Resistant Chronic Myelogenous Leukemia Research Articles

Vorinostat Synergistically Potentiates MK-0457 Lethality in Chronic Myelogenous Leukemia (CML) Cells Sensitive and Resistant to Imatinib Mesylate.

MK-0457/VX-680 was developed as a pan-aurora kinase inhibitor that induces apoptosis in tumor cells and suppresses tumor growth in vivo. Recently, it was found that MK-0457 is also a potent inhibitor of both wild type and mutated Bcr/Abl kinases, including the T315I mutant which confers resistance to first- (i.e., imatinib mesylate/IM) and second-generation kinase inhibitors (e.g., dasatinib and nilotinib). Moreover, MK-0457 exhibits significant activity in patients with T315I phenotype-refractory CML or Ph-positive ALL. Here we examined interactions between MK-0457 and the histone deacetylase inhibitor (HDACI) vorinostat/SAHA in Bcr/Abl+ leukemia cells, including those resistant to IM through various mechanisms, particularly the T315I mutation. Co-administration of non- or subtoxic concentrations of vorinostat (0.5–2 μM) increased the lethality of MK-0457 (5–100 nM) in a highly synergistic manner in K562 and LAMA84 cells. Similar interactions were observed in primary CD34+ CML cells, whereas the combination largely spared normal bone marrow mononuclear cells. Moreover, the MK-0457/vorinostat regimen markedly induced cell death in IM-resistant K562 cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, a phenomenon accompanied by Lyn inactivation. Notably, the regimen was highly active against BaF/3 cells bearing various Bcr/Abl point mutations (i.e., T315I, E255K, and M351T) conferring resistance to IM. These events were associated with inactivation and downregulation of wild type and mutated Bcr/Abl (particularly T315I), reduced expression of phospho-CrkL, and vorinostat-mediated interruption of interactions between Bcr/Abl and Hsp90. Moreover, exposure of cells to marginally toxic doses of MK-0457 resulted in accumulation of cells with greater than 4N DNA content, resulting from induction of endoreduplication. Co-administration of vorinostat strikingly enhanced aurora kinase inhibition by MK-0457, reflected by markedly diminished expression of phospho-histone H3 (Ser 10), accompanied by preferential killing of polyploid cells. Interestingly, vorinostat also interacted with a selective inhibitor of aurora kinase A and B, at concentrations that inhibited histone H3 phosphorylation, to potentiate apoptosis in CML cells without modifying expression of phospho-Bcr/Abl or -CrkL. These results suggest that aurora kinase inhibition plays a functional role in synergistic interactions between HDACI and aurora kinase inhibitors. Finally, vorinostat markedly induced expression of the pro-apoptotic BH3-only protein Bim in various CML cell lines, including BaF/3 cells expressing the T315I Bcr/Abl mutation, as well as in primary CML cells. Knockdown of Bim by siRNA significantly protected K562 cells from apoptosis induced by vorinostat but not MK-0457. Furthermore, siRNA-mediated Bim downregulation blocked induction of Bim expression by vorinostat and dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. In addition, blockade of apoptosis by Bim siRNA did not modify downregulation of Bcr/Abl or phospho-histone H3 in cells exposed to the vorinostat/MK-0457 regimen, arguing against the possibility that the latter phenomena are secondary to activation of apoptotic cascade. Together, these findings indicate that vorinostat dramatically increases MK-0457 lethality in IM-sensitive and -resistant CML cells, and suggest that inactivation of Bcr/Abl kinase and aurora kinases, as well as Bim induction by vorinostat, contribute to this phenomenon.

Successful Overcoming of Imatinib Resistance by Synergistic Effect of Imatinib and Simvastatin in Imatinib Resistant Chronic Myelogenous Leukemia Cell Line.

Statins (HMG-CoA reductase inhibitors) are known to show antiproliferative effects and are anticipated as a potential drug in the treatment of malignancies including acute myelogenous leukemia (AML) and multiple myeloma (MM). Using cell proliferation assays, we determined the effects of simvastatin in CML cells. In the current study we show that simvastatin inhibits the proliferation of CML cell lines (The IC50 to simvastatin of K562, Kcl22, and LAMA84 were 13 uM, 19 uM, and 31 uM, respectively). Moreover, synergistic interactions between simvastatin and imatinib were observed in imatinib-resistant CML cell lines (K562-r, KCL22-r, and LAMA84-r). The cytotoxic effect of simvastatin occurred via the induction of apoptosis and inhibited Bcr-Abl tyrosine kinase (TK) activity, as evidenced by annexin V assay and Western blot analyses. Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Mean combination index (CI) values at 50%, 75%, and 90% growth inhibition (IC50, IC80) derived from combined treatment experimentsCell lineDrug combinationDrug ratioCI: IC50CI: IC75CI: IC90K562-sImatinib + Simvastatin1:400.61+++0.49+++0.40+++K562-rImatinib + Simvastatin1:100.60+++0.46+++0.41+++Kcl22-sImatinib + Simvastatin1:1000.96±1.32−1.83− −Kcl22-rImatinib + Simvastatin1:10.42+++0.35+++0.30+++LAMA84-sImatinib + Simvastatin1:2001.20−1.0±1.0±LAMA84-rImatinib + Simvastatin1:200.99±0.8+0.84+Gradual levels of synergy are marked as recommended by the manufacture of the Calcusyn software: +++, strong synergism; ++, moderate synergism; + slight synergism; ±, nearly additive; −, slight antagonism: and − −, moderate antagonism [Display omitted]

A phase II study of nilotinib administered to patients with imatinib resistant or intolerant chronic myelogenous leukemia (CML) in chronic phase (CP), accelerated phase (AP) or blast crisis (BC) who also failed dasatinib

7038 Background: Nilotinib and dasatinib are two new second generation tyrosine kinase inhibitors used in the treatment of imatinib-resistant/intolerant CML. There is limited data regarding the efficacy and safety of these therapies when given sequentially. Methods: This phase II open-label study was designed to evaluate the safety and efficacy of nilotinib at a dose of 400mg BID in CML-CP, -AP, and -BC pts who previously received and either failed or were intolerant to imatinib and dasatinib. Results: A total of 42 pts are reported - CP (16), AP (9), and BC (17 total; 13 myeloid, 4 lymphoid). Overall 10 (24%) pts had extramedullary disease at baseline. For all pts, median time from first diagnosis was 16.8 (0.6–265) months. The median duration of nilotinib exposure was 81 days with median dose intensity of 800mg/day. A total of 13 (31%) pts with dasatinib failure remain on treatment, 29 (69%) discontinued (6 for AEs, 16 for disease progression). CP: Of the 16 pts, 5 (31%) had a MCyR (3 complete, 2 partial). Complete hematologic response (CHR) was reported in 5/13 (39%) pts without CHR at baseline. Disease progression only occurred in 2 pts. AP: 2/9 (22%) pts had a return to chronic phase (RTC), 6 pts were not evaluable and there was 1 death. Disease progression occurred in 5 pts. BC: 3/17 (18%) achieved CHR, 1 (6%) had RTC, and 4 (24%) pts had disease progression. For all pts, the most common Grade 3/4 AEs reported were thrombocytopenia (26%), neutropenia (24%), and anemia (7%) pts. Conclusions: Nilotinib has significant clinical activity in CML-CP, AP, and BC patients who have failed imatinib and dasatinib. Nilotinib is safe and well tolerated, consistent with its kinase selectivity profile. No significant financial relationships to disclose.

Overview of Second-Generation Tyrosine Kinase Inhibitors for Patients With Imatinib-Resistant Chronic Myelogenous Leukemia

Imatinib mesylate (Gleevec, Novartis Pharmaceuticals Corporation, East Hanover, NJ) has revolutionized the treatment of chronic myelogenous leukemia (CML) with marked improvement in survival in all three phases--chronic, accelerated, and blast. Most patients with CML now receive imatinib, which produces complete cytogenetic response in more than 80% of patients. About 10% of patients who initially respond to imatinib subsequently develop resistance. Mechanisms of imatinib resistance in CML include amplification, mutations, and additional chromosomal aberration. To date, more than 30 mutations have been identified in imatinib-resistant CML. Dasatinib and AMN107, second-generation tyrosine kinase inhibitors, are highly effective therapies for patients with CML experiencing imatinib resistance and mutation and offer new options for patients who do not achieve an optimal response to imatinib therapy. Studies found that dasatinib and AMN107 form tighter bonds, overcoming imatinib resistance and producing complete hematologic and cytogenetic remissions. Long-term observations are needed to determine the effectiveness of the treatment. Primary care providers need to follow patients receiving first- or second-generation tyrosine kinase inhibitors because unforeseen toxicity may surface, requiring accurate assessment, evaluation, and management. Oncology nurses will be actively involved in the symptom management of patients. Providing guidelines for symptom management and advanced knowledge of specific test results for monitoring CML may increase positive outcomes.

Generation of Highly Efficient Recombinant Adeno-Associated Virus (rAAV) Vectors Targeted to Chronic Myelogenous Leukemia (CML) - and CD34+ Progenitor Cells Obtained by the „AAV Random Peptide Library“ Method.

Resistance of chronic myelogenous leukemia (CML) to imatinib is a serious clinical challenge. In vivo targeted gene therapy may be a strategy to overcome resistance. For gene therapy of CML standard rAAV2-based vectors lack the required gene transfer efficiency as well as the in vivo selectivity. An advancement in vector development (Muller et al., Nat. Biotech., 2003) now allows the generation of rAAV capsid mutants that offer higher target cell specificity and efficiency. We used the “AAV random peptide library” method to generate capsid mutants efficient and specific for CML cell lines, primary CML cells and primary human peripheral blood progenitor cells PBPC. After previously applying this method successfully on CML cell lines, an effective vector for CML cell lines could be obtained. On primary human CML and PBPC the new vector was more efficient than rAAV2 but still at an unsatisfying level. To increase the gene transfer on primary cells we applied the “AAV library” on primary human CML and PBPC. A total of 44 different mutant capsid clones were obtained, most of them at three or less copies. Four clones were observed >10x to 39x and were selected for further testing. To determine efficiency and specificity, a panel of leukemic (CML, imatinib-sensitive and -resistant, and AML) and other solid tumor cell lines were transduced (moi 100). On a panel of leukemic cell lines similar or higher gene transfer efficiency of the rAAV capsid mutants was observed compared to standard rAAV2 vectors. The higher transduction efficiency in imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S was particularly striking, as the difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S (table 1). On solid tumor cell lines rAAV2 was more efficient, confirming the higher specificity of our capsid mutants. Compared to our previous work (<1% GFP+ cells), the new capsid mutants now result in efficient gene transfer into the AML cell line KG1a, and to lesser extend the CML cell line EM3. For both cell lines gene transfer with the capsid mutants was readily more efficient than with standard rAAV2 vectors. Preliminary data on primary PBPC showed a uniformly higher transduction efficiency for all capsid mutants (%GFP_mut1-4: 13.6% ± 2.2% vs %GFP_AAV2: 5.3%). Experiments with primary human CML cells are ongoing. Using the “AAV random peptide library” method on primary cells, we were able to generate rAAV vectors with higher transduction efficiency on leukemic cell lines and primary PBPC than standard rAAV2 vectors. The development of these highly efficient AAV mutant capsid vectors holds promise for future development.Gene transfer efficiency (%GFP+ cells)Cell lineCapsid mutantStandard rAAV2LAMA84-S78.2 ± 7.99.2 ± 2.5LAMA84-R97.8 ± 0.393.0 ± 0.2HL-6054.0 ± 5.457.6 ± 8.2BV17348.4 ± 7.430.6 ± 1.8EM33.9 ± 0.70.2 ± 0.2KG1a24.7 ± 0.712.8 ± 5.0Data are given as mean ± SD

Computationally Designed, Small Molecule Inhibitors of Tubulin Inhibit the Growth of Gleevec Resistant Chronic Myelogenous Leukemia Cells.

The abl tyrosine kinase inhibitor (TKI) imatinib mesylate has revolutionized treatment of Chronic Myelogenous Leukemia (CML). Nonetheless, for the small percentage of chronic phase patients in whom resistance to imatinib develops, and for patients in accelerated phase/blast crisis, disease management is problematic. Second generation TKI may address the resistance issue, but serious cardiotoxicity might be a concern for all TKIs. We are therefore investigating other candidates for rationally directed CML therapy. Herein we report that targeting tubulin with computationally designed small molecules may prove useful for the treatment of TKI resistant CML cells. Tubulin inhibitors were obtained from Locus Pharmaceuticals, Blue Bell, PA. They were designed using a method that computes virtual inhibitor molecule binding efficiency after integrating free energy calculations from all chemically possible combinations of molecule fragment poses. Molecules that perform well in in silico screens are then synthesized for biological testing. We evaluated 3 such molecules (LP-261, LOC-011294, and LOC-011423), and 2 control compounds (LOC-007708, LP-590), on K562 human leukemia cells, murine BaF3 cells expressing wild type bcr-abl, or the Y253F, T315I, E255K, H296P and M351T kinase domain mutations (gift from B. Druker, Portland, Oregon), and on consenting donors of normal, and CML, bone marrow cells. LP-261, LOC-011294, and LOC-011423 all had significant activity in K562 cells and BaF3 cells expressing wild type bcr-abl. Inhibition of cell growth in these lines was ~90% when employed at concentrations of > 100nM. More importantly in BaF3 cells expressing each of the mutant abl kinases, including T315I, growth inhibition was also ~90%. Moreover, LP-261, and LOC-011294 were also highly effective against primary cells obtained from patients with chronic phase and blast crisis CML. Treatment of primary CML cells with LP-261 resulted in >80% inhibition of proliferation in all five CML patient samples when compared to control cells. LOC-011294 inhibited cell proliferation by >80% in 3 out of 5 primary patient samples, and by ~50% in one other. Additional testing revealed that LP-261 was not a substrate for the p-glycoprotein multi-drug resistance porter, and that it is orally bioavailable. Neither LOC-007708, (a selective inhibitor of p38 kinase), nor LP-590 (an inhibitor of p38, Flt-3 and tie-2 kinases) inhibited proliferation of the cells expressing mutated bcr-abl. Since these kinases are not thought to influence bcr-abl driven cell growth, these results were expected. In contrast, MOLM14 cells, which overexpress Flt-3, were profoundly inhibited by LP-590. Disease specificity is suggested by the fact that none of these compounds had any effect on growth of acute lymphoid leukemia (ALL) patient samples. To be clinically useful, it was important to demonstrate that normal CD34+ were less sensitive to the growth inhibitory effects of these compounds. For this purpose, normal CD34+ cells were exposed to LP-261, LOC-011294, and LOC-011423 for 24 hours at concentrations shown to inhibit CML cell growth and then plated in methylcellulose with cytokines. Under these conditions, no significant inhibition of CFU-GM, CFU-E, or BFU-E in comparison to control cells was shown. These results suggests that rationally designed anti-tubulin small molecules, alone, or in combination with other active agents, may prove quite useful for treating kinase inhibitor resistant, as well as de novo, CML. This hypothesis may be tested in the near future as an IND has been filed for LP-261.

BCR-ABL P-Loop Deletion Detected in Imatinib-Resistant CML Patients Corresponds to Splice Variant Associated with L248V Point Mutation, Retains BCR-ABL Kinase Activity, and Responds to Dasatinib Treatment.

Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch.The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

Cotreatment with Vorinostat (Suberoylanilide Hydroxamic Acid) Enhances Activity of Dasatinib (BMS-354825) against Imatinib Mesylate–Sensitive or Imatinib Mesylate–Resistant Chronic Myelogenous Leukemia Cells

We determined the effects of vorinostat [suberoylanilide hydroxamic acid (SAHA)] and/or dasatinib, a dual Abl/Src kinase (tyrosine kinase) inhibitor, on the cultured human (K562 and LAMA-84) or primary chronic myelogenous leukemia (CML) cells, as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and kinase domain-mutant forms of Bcr-Abl. Following exposure to dasatinib and/or vorinostat, apoptosis, loss of clonogenic survival, as well as the activity and levels of Bcr-Abl and its downstream signaling proteins were determined. Treatment with dasatinib attenuated the levels of autophosphorylated Bcr-Abl, p-CrkL, phospho-signal transducer and activator of transcription 5 (p-STAT5), p-c-Src, and p-Lyn; inhibited the activity of Lyn and c-Src; and induced apoptosis of the cultured CML cells. Combined treatment of cultured human CML and BaF3 cells with vorinostat and dasatinib induced more apoptosis than either agent alone, as well as synergistically induced loss of clonogenic survival, which was associated with greater depletion of Bcr-Abl, p-CrkL, and p-STAT5 levels. Cotreatment with dasatinib and vorinostat also attenuated the levels of Bcr-AblE255K and Bcr-AblT315I and induced apoptosis of BaF3 cells with ectopic expression of the mutant forms of Bcr-Abl. Finally, cotreatment of the primary CML cells with vorinostat and dasatinib induced more loss of cell viability and depleted Bcr-Abl or Bcr-AblT315I, p-STAT5, and p-CrkL levels than either agent alone. As shown here, the preclinical in vitro activity of vorinostat and dasatinib against cultured and primary CML cells supports the in vivo testing of the combination in imatinib mesylate-sensitive and imatinib mesylate-resistant CML cells.

Imatinib resistant chronic myelogenous leukemia, BCR-ABL positive by chromosome and FISH analyses but negative by PCR, in a child progressing to acute basophilic leukemia: cytogenetic follow-up

The case of an 11-year-old child with adult-type chronic myeloid leukemia, Philadelphia ( BCR-ABL) positive, reverse transcription–polymerase chain reaction negative for the major, minor, and micro breakpoints is presented. In the course of 3 years, the child failed to respond to treatment with hydroxyurea, refused all therapy for 6 months, was intolerant to α-interferon and progressed, while on imatinib, to acute basophilic leukemia. Subsequently he underwent successful bone marrow transplantation. A secondary cytogenetic clonal evolution, i(17q), developed during hydroxyurea treatment and a tertiary clonal evolution, +8, was detected during imatinib treatment. It is not clear to what extent the several factors (undefined BCR-ABL breakpoint, treatment avoidance, and initial treatment choices, alone or in combination) played a role in the imatinib relapse and resistance and in the disease progression. We conclude that close follow-up with frequent bone marrow sampling is crucial in order to monitor such patients for early relapse and prompt referral for bone marrow transplant.

Loss of p53 impedes the antileukemic response to BCR-ABL inhibition

Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers, although antitumor responses are often heterogeneous, and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug, we studied the action of imatinib (STI-571, Gleevec) against murine cells and leukemias expressing BCR-ABL, an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53, which can accompany disease progression in human CML, impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly, p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution.

Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(ϕ)-LMC GROUP)

The emergence of ABL point mutations is the most frequent cause for imatinib resistance in chronic myelogenous leukemia (CML) patients and can occur during any phase of the disease; however, their clinical impact remains controversial. In this study, we retrospectively analyzed the predictive impact of 94 BCR-ABL kinase domain mutations (18 T315I, 26 P-loop, 50 in other sites) found in 89 imatinib-resistant CML patients. At imatinib onset, 64% of patients (57/89) were in chronic phase (CP), 24% (21/89) in accelerated phase (AP) and 12% (11/89) in blastic phase (BP). T315I and P-loop mutations were preferentially discovered in accelerated phase of BP CML, and other types of mutations in CP (P=0.003). With a median follow-up of 39.2 months (6.3-67.2), since imatinib initiation, overall survival (OS) was significantly worse for P-loop (28.3 months) and for T315I (12.6 months), and not reached for other mutations (P=0.0004). For CP only, multivariate analysis demonstrated a worse OS for P-loop mutations (P=0.014), and a worse progression-free survival (PFS) for T315I mutations (P=0.014). Therefore, P-loop and T315I mutations selectively impair the outcome of imatinib-resistant CML patients, in contrast to other mutations, which may benefit from dose escalation of imatinib, able to improve or stabilize disease response.

Adaphostin-induced oxidative stress overcomes BCR/ABL mutation-dependent and -independent imatinib resistance

AbstractThe BCR/ABL kinase has been targeted for the treatment of chronic myelogenous leukemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML and Ph+ acute lymphoblastic leukemia (ALL) are often resistant. In particular, mutation of the T315 residue in the bcr/abl activation loop renders cells highly resistant to imatinib and to secondgeneration kinase inhibitors such as BMS-354825 or AMN107. Adaphostin is a tyrphostin that was originally intended to inhibit the BCR/ABL kinase by competing with its peptide substrates. Recent findings have in addition implicated reactive oxygen species (ROS) in the cytotoxic mechanism of adaphostin. In view of this unique mode of action, we examined the effects of adaphostin on numerous imatinib-resistant leukemia models, including imatinib-resistant CML and Ph+ ALL cell lines, cells harboring point mutations in BCR/ABL, and specimens from imatinib-resistant CML patients, using assays for intracellular ROS, apoptosis, and clonogenicity. Every model of imatinib resistance examined remained fully sensitive to adaphostin-induced cell death. Collectively, these data suggest that ROS generation by adaphostin overcomes even the most potent imatinib resistance in CML and Ph+ ALL. (Blood. 2006;107: 2501-2506)

Heat Shock Protein 70 over Expression Is Associated to Imatinib Resistance in Chronic Myelogenous Leukemia.

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood , 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p < 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p < 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.

Treatment of Imatinib-Sensitive and -Resistant Chronic Myelogenous Leukemia Cells with a Combination of Imatinib and Farnesyltransferase Inhibitors.

Farnesyl protein transferase inhibitors (FTIs) represent a new class of anticancer agents. We were interested whether the farnesyltransferase inhibitors L-744,832 and LB42918 alone and in combination with imatinib are active in imatinib-sensitive and -resistant CML cells. LB42918 was more potent with ED50 = 4 ± 1 μM (mean ± S.D., n = 3) in the BCR-ABL+ imatinib-sensitive cell line EM-3 vs. ED50 = 25 ± 2 μM for L-744,832 after 48 hrs of treatment and assessment by MTT assay. The growth of imatinib-sensitive K562 and LAMA84 cells was not measurably inhibited by L-744,832 doses up to 25 μM. Similarly, the growth of K562 was not affected by 25 μM of LB42918. However, for LB42918 in LAMA84 cells an ED50 value of 30 ± 11 μM could be determined. In the imatinib-sensitive cell line EM-3, combination index values (CI) obtained using the method of Chou and Talalay indicated synergistic effects following simultaneous treatment with imatinib and FTI (CI = 0.5 ± 0.2 and 0.7 ± 0.2 at ED75 for imatinib + L-744,832 and imatinib + LB42918 respectively). In the cell imatinib-sensitive and -resistant lines K562 and LAMA84 a trend to lower ED50 values of imatinib was determined when up to 16 μM of L-744,832 or LB42918 were added, indicating potentiation of imatinib activity. Annexin V / propidium showed a strong increase of the apoptotic cell fraction in all imatinib-sensitive and an increase in the imatinib-resistant cells treated for 48 hrs by the combination imatinib + L-744,832 or imatinib + LB42918 as compared to treatment with each drug alone. Growth inhibition of CFU-GM colonies of primary imatinib-sensitive CML cells obtained from 4 patients is stronger after treatment with different concentrations of the combination of both drugs than after monotherapy with either imatinib or L-744,832. On the basis of the observed potentiation effects FTIs may find their place as supplement for CML patients on imatinib treatment.

Targeting the Kinase Activity of the BCR-ABL Fusion Protein in Patients with Chronic Myeloid Leukemia

Imatinib mesylate is a major advance in the therapy of patients with chronic myelogenous leukemia (CML). Imatinib mesylate binds to the inactive conformation of BCR-ABL tyrosine kinase suppressing the Philadelphia chromosome positive clone in CML. Clinical studies have yielded impressive results in all phases of CML. With higher rates of complete cytogenetic response with imatinib, molecular monitoring of disease is now advisable in assessing response and determining prognosis. Emergence of resistance to imatinib may be manifest at the hematologic, cytogenetic, or molecular levels in patients who remain in chronic phase, or may be evidenced by the development of more advanced CML phases. Resistance and eventual clinical failure of imatinib occurs in most patients with blastic phase disease. Resistance may occur at the level of Bcr-Abl, with reduction or loss of imatinib effectiveness as a kinase inhibitor, or, despite retention of its inhibitory ability, with changes in the ability to deliver an effective dose at the cellular level, and/or, the leukemia becoming less dependent on Bcr-Abl. The various mechanisms underlying these differing, non-mutually exclusive, mechanisms of resistance must be understood to develop corresponding therapeutic remedies. We review the current data on imatinib in CML, the criteria for diagnosis of imatinib resistance, and the mechanisms that underlie such resistance in CML.

Loss of P-glycoprotein expression in hematopoietic stem cells does not improve responses to imatinib in a murine model of chronic myelogenous leukemia

Selective inhibition of the BCR/ABL tyrosine kinase by imatinib has become a first-line therapy for chronic myelogenous leukemia (CML). However, BCR/ABL-positive progenitors often persist despite treatment, and relapse associated with resistance to imatinib has been described in many patients with advanced disease. Drug efflux by P-glycoprotein (P-gp), as well as point mutations in BCR/ABL oncoprotein, has been implicated in the mechanism of resistance to imatinib. In this study, we established a murine transplantation model of CML-like myeloproliferative disease using Mdr1a/1b-null mice and analyzed the effects of loss of P-gp on resistance to imatinib. We found that mice transplanted with Mdr1a/1b-null bone marrow (BM) that had been transduced with a BCR/ABL retroviral vector displayed similar responses to imatinib, compared with those transplanted with BCR/ABL-transduced wild-type BM. In the absence of P-gp, the incidence and latency of disease in secondary recipients was not changed in imatinib-treated mice, relative to wild-type controls. Furthermore, K562 cells engineered to overexpress P-gp remained sensitive to imatinib-induced growth inhibition and cell death. Together, our findings suggest that P-gp expression in hematopoietic stem cells does not significantly contribute to imatinib resistance in CML.

Malignant Transformation of Human Cells by Constitutive Expression of Platelet-derived Growth Factor-BB

Platelet-derived growth factors (PDGFs) comprise a family of growth factors strongly implicated in human oncogenesis. A number of human tumors overexpress PDGF family members or have translocations activating PDGF receptors. Whereas the epidemiologic evidence implicating PDGF in human tumors is strong, malignant transformation of human cells by overexpression of PDGF has not been demonstrated. We have previously developed a human cell line by the sequential introduction of large T cells and telomerase, and we have demonstrated that these cells express functionally active PDGF receptor (PDGFR) beta. In order to determine whether growth factor-mediated transformation of human cells could occur, these cells were transduced with a retrovirus encoding PDGF-BB. Constitutive expression of PDGF-BB led to malignant transformation in nude mice. This is the first demonstration of constitutive signaling causing malignant transformation of human cells. Some of the changes that occur because of constitutive growth factor expression can be reversed by the clinically approved tyrosine kinase inhibitor Glivec, whereas other changes are not reversible by tyrosine kinase inhibitors. Our model allows the assessment of epigenetic changes that occur during human carcinogenesis. In addition, these studies provide insight into the clinical failure of tyrosine kinase inhibitors as monotherapy for advanced malignancy.

Src Family Kinases as Regulators of Angiogenesis: Therapeutic Implications

Despite its discovery nearly a century ago, the functions of the Src family of protein tyrosine kinases (SFKs) remain incompletely understood. While much has been learned regarding the functions of Src family kinases in the last few years, new roles for Src, particularly in promoting the progression of cancer towards the metastatic phenotype, continue to emerge. SFKs, through their functions as kinases and adapter proteins in signaling complexes, regulate such diverse cellular events as proliferation, migration, cell cycle control, and apoptosis. In tumor cells, the kinase activity of Src is frequently activated, with greater increases during progressive stages. Likewise, resistance to chemotherapy also corresponds with Src kinase activity. Thus, Src activation is predictive of poor prognosis in several tumors. Recently, selective SFK inhibitors are showing promise in clinical trials in imatinib mesylate (Gleevec, Novartis) resistant chronic myelogenous leukemia. However, in vitro studies have suggested that Src inhibitors may hold promise in the treatment of solid tumors such as colon and pancreatic cancer in which new therapeutic inhibitors are desperately needed. This review will summarize briefly the structure and function of Src and the evidence for Src in promoting tumor progression and metastasis. As recent work in this laboratory and others has demonstrated that Src is a regulator of expression of diverse pro-angiogenic factors produced by tumor cells, and a regulator of the endothelial cells that respond to these factors, this review will focus on the role of Src in angiogenesis and potential roles of Src inhibitors as antiangiogenic agents. Keywords: src kinases, angiogenesis, targeted therapy, chemotherapy, metastasis

The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.

BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.

Targeting BCR-ABL and Its Downstream Signaling Cascade as Therapy for Chronic Myelogenous Leukemia.

BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of chromosomal translocation in chronic myelogenous leukemia (CML). The tyrosine kinase activity of BCR-ABL activates signaling cascades that induce cytokine independence and transformation of myeloid progenitor cells. Targeted inhibition of this kinase with specific inhibitors (imatinib or BMS-354825) is a very effective therapy for some CML patients but resistance to these agents (through point mutations and other mechanisms) leads to advanced disease with very few therapeutic options. An alternate therapeutic strategy is to reduce BCR-ABL expression or its critical downstream signaling elements important for transformation. We examined BCR-ABL signaling elements and gene expression changes that occur in CML cells following kinase inhibition by imatinib in newly established imatinib sensitive and resistant cells to identify critical signaling elements involved in CML cell death. Imatinib rapidly and progressively suppressed c-myc expression in imatinib sensitive but not resistant cells prior to the onset of apoptosis. These results suggested that c-myc expression was regulated by BCR-ABL signaling and may play a role in CML tumorigenicity. To confirm a role for c-myc in CML cell growth and/or survival, c-myc expression was specifically down-regulated by siRNA using a novel electroporation instrument (AMAXA) that permits high level gene transfer with limited toxicity in CML cell lines. Jak2 siRNA was used as a control. c-myc, but not Jak2 siRNA, suppressed c-myc expression and cell growth and survival in both imatinib sensitive and resistant CML cells, suggesting that targeted suppression of c-myc may have therapeutic activity against both kinase inhibitor sensitive and resistant CML cells. Since the tyrphostin AG490 was previously shown to inhibit c-myc expression in CML cells through its inhibitory effects on Jak2, we screened a series of > 200 AG490 derivatives for their ability to rapidly reduce c-myc expression in hematological malignancies. After several rounds of testing we synthesized an agent (WP-1066) capable of rapid c-myc downregulation (beginning 1–5 min after treatment with 1–2 microM) but poor Jak2 kinase inhibitory activity (IC50 > 100 microM). These results suggested a more direct effect of WP-1066 on c-myc protein expression than AG490 and mechanistic studies suggest that WP-1066 reduces c-myc protein stability but does not affect c-myc gene expression. In BCR-ABL expressing cells WP-1066 rapidly reduced c-myc protein levels in CML cells and inhibited the growth and survival of cell lines or patient specimens expressing wild-type or mutant forms of BCR-ABL that effect tyrosine kinase inhibitory activity (T315I in BV-173R cells). Equal concentrations of imatinib or WP-1066 reduced BCR-ABL activation and downstream signaling (Stat5 phosphorylation) in CML cells. However, WP-1066 differed from imatinib in its ability to downregulate BCR-ABL protein expression without affects on c-abl or Stat5 expression. Similar results were obtained in clinical specimens taken from patients with BCR-ABL point mutations that mediate imatinib (or BMS-354825) resistance. Nude mouse studies demonstrated that WP-1066 reduced the growth of K562 tumors to an extent similar to that of imatinib. Together these results suggest that WP-1066 downregulates BCR-ABL and c-myc expression, induces apoptosis in CML cells expressing wild-type or mutant BCR-ABL and may have therapeutic activity in imatinib (or BMS-354825) resistant CML tumors.