine cell lines are insensitive to IM because of point mutations in the BCR-ABL kinase domain (Ba/F3p210Y253F, Ba/F3p210D276G and Ba/F3p210T315I). Conclusions: Reduction of Survivin expression improves the efficacy of IM and increases the sensitivity of IM-resistant CML cells to hydroxyurea. Survivin may represent an attractive therapeutic target for both IM-sensitive and IM-resistant CML patients. No significant financial relationships to disclose.

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Lyn Kinase Overexpression Is One of the Mechanisms of Resistance to Nilotinib In Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v112.11.3181.3181 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3181-3181 ◽

Cited By ~ 2

Author(s):

Francois-Xavi er Mahon ◽

Sandrine Hayette ◽

Valerie Lagarde ◽

Franck E Nicolini ◽

Francis Belloc ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Activity ◽

Resistant Cell ◽

Resistant Cell Line ◽

Over Expression ◽

Abl Tyrosine Kinase ◽

Lyn Kinase

Abstract Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress. In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.

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Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽

10.1182/blood.v106.11.438.438 ◽

2005 ◽

Vol 106 (11) ◽

pp. 438-438 ◽

Cited By ~ 1

Author(s):

Xiaoyan Jiang ◽

Kyi Min Saw ◽

Allen Eaves ◽

Connie Eaves

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Point Mutations ◽

Chronic Phase ◽

Kinase Domain ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

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Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3116-3123.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3116-3123

Author(s):

J B Konopka ◽

O N Witte

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Myelogenous Leukemia ◽

Gene Products ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Assay Conditions

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.

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The Protein Tyrosine Phosphatase STS-1 Interacts with Bcr-Abl and Impairs the Response of Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) to Tyrosine Kinase Inhibitors

Blood ◽

10.1182/blood.v116.21.3236.3236 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3236-3236

Author(s):

Marcus Liebermann ◽

Daniela Hoeller ◽

Susanne Badura ◽

Tamara Tesanovic ◽

Hubert Serve ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Signalling Pathways ◽

Abl Kinase ◽

Clinical Resistance

Abstract Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

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Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3116 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3116-3123 ◽

Cited By ~ 155

Author(s):

J B Konopka ◽

O N Witte

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Myelogenous Leukemia ◽

Gene Products ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Assay Conditions

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Activity and Induction of Apoptosis of the Specific Tyrosine Kinase Inhibitor AMN 107 in bcr-abl + Cell Lines and in Imatinib Resistant Primary Cells from CML Patients.

Blood ◽

10.1182/blood.v104.11.762.762 ◽

2004 ◽

Vol 104 (11) ◽

pp. 762-762 ◽

Cited By ~ 6

Author(s):

Philipp Le Coutre ◽

Gökben Baskaynak ◽

Ingo Tamm ◽

Jörg Westermann ◽

Justus Duyster ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Lines ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Clonal Evolution ◽

Point Mutations ◽

Primary Cells ◽

Tyrosine Kinase Activity ◽

Imatinib Resistant ◽

Induction Of Apoptosis

Abstract The selective tyrosine kinase inhibitor imatinib eradicates bcr-abl+ cells in chronic myeloid leukemia patients (pts). Although a previous clinical trial showed superiority of an imatinib therapy over an interferon-α containing regimen, a significant number of pts eventually relapse with leukemia because of either point mutations within the imatinib-binding site, amplification of the Philadelphia chromosome or other mechanisms, e.g. clonal evolution. AMN107 (Novartis Pharma AG) is a new anilino-pyrimidine derivative (MW: 529.5) structurally related to imatinib. AMN107 was tested in three human bcr-abl positive lines (K562, KCL-22, Lama-84) and in primary cells derived from two bcr-abl + CML pts who were resistant to imatinib, as well as in one newly diagnosed chronic phase patient. In all pts sequencing of the bcr-abl kinase domain excluded any point mutations, but cytogenetic analysis of the bone marrow revealed clonal evolution in the resistant pts including t(1;5) and t(3;21) translocations, trisomy of chromosome 8 and monosomy of chromosome 7. Determination of the proliferative activity by XTT-assay in cell lines demonstrated a decrease of the IC50 in imatinib versus AMN107 treated samples from 0.08μM to 0.0075μM in Lama 84, from 0.25μM to 0.08μM in K562 and from 0.45μM to 0.03 in KCL-22 cells. No activity of either compound was observed in the bcr-abl negative HL-60 and KG-1 cells. In primary cells from imatinib-resistant pts, a decrease of the IC50 in imatinib versus AMN107 treated peripheral blood cells from 0.75μM to 0.1μM and from 4 to 0.4μM was detected. In addition, in primary cells from one newly diagnozed CML patient the IC50 of AMN107 (2.5μM) was reduced when compared to imatinib (5μM). Immunoblotting showed that in LAMA84 cells a concentration of 0.01μM AMN107 completely inhibited the tyrosine kinase activity as detected by use of an anti-phosphotyrosine antibody in contrast to almost 5μM in imatinib treated samples. Further, induction of apoptosis was detected using annexin V and propidium iodide by double fluorescence. After 48 hours of incubation with either 0.25 μM imatinib or 0.005 μM AMN107 induction of early apoptosis was detected in 8.8% of imatinib treated and 26% of AMN107 treated cells. Finally, HPLC analysis in HL-60 cells showed increased uptake by 1,5 fold for AMN107 when compared to imatinib. In addition, in MDR1 over-expressing CCRF cells co-culture with either AMN107 or imatinib revealed elevated AMN107 levels (3.7 fold) indicating that this substance is less susceptible to MDR1 driven resistance than imatinib. Conclusions: 1. AMN107 showed elevated activity when compared to imatinib in bcr-abl + cell lines and primary cells derived from imatinib resistant leukemic pts. 2. Complete inhibition of the bcr-abl tyrosine kinase activity and induction of apoptosis was achieved at lower concentrations in AMN107 treated samples when compared to imatinib. 3. Preliminary data indicate favourable cellular uptake of AMN107 when compared to imatinib. 4. AMN107 may be useful in the treatment of bcr-abl + leukemic pts.

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c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.7054 ◽

1996 ◽

Vol 16 (12) ◽

pp. 7054-7062 ◽

Cited By ~ 26

Author(s):

J Zhu ◽

S K Shore

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Cellular Transformation ◽

Sh3 Domain ◽

Cellular Protein ◽

Glutathione S Transferase ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Domain 3

The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase. Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.

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Influence of chelidonine, an inhibitor of tubulin polymerisation on tyrosine kinase activity in normal, transformed and malignant cell lines

Biomedical Research ◽

10.2220/biomedres.25.27 ◽

2004 ◽

Vol 25 (1) ◽

pp. 27-33 ◽

Cited By ~ 2

Author(s):

Annie JOUBERT ◽

Mona-Liza LOTTERING ◽

Annie PANZER

Keyword(s):

Tyrosine Kinase ◽

Cell Lines ◽

Kinase Activity ◽

Malignant Cell ◽

Tyrosine Kinase Activity

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A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation

Blood ◽

10.1182/blood-2006-04-016113 ◽

2006 ◽

Vol 108 (5) ◽

pp. 1716-1723 ◽

Cited By ~ 45

Author(s):

Lynn McCallum ◽

Susan Price ◽

Nathalie Planque ◽

Bernard Perbal ◽

Andrew Pierce ◽

...

Keyword(s):

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Direct Consequence ◽

Suppressor Gene ◽

Hematopoietic Stem ◽

Protein Levels ◽

Abl Kinase ◽

Abl Tyrosine Kinase ◽

Ccn3 Expression

Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

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