Imatinib-resistant Chronic Myeloid Leukemia Research Articles

Detection of single nucleotide insertion of BCR/ABL region in imatinib-resistant human myelogenous leukemia SR-1 cells

Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.

Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells

Although imatinib mesylate has revolutionized the treatment of chronic myeloid leukaemia (CML), resistance to the drug, manifesting as relapse after an initial response or persistence of disease, remains a therapeutic challenge. In order to overcome this, alternative or additional targeting of signaling pathways downstream of Bcr-Abl may provide the best option for improving clinical response. Bisphosphonates, such as zoledronate, have been shown to inhibit the oncogenicity of Ras, an important downstream effector of Bcr-Abl. In this study, we show that zoledronate is equally effective in inhibiting the proliferation and clonogenicity of both imatinib-sensitive and -resistant CML cells, regardless of their mechanism of resistance. This is achieved by the induction of S-phase cell cycle arrest and apoptosis, through the inhibition of prenylation of Ras and Ras-related proteins by zoledronate. The combination of imatinib and zoledronate also augmented the activity of either drug alone and this occurred in imatinib-resistant CML cells as well. Since zoledronate is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.

Decitabine has single-agent activity in imatinib-resistant CML

Decitabine has single-agent activity in imatinib-resistant CML

RNAi-mediated knockdown of P-glycoprotein using a transposon-based vector system durably restores imatinib sensitivity in imatinib-resistant CML cell lines

Resistance to therapeutic drugs is a frequent phenomenon in hematologic malignancies, causing treatment failure in patients with leukemias and lymphomas. Overexpression of the multidrug-resistance gene (MDR-1) and its translational product P-glycoprotein (PgP) represents one mechanism of fatal drug resistance. We constructed a nonviral, transposon-based vector system for the stable knockdown of PgP in chronic myeloid leukemia cell lines resistant to imatinib and doxorubicin. Using this strategy, PgP expression was completely knocked down 72 hours after vector inoculation and lasted for several months. Cellular efflux of the PgP substates rhodamine and doxorubicin was abolished. Vector-treated cells were resensitized to imatinib- and doxorubicin-induced cell death. Using chronic myeloid leukemia as a model, we show that PgP-mediated resistance to imatinib and anthracyclines can be durably reversed by nonviral, transposon-based knockdown of PgP in malignant cells.

AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has significant activity in imatinib-resistant bcr-abl positive chronic myeloid leukemia (CML)

3014 Background: AMN107, a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl, is 10 to 50 fold more potent than imatinib against Bcr-Abl expressing cell lines, including most imatinib-resistant Bcr-Abl mutants. The increased potency and broader activity of AMN107, relative to imatinib, may result in clinical benefit for patients (pts) with CML. Methods: In a Phase I/II study of AMN, 52 of 67 total pts (other 15 had lymphoid disease) had imatinib resistant CML in different phases. Results: Median age was 62 years (range 15–83). CML phase was: chronic (CP) 2 pts, accelerated (AP) 35 pts; myeloid blastic (BP) 15 pts. AMN daily doses were (mg/day): 50 - 6 pts, 100 - 5 pts, 200 - 7 pts, 400 - 9 pts, 600 - 1 pt, 800 - 14 pts, 1200 - 9 pts, 400 mg BID - 1 pt. Patients have been treated for 12 to 200 days. In the overall cohort, possible AMN-related Grade 3 or 4 hematologic adverse events were observed in 5/67 pts (3 responded) at doses 200 mg or more: G3 thrombocytopenia (1 pt), G4 thrombocytopenia (1 pt), G4 thrombocytopenia, G3 anemia, G4 neutropenia (1 pt), G4 neutropenia, G4 thrombocytopenia, (1 pt), and G3 neutropenia (1 pt). Possible AMN-related non-hematologic Grade 3/4 events observed: 600 mg, G3 skin rash (1 pt); ≥ 800 mg, G3 SGOT and/or SGPT elevations (2 pts), G3 indirect hyperbilirubinemia (2 pts); 1200 mg, G2 pancreatitis (1 pt). In AP, 19/30 evaluated pts (63%) responded: 11 CHR, 7 return to chronic phase (RTC) and 1 marrow CR (MR). 6/30 evaluated pts (20%) had cytogenetic (CG) responses: complete 2, partial (Ph 1–34%) 2, minor (Ph 35–90%) 2. In myeloid BP, 7/12 evaluated pts (58%) responded: 2 CHR, 5 RTC; and 1/12 (8%) had a CG response. 1 pt in CP had CHR and a CGPR. Mutational data available in 36 pts showed 9 mutations in 15 pts: G250E(4); M351T (4); E355G (2); E459Q, Y253F, F317L, F359V, T315I, M244V (1 each). 12/15 pts with mutations responded to AMN. PK studies showed steady-states reached by day 8. Both Cmax and AUC were dose proportional from 50 to 400 mg with a median Tmax of 3 hours but were not increased with 800 - 1200 mg/daily. To increase exposure, a BID schedule is being explored. The half-life was 16 hours. Conclusions: AMN107 has significant activity in imatinib-resistant CML. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis

Correlation of clinical response to BMS-354825 with BCR-ABL mutation status in imatinib-resistant patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-associated acute lymphoblastic leukemia (Ph+ ALL)

6521 Background: Relapse of CML on imatinib is most often associated with point mutations in the BCR-ABL kinase domain. BMS-354825 is a novel ABL kinase inhibitor with ≥ 300-fold greater potency than imatinib and preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants tested. Unlike imatinib, BMS-354825 binds the ABL kinase domain in both active and inactive conformations, and is thus predicted to have significant activity in most imatinib-resistant CML and Ph+ ALL cases. Methods: CA180002 is a phase I, dose-escalation study in CML and Ph+ ALL patients resistant or intolerant to imatinib. Prior to therapy, peripheral blood samples were collected for mutation analysis for correlation with response. Response was assessed by standard methods. Sixty-three patients are currently evaluable [chronic phase=36, accelerated phase=8, blast phase/Ph+ ALL=19]. Results: Seventeen different imatinib-resistant point mutations in the BCR-ABL kinase domain were identified in 67% of patients prior to therapy. Complete hematologic remission has been observed in patients harboring each of these mutations except T315I (5), F317L (1), and D276G (1). The two patients with F317L and D276G have had partial responses and remain on study. The T315I mutation is highly resistant to BMS-354825 in experimental systems. Nine patients have gone off study due to progressive disease. Of these, three had T315I detected prior to treatment and two patients had the T315I mutation at the time of disease progression. Assessment of BCR-ABL kinase inhibition by analysis of phospho-CRKL in patient samples was correlated with response, and loss of BCR-ABL kinase inhibition was correlated with relapse. Conclusions: Clinical activity of BMS-354825 in imatinib-resistant CML and Ph+ ALL is observed in patients with a wide range of imatinib-resistant BCR-ABL kinase domain mutations. The exception to date is T315I, which is cross-resistant to both drugs, as predicted from preclinical studies. This works demonstrates the utility of molecular assessment of tumor early in the clinical evaluation of a targeted inhibitor. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bristol-Myers Squibb Bristol-Myers Squibb Bristol-Myers Squibb Bristol-Myers Squibb Bristol-Myers Squibb

ABL mutations in imatinib-resistant CML indicate worse prognosis

ABL mutations in imatinib-resistant CML indicate worse prognosis

A phase I study of BMS-354825 in patients with imatinib-resistant and intolerant chronic phase chronic myeloid leukemia (CML): Results from CA180002

6519 Background: Resistance to imatinib in CML patients is often associated with Bcr-Abl point mutations which interfere with imatinib binding. BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with more than 300-fold greater potency than imatinib and has preclinical activity against 14 of 15 imatinib resistant Bcr-Abl mutants (Shah et al, Science, 305:399, 2004). Methods: CA180002 is a phase I, dose-escalation study of BMS-354825 in CML patients with hematologic resistance or intolerance to imatinib. Intrapatient dose escalation is permitted. Patient samples are analyzed for pharmacokinetics (PK), Bcr-Abl kinase domain mutations, and CRKL, HCK, and LYN phosphorylation. Results: 36 chronic phase CML patients have been treated (31 resistant, 5 intolerant) with total daily doses ranging from 15 - 180 mg (given as single or divided doses). Mutations associated with imatinib resistance were identified in 27 patients. Thirty-one patients (86%) have had complete hematologic response (CHR). Only 2 patients have had progression of their disease. Thirteen of 29 patients had cytogenetic improvement including 5 complete and 4 partial cytogenetic responses (i.e. major cytogenetic responses (MCyR) in 9 of 29 patients. MCyR rate = 31%). Hematologic and cytogenetic responses have occurred regardless of mutation status, or whether the mutation was in the P-loop, catalytic or activation region, with the exception of one patient who progressed with T315I. Responses are durable, and 33 of 36 patients remain on study for 1+ to 13+ months. BMS-354825 has been well tolerated. Three patients had grade 4 thrombocytopenia and 2 patients had grade 4 neutropenia, all of which were reversible and easily managed with dose modification. One patient developed a duodenal ulcer possibly related to BMS-354825. Mild QTc prolongation has been noted. Conclusions: BMS-345825 appears to be safe and effective therapy for patients with imatinib-resistant and imatinib-intolerant CML producing hematologic and cytogenetic responses. The phase I study continues to accrue patients to determine the maximally tolerated dose. No significant financial relationships to disclose.

Activity of AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, in imatinib-resistant bcr-abl positive lymphoid malignancies

3015 Background: AMN107, a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl. AMN107, is 10- to 50-fold more potent than imatinib against Bcr-Abl expressing cell lines, and is effective against most cell lines expressing imatinib-resistant Bcr-Abl mutants. In two p190-Bcr-Abl ALL cell lines, AMN107 was 30–40 times more potent than imatinib in inhibiting cellular proliferation and in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. Methods: In an ongoing phase I study, AMN107 was given orally, once or twice daily dosing schedule. Results: Fifteen of 67 total pts entered had lymphoid disease: lymphoid blastic phase CML (CML LBP) 7 pts; Ph+ acute lymphocytic leukemia (Ph+ ALL) 8 patients. Median age was 53 years (range 28–80). AMN doses were: QD dose cohorts (mg/day); 50 (1 pt), 100 (2 pts), 200 (3 pts), 400 (1 pt), 600 (2 pts), 800 (4 pts), 1200 (1 pt); BID dose cohort; 400 mg BID (1 pt). Pts have been treated for 15 to 89 days. In the overall cohort, possible AMN-related Grade 3 or 4 hematologic adverse events were observed in 5/67 patients (3 responded) at doses ≥ 200 mg: Possible AMN-related non-hematologic Grade 3/4 events were: 600 mg, G3 skin rash (1 pt); ≥ 800 mg, G3 SGOT and/or SGPT elevations (2 pts), G3 indirect hyperbilirubinemia (2 pts), and at 1200 mg, G2 pancreatitis (1 pt). Clinical activity has been observed in 5 of the 14 with lymphoid disease treated with doses of ≥ 600 mg: hematological improvement (1 pt, Ph+ ALL), peripheral responses (2 pts, LBP), and bone marrow responses (marrow response/no evidence of leukemia, 2 pts, LBP) 1 pt with LBP had a cytogenetic CR. Mutational data are available for 3 of the responding patients: Y253H (hematologic improvement), T315I (peripheral response), and E355G (marrow response). Pharmacokinetic (PK) studies showed steady state was generally reached by day 8. Both Cmax and AUC were dose proportional from 50 to 400 mg with a median Tmax of 3 hours. The apparent half-life was 16 hours. Conclusions: AMN107 has significant activity in patients treated with ≥ 600 mg per day in imatinib-resistant CML LBP or Ph+ ALL Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis

Synergistic activity of imatinib and 17-AAG in imatinib-resistant CML cells overexpressing BCR-ABL – Inhibition of P-glycoprotein function by 17-AAG

Overexpression of BCR-ABL and P-glycoprotein (Pgp) are two of the known mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we investigated the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat-shock protein 90 (Hsp90) inhibitor, on different imatinib-sensitive and imatinib-resistant CML cell lines. In imatinib-sensitive cells, combination index (CI) values obtained using the method of Chou and Talalay indicated additive (CI=1) or marginally antagonistic (CI>1) effects following simultaneous treatment with imatinib and 17-AAG. In imatinib-resistant cells both drugs acted synergistically (CI<1). In primary chronic-phase CML cells additive or synergistic effects of the combination of imatinib plus 17-AAG were discernible. Annexin V/propidium iodide staining showed that the activity of imatinib plus 17-AAG is mediated by apoptosis. Combination treatment with imatinib plus 17-AAG was more effective in reducing the BCR-ABL protein level than 17-AAG alone. Monotherapy with 17-AAG decreased P-glycoprotein activity, which may increase intracellular imatinib levels and contribute to the sensitization of CML cells to imatinib. The results suggest that combination of imatinib and 17-AAG may be useful to overcome imatinib resistance in a clinical setting.

Dasatinib: the emerging evidence of its potential in the treatment of chronic myeloid leukemia

Current therapy options for chronic myeloid leukemia (CML) include conventional chemotherapy, allogeneic stem cell transplant, interferon-alfa, and imatinib mesylate, which has recently achieved gold standard status. Although the majority of patients initially respond well to treatment with imatinib, wider clinical experience with this drug has resulted in the development of imatinib resistance being increasingly documented. There is therefore an unmet medical need for novel therapies to override imatinib resistance in CML. This review summarizes the emerging evidence for the potential use of dasatinib in the treatment of imatinib-resistant CML. Dasatinib is a novel small molecule that has shown potent antileukemic activity in imatinib-resistant cell lines, malignant marrow cells isolated from patients with imatinib-resistant CML, and in mouse xenograft models of imatinib-resistant CML. Preliminary data from an initial phase I dose escalation trial have been encouraging, indicating that dasatinib is generally well tolerated and produces hematologic and cytogenetic responses in patients with imatinib-resistant CML in all phases of the disease. The maximum tolerated dose (MTD) has not yet been reached, and dose escalation continues to determine the dose range that yields optimal results. Although dasatinib is still in the early stages of development, the potential impact of this molecule on the treatment of CML could be revolutionary, not only providing a much needed treatment option for patients with imatinib-resistant CML, but also, combined with imatinib, could possibly prove useful in delaying the onset of resistance to treatment. Furthermore, combined with other agents active in CML, dasatinib could have potential utility in purging residual leukemic cells in patients whose disease is controlled by imatinib.

Targeted therapy for the treatment of imatinib-resistant chronic myeloid leukemia: A pharmacogenetic analysis

Background/Aims Chronic myeloid leukemia (CML) is driven by the enzymatic activity of BCR-ABL, a tyrosine kinase that is selectively inhibited by imatinib. However, a subset of patients develop resistance. BMS-354825 retains in vitro activity against 14 out of 15 imatinib-resistant forms of BCR-ABL in the nanomolar concentration range. BMS-354825 is currently undergoing analysis in a phase I clinical trial of imatinib-resistant or -intolerant CML cases. We sought to determine if a correlation exists between specific kinase domain mutation and response to BMS-354825. Methods Fifteen patients were included in the analysis. All had a diagnosis of Philadelphia chromosome-positive CML, and were either imatinib-resistant or -intolerant. Sequencing of the BCR-ABL kinase domain was performed prior to treatment. The achievement of a complete hematologic response was correlated with BCR-ABL genotype. Results Preliminary analysis suggests a strong correlation between response and absence of the T315I mutation, and no significant response in cases of the T315I mutant. These data corroborate our preclinical results, and identify the T315I mutation as a potentially major component of clinical resistance to BMS-354825. Conclusions These studies suggest an evolving role for patient-specific therapy based upon the specific genotype of the target gene in human cancers treated with the burgeoning class of small molecule kinase inhibitors, and warrant confirmatory analysis in a larger cohort of patients. Clinical Pharmacology & Therapeutics (2005) 77, P17–P17; doi: 10.1016/j.clpt.2004.11.066

Hematologic and Cytogenetic Responses in Imatinib-Resistant Accelerated and Blast Phase Chronic Myeloid Leukemia (CML) Patients Treated with the Dual SRC/ABL Kinase Inhibitor BMS-354825: Results from a Phase I Dose Escalation Study.

BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with 100-fold greater potency to inhibit BCR-ABL in vitro than imatinib and has in vitro and in vivo preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants (Shah et al, Science, 305:399, 2004). Here we report the phase I clinical results of BMS-354825 in Philadelphia chromosome positive accelerated phase (AP) and blast phase (BP) CML patients who had hematologic progression or intolerance while being treated with imatinib. As of Aug 6, 2004, 17 patients (6 with AP; 11 with BP) have been treated in 3 cohorts with doses ranging from 35 mg BID (1 patient) to 70 mg BID of BMS-354825. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Consistent, rapid and sustained inhibition of LYN kinase, a member of the SRC family of tyrosine kinases, has been demonstrated.Of the 11 BP patients, 7 have had hematologic response: 3 complete hematologic response (CHR), 2 ‘no evidence of leukemia' (NEL), and 2 ‘return to chronic phase' (RTC). Three additional patients have had significant hematologic improvement despite being on treatment only a short period of time (10–23 days). One patient with extramedullary disease was stable. Cytogenetic data is available for 8 of the 11 BP patients. Four patients had major cytogenetic response, 2 patients had a minor cytogenetic response and 2 patients had no response. BCR-ABL mutation data is available for 2 patients: one patient did not have a mutation and one patient who had a non-sustained CHR was found to have a E355G mutation.Three of 6 AP patients have had hematologic response: 2 CHRs and 1 NEL. Two patients are too early to assess. One patient demonstrated resistance to BMS-354825 due to a T315I mutation in BCR-ABL found in 8 of 10 clones. This mutation confers resistance to BMS-354825 in preclinical studies. BCR-ABL mutation status is available for 3 additional AP patients: 2 patients had no mutations identified and 1 patient in CHR had M351T/A imatinib-resistant mutations. Of 3 patients for whom early cytogenetic data is available, 1 had a minor cytogenetic response (40% Ph+).To date BMS-354825 has been very well tolerated. Two patients in blast phase had evidence of mild tumor lysis syndrome. Dose escalation is continuing and phase II studies in chronic, accelerated and blast phase CML are currently being initiated. Further studies are required to establish LYN's potential role in imatinib-resistant CML. The clinical data demonstrate that BMS-354825 can frequently override imatinib resistance in advanced CML, and provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant accelerated and blast phase CML.

Major Cytogenetic Responses to BMS-354825 in Patients with Chronic Myeloid Leukemia Are Associated with a One to Two Log Reduction in BCR-ABL Transcript.

The success of imatinib for the treatment of chronic myeloid leukemia (CML) has created a need for a sensitive and accurate method of monitoring disease response and burden. Quantitative PCR (Q-PCR) has been previously shown to correlate well with cytogenetic response in patients treated with imatinib, whereby a one-log reduction in BCR-ABL transcript level corresponded well with attainment of a major cytogenetic response (MCyR) and a two-log reduction correlated with a complete cytogenetic response (CCyR) (Branford et al, Leukemia 17:2401, 2003). BMS-354825 is a novel orally bioavailable SRC/ABL kinase inhibitor with impressive activity against imatinib-resistant BCR-ABL mutant isoforms in vitro (Shah et al, Science 305:399, 2004). The compound is presently undergoing evaluation in phase I clinical trials (see Sawyers et al, Talpaz et al, abstracts submitted for this meeting). We sought to address whether cytogenetic responses in patients treated with BMS-354825 correlated with reduction in BCR-ABL transcript levels as determined by Q-PCR. Of 13 evaluable imatinib-resistant/intolerant patients with chronic phase CML treated at UCLA, four have attained a MCyR. Achievement of MCyR corresponded with a one to two-log reduction in BCR-ABL transcript as assessed by Q-PCR. In the majority of cases, a substantial reduction in BCR-ABL transcript was detected four weeks after initiation of BMS-354825. Overall, the median reduction in BCR-ABL transcript level after four weeks of therapy was 32%. Three of these patients had developed resistance to imatinib, and two harbored the common imatinib-resistant M351T mutation. Of the nine patients who have failed to achieve a MCyR, none have achieved a one log reduction in BCR-ABL transcript level. We conclude that similar to imatinib, BMS-354825-associated MCyR in chronic phase CML is highly associated with a one to two-log reduction in BCR-ABL transcript level. Furthermore, Q-PCR offers a rapid and reliable method to assess for disease response in this setting, which promises to be of significant clinical value. Although the maximal tolerated dose of BMS-354825 has yet to be determined, the compound is clearly capable of substantially reducing disease burden in patients with imatinib-resistant CML. Updated Q-PCR data from all chronic, accelerated, and blast crisis-phase patients on study at UCLA will be presented.

Triptolide-Induced Apoptosis Is Dependent on Caspase Activation and NFκB Signaling and Mediated by XIAP and Survivin Downregulation through the Mitochondrial Pathway in Leukemic Cells.

Triptolide, an immunosuppressor isolated from the Chinese herb, Tripterygium wilfordii Hook. F, has recently shown anti-tumor activities in a broad range of solid tumors. We examined its effects on leukemic cells and investigated mechanisms of apoptosis. Triptolide, at less than 100 nM, arrested cell growth and potently induced cell death in myeloid and lymphoid leukemic cells tested, including OCI-AML3, U937, Jurkat, KBM5, and K562 cells. In OCI-AML3 cells, triptolide induced caspase 3 activation, PARP cleavage and annexin V positivity with an IC50 of about 30 nM, at 24 hrs, all of which were inhibited by a general caspase inhibitor suggesting caspase dependent cell death. However, Triptolide-induced cell growth arrest was not affected by caspase inhibition. Treatment of OCI-AML3 cells with triptolide decreased XIAP and survivin expression, but did not affect Bcl2 and BclXL levels. Forced overexpression of XIAP attenuated Triptolide-induced cell death. Triptolide induced Bid cleavage, but Jurkat cells deficient in caspase 8 were only slightly less sensitive to triptolide than the wild-type counterpart indicating that Triptolide-induced cell death is caspase 8 independent. Jurkat cells deficient in receptor interacting protein (RIP) and therefore deficient in NFκB activation were resistant to Triptolide demonstrating that NFκB signaling is essential for Triptolide-induced cell death. Triptolide treatment induced cytosolic release of cytochrome C and loss of mitochondrial membrane potential, overexpression of Bcl2 effectively suppressed apoptosis induced by Triptolide, and caspase 9 knockout MEF cells were resistant to Triptolide suggesting criticality of the mitochondrial pathway. The antioxidants GSH (5 mM) and vitamin C (150 μM) did not protect from apoptotic cell death induced by Triptolide. In addition, Triptolide-induced apoptosis of blast crisis CML KBM5 cells was independent of their sensitivity or resistance to Imatinib: Triptolide killed Imatinib resistant KBMSTI cells as effectively as Imatinib sensitive KBM5 cells. Ex vivo studies showed that Triptolide also induced cell death in primary AML blasts. Collectively, our studies demonstrate that Triptolide potently induces caspase-dependent apoptosis and arrests cell growth in leukemic cells. Triptolide-induced cell death is dependent on NFκB signaling, and mediated by downregulation of XIAP and survivin through the mitochondrial pathway. The potent anti-leukemic activity of Triptolide in vitro warrants further investigation of this compound for the treatment of leukemia and other malignancies. This drug may also be potentially useful in overcoming Imatinib resistance in CML and Philadelphia chromosome positive ALL.

Effects of Adaphostin, a Novel Tyrphostin Inhibitor, in Diverse Models of Imatinib Mesylate Resistance.

The impetus for kinase inhibition as a therapeutic endpoint in leukemia research has been driven largely by the success of imatinib mesylate in chronic myelogenous leukemia. However, while imatinib mesylate has been extremely effective in the treatment of chronic phase CML, resistant disease often emerges in both Philadelphia positive ALL and aggressive phase CML patients. Adaphostin is a tyrphostin inhibitor originally developed to target the bcr/abl kinase and demonstrates selectivity in CML patient isolates. Here we show that in vitro, adaphostin is cytotoxic in numerous models that are resistant to imatinib. Features of adaphostin action in bcr/abl containing cells include apoptosis induction via an oxidant dependent mechanism and degradation of bcr/abl protein. Cell lines containing point mutations in p210 bcr/abl which confer imatinib resistance exhibit intracellular peroxide production and DNA fragmentation in response to adaphostin. Bcr/abl degradation also occurs but can be dissociated from peroxide production and subsequent apoptosis signaling. Similar data has also been obtained in cell lines containing p190 bcr/abl. Studies extending these observations into mononuclear and polymorphonuclear cells from imatinib resistant CML patients and Ph+ ALL patients confirm the same mechanism in primary clinical isolates. Collectively, these data suggest that reactive oxygen species generation by adaphostin bypasses imatinib resistance in CML and Ph+ ALL. Further studies dissecting the mechanism of ROS generation by adaphostin in imatinib resistant cells may lead to the identification of new therapeutic targets.

Imatinib-Resistant BCR-ABL Mutations Alter Oncogenic Potency, Kinase Activity and Substrate Selection.

Point mutations in the kinase domain of BCR-ABL are the most common cause of resistance to imatinib therapy in CML. Y253H, E255K, T315I, and M351T are among the most frequently reported point mutations in imatinib-resistant CML patients. Data from our lab and others has shown that T315I and E255K are detectable in some CML patients prior to imatinib therapy using relatively insensitive methods, whereas other mutations have only been detected after initiation of imatinib therapy. Additionally, some mutations persist while others become undetectable after discontinuation of imatinib treatment. Finally, mutations in the P-loop (phosphate binding loop) are associated with a worse clinical prognosis. Collectively, these epidemiological observations suggest the following model: some BCR-ABL point mutations confer gain-of-function relative to wild-type BCR-ABL that leads to excess proliferation, thereby explaining detection prior to imatinib-mediated selection. Conversely, mutations whose prevalence declines relative to wild-type BCR-ABL after cessation of imatinib therapy are hypomorphic, loss-of-function mutants. Here we provide biochemical and genetic evidence supporting this model. Primary murine bone marrow transformation assays using BCR-ABL-expressing retroviruses indicate that certain mutants (Y253H, E255K, T315I) indeed have greater oncogenic potency whereas other mutants have reduced transforming capacity when compared to wild-type BCR-ABL. To elucidate the molecular basis for the variable phenotypes of the different BCR-ABL mutants, we first examined the kinase activity of wild-type versus mutant full-length BCR-ABL. Utilizing GST-CrkL as a BCR-ABL specific substrate and myelin basic protein as a general kinase substrate, as well as by measuring levels of autophosphorylated BCR-ABL, we found that the kinase activities of Y253H, E255K, and T315I were essentially identical to wild-type BCR-ABL and the kinase activity of M351T was decreased approximately 2-fold. Therefore, altered intrinsic kinase activity may account for the variable transforming ability of some (M351T) but not all (Y253H, E255K, T315I) BCR-ABL mutants. We next looked for biochemical differences in the signaling properties of these BCR-ABL mutants. Wild-type and mutant BCR-ABL proteins were immunoprecipitated from stably expressing IL-3-independent Ba/F3 cells, then kinase assays were performed to determine if the mutants bind and subsequently phosphorylate different substrates. These experiments show that certain mutants (Y253H, T315I, M351T) bind and phosphorylate substrates distinct from wild-type p210 BCR-ABL. The identity of these substrates is currently under investigation by mass spectrometry, as these substrates have the potential to activate alternative signaling pathways. We conclude that certain BCR-ABL mutations, in addition to conferring imatinib-resistance, also influence the oncogenicity of BCR-ABL by altering intrinsic kinase activity or substrate selection. These studies provide molecular insight into why certain mutations are associated with a poor prognosis, and elucidation of the alternative signaling pathways activated by these mutants may provide novel therapeutic targets for imatinib-resistant CML.

BMS-354825 Potently Inhibits the Kinase Activity of KIT Activation Loop Mutations Associated with Systemic Mastocytosis and Induces Apoptosis of Mastocytosis Cell Lines.

Activating mutations of the activation loop (AL) of KIT are reported in several human neoplasms including a subset of patients with AML or systemic mast cell disorders. The small molecule tyrosine kinase inhibitor, imatinib, is a potent inhibitor of wild type KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GI stromal tumors. However, KIT AL mutations such as D816V that are typically found in AML and systemic mast cell disease are insensitive to imatinib (IC50 > 5–10 micromolar). In addition, acquired AL mutations are associated with imatinib resistance in GI stromal tumors. Experimental models of these diseases suggests that targeted inhibition of AL-mutant KIT in such cells would result in decreased proliferation and induction of apoptosis. BMS-354825 is a synthetic small-molecule ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomoloar range. In addition, BMS-354825 also inhibits many of the acquired BCR-ABL mutations associated with imatinib-resistant CML. Notably, BMS-354825 binds to the ATP-binding site in ABL regardless of the conformation of the AL and is therefore able to potently inhibit the kinase activity of AL-mutants of BCR-ABL. BMS-354825 is currently being tested in phase I studies for safety and efficacy in the treatment of imatinib-resistant CML as well as for various solid tumors. We investigated the inhibitory potential of BMS-354825 against KIT AL-mutants expressed by different mastocytosis/mast cell leukemia cell lines. We first examined the activity of BMS-354825 against the kinase activity of wild-type KIT kinase expressed by M07e cells. BMS-354825 potently inhibited the kinase activity of SLF-dependent M07E cells with an IC50 of 1–10 nM for autophosphorylation and cellular proliferation. In contrast, the IC50 for inhibition of GM-CSF-dependent proliferation was ~5 micromolar. BMS-354825 also potently inhibited the proliferation of the human mast cell leukemia cell line HMC-1 that expresses a juxtamembrane KIT V560G mutant kinase (IC50 1–10 nM). Notably, BMS-354825 inhibited autophosphorylation of KIT D816V with an IC50 of ~100 nM. Similar potency for inhibition of proliferation and induction of apoptosis (IC50 150–400 nM) was seen using three different cell lines expressing human D816V or the homologous murine D814Y KIT kinase. We conclude that BMS-354825 potently inhibits the kinase activity of wild-type KIT and imatinib-resistant KIT AL-mutant kinases associated with AML and systemic mast cell disorders. If BMS-354825 proves to be safe and tolerable in ongoing phase I trials, it should be further tested in clinical studies targeting patients with human malignancies associated with imatinib-resistant KIT AL-mutations.

Hematologic and Cytogenetic Responses in Imatinib-Resistant Chronic Phase Chronic Myeloid Leukemia Patients Treated with the Dual SRC/ABL Kinase Inhibitor BMS-354825: Results from a Phase I Dose Escalation Study.

Disease relapse in CML patients treated with imatinib is often associated with mutations in the BCR-ABL gene that interfere with the ability of imatinib to block BCR-ABL kinase activity. BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with more than 100-fold greater potency than imatinib that has in vitro and in vivo preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants (Shah et al, Science, 305:399, 2004). Here we report the phase I clinical results of BMS-354825 in Philadelphia chromosome positive (Ph+) CML patients in chronic phase with hematologic progression or intolerance while being treated with imatinib. To date (Aug 6, 2004), 29 patients have been treated on 9 cohorts with doses ranging from 15 to 180 mg of BMS-354825 per day given in single or divided doses for 5–7 days per week, for up to 9 months. Similar to imatinib, BMS-354825 has been well tolerated in all patients, with a single episode of grade 4 thrombocytopenia as the only potential drug related adverse event. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Serum levels well above the concentration required to block CML cell proliferation in vitro have been readily achieved without side effects. Pharmacodynamic studies demonstrate greater than 50 percent inhibition of phosphorylation of the BCR-ABL substrate CRKL and the SRC kinase Lyn, consistent with the serum concentrations observed in pharmacokinetic studies. Patients not receiving optimal clinical benefit were escalated to the next higher dose for which safety parameters were available, thereby allowing a preliminary assessment of clinical activity. To date, 26 patients (22 with imatinib resistance, 4 with imatinib intolerance; average CML duration 6.1 years) have been followed for greater than 4 weeks and are eligible for assessment of hematologic benefit. 22 patients had detectable BCR-ABL kinase domain mutations prior to starting BMS-354825. All 26 patients have been treated with doses of 35 mg per day or greater and have had clinical benefit, including 19 with complete hematologic responses (73%). Of the 7 partial responders, two subsequently had disease progression, one of whom had expansion of a CML subclone containing the imatinib-resistant T315I mutation in BCR-ABL, which also confers resistance to BMS-354825 in preclinical studies. The other 5 partial responders are now being treated with higher doses to attempt conversion to complete hematologic response. 11 of 21 patients (52%) treated for greater than 3 months have cytogenetic benefit, including 6 major (1–35% Ph+), 1 minor (36–65% Ph+) and 4 minimal (66–95% Ph+) cytogenetic responses. One patient has achieved complete cytogenetic response. Dose escalation continues, and phase II studies in chronic, accelerated and blast crisis CML are currently being initiated. These data provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant chronic phase CML.

A New Abl Kinase Inhibitor (AMN107) Has In Vitro Activity on CML Ph+Blast Cells Resistant to Imatinib.

Imatinib mesylate (STI571), an inhibitor of the bcr/abl tyrosine kinase, is rapidly becoming the first-line therapy for chronic myeloid leukemia (CML). Imatinib has proved remarkably effective at reducing the number of leukemia cells in individual CML patients and promises to prolong life substantially in comparison with earlier treatments. However, the development of resistance to this drug is a frequent setback, particularly in patients in advanced phases of the disease. Therefore, new inhibitors of bcr/abl are needed. Very recently, a new bcr/abl inhibitor, AMN107, has been synthesized. We have tested AMN107 on leukemic cell lines and on blasts isolated from imatinib-resistant CML patients. Western blot analysis with phosphospecific antibodies revealed that in K562 cells AMN107 (10 nM) down-regulated phosphorylation of bcr/abl Tyr177, while the phosphorylation levels of Tyr412 were unaffected. This finding seems particularly important because recent evidence has demonstrated that the signaling pathway emanating from Tyr177 plays a major role in the pathogenesis of CML. Indeed, phosphorylated Tyr 177 forms a high-affinity binding site for the SH2 domain of the adapter protein Grb2. The main effectors of Brb2 are Sos and Ras, however Grb2 also recruits the scaffolding adapter protein Gab2 to bcr/abl via a Grb2,Gab2 complex. Which results in activation of the PI3K/Akt and Erk signalling networks.In contrast, STI571, even if used at 200 nM, did not diminish phosphorylation of bcr/abl Tyr177. At 10 nM AMN107 blocked K562 cells in the G1 phase of the cell cycle. To obtain the same effect with imatinib, a 200 nM concentration was required. AMN107 did not affect cell cycle progression of bcr/abl-negative cell lines such as HL60 and NB4, even if the concentration was raised to 200 nM. AMN107 (5 μM for 24 h) significantly increased apoptosis rate in CML blasts isolated from patients resistant to the same concentration of imatinib. Therefore, AMN107 might represent a new bcr/abl selective inhibitor useful for overcoming imatinib resistance.