Imatinib-resistant Chronic Myeloid Leukemia Research Articles

BCR-ABL isoforms associated with intrinsic or acquired resistance to imatinib: more heterogeneous than just ABL kinase domain point mutations?

Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mutation status in two independent Nordic populations (n = 77) of imatinib-resistant CML patients. We detected BCR-ABL transcripts containing point mutations of residues in the P-loop, A-loop and other kinase domain residues in 32 patients (42%). In contrast to previous data, mutations in BCR-ABL were as frequently found in patients with primary resistance (56%) as with secondary resistance (53%). No T315I mutations were found in the study cohort. BCR-ABL splice variants were identified in a significant number of our cases (19%): BCR-ABL transcripts of variable length; a variant fusion transcript joining BCR exon 14 sequences to ABL exon 4; partial, in-frame-deletion of exon 4 due to induction of a cryptic splice site by the L248V and finally, alternative splicing of ABL exon 7 sequences. Though the majority of splice variants observed in this study do not encode functional proteins, alternative splicing appears to represent a common phenomenon in the biology of CML. We conclude that Abl KD point mutations represent a major mechanism of imatinib resistance. Other sequence irregularities were also detected, but their significance in conferring resistance is unclear. Diagnostic strategies looking for imatinib-resistant clones should be designed to detect a broader profile of BCR-ABL variants than just point mutations.

Dasatinib in the Treatment of Chronic Myeloid Leukemia

The recognition that the BCR-ABL gene and corresponding protein with deregulated tyrosine kinase (TK) activity is crucial for malignant transformation in chronic myeloid leukemia (CML), led to the synthesis of the smallmolecule drugs designed to interfere with BCR-ABL TK activation. The first tyrosine kinase inhibitor (TKI) was imatinib mesylate, introduced into clinical practice in 1998, which became the first choice drug in chronic phase CML. However, approximately 20-25% of patients initially successfully treated with imatinib will need alternative therapy because of unsatisfactory therapeutic results due to drug resistance. The risk of resistance is even higher in patients in advanced phases of CML. Resistance to imatinib is in about 35%-45% attributed to selection of clones expressing mutant forms of BCRABL which impair imatinib binding but preserve the kinase activity. The availability of second-generation TKIs has provided a new therapeutic option for patients with imatinib resistance. Among them, dasatinib is the first to be approved in the US and European Union for CML patients resistant or intolerant to imatinib. This drug is a dual SRC/ABL kinase inhibitor, 325-fold more potent than imatinib against cells expressing wild-type BCR-ABL and active in most clinically relevant BCR-ABL mutations, except highly resistant T315I. Several clinical trials have demonstrated that dasatinib is effective and generally well tolerated in imatinib resistant or intolerant CML and represents a promising therapeutic option for these patients. Keywords: Chronic myeloid leukemia, tyrosine kinase inhibitors, dual SRC/ABL kinase inhibitor, dasatinib, imatinib, AGP, A-loop, AP, ATP, AUC, BP, CCyR Comp, CFU, CHR, CML, CMoR, CP, CyR, EGFR, HR, IC50, JNK, MAPK, MCyR, MMoR, OS, P-gp, PCyR, PDGFR, PFS, Ph, PI3K, P-loop, SFKs, STAT5, TK, TKI, piperazin-1-yl-2-methylpyrymidin-4-ylamino thiazole-5-carboxamide, family kinase (SFks), ABL kinase domain, BCR-ABL mutants, F317L, Threonine315, M351T, colony-forming-unit (CFU) assay, IC50 0.2 nM), Lck (IC50 1.1 nM), Src (IC50 0.55 nM), Yes (IC500.41 nM), hOCT1 (human organic cation transporter 1), CYP3A4 enzyme

Seeking the causes and solutions to imatinib-resistance in chronic myeloid leukemia

Although only 5000 new cases of chronic myeloid leukemia (CML) were seen in the United States in 2009, this neoplasm continues to make scientific headlines year-after-year. Advances in understanding the molecular pathogenesis coupled with exciting developments in both drug design and development, targeting the initiating tyrosine kinase, have kept CML in the scientific limelight for more than a decade. Indeed, imatinib, a small-molecule inhibitor of the leukemia-initiating Bcr-Abl tyrosine kinase, has quickly become the therapeutic standard for newly diagnosed chronic phase-CML (CP-CML) patients. Yet, nearly one-third of patients will still have an inferior response to imatinib, either failing to respond to primary therapy or demonstrating progression after an initial response. Significant efforts geared toward understanding the molecular mechanisms of imatinib resistance have yielded valuable insights into the cellular biology of drug trafficking, enzyme structure and function, and the rational design of novel small molecule enzyme inhibitors. Indeed, new classes of kinase inhibitors have recently been investigated in imatinib-resistant CML. Understanding the pathogenesis of tyrosine kinase inhibitor resistance and the molecular rationale for the development of second and now third generation therapies for patients with CML will be keys to further disease control over the next 10 years.

The Multi-Kinase/ABL Inhibitor R763/AS703569 Induces DNA Endoreduplication and Apoptosis In Imatinib-Resistant CML Cells and Synergizes with Nilotinib, Dasatinib, and the Plk-1 Inhibitor BI 2536, In Producing Growth Inhibition.

AbstractAbstract 3394Resistance to imatinib is a major clinical problem and challenge in advanced chronic myeloid leukemia (CML). In most patients, drug-resistant mutants of BCR/ABL are detectable. Although most of these mutants still are responsive to second generation BCR/ABL kinase inhibitors (KI) such as nilotinib or dasatinib, drug responses are often short-lived. The BCR/ABL mutant T315I confers resistance against all available BCR/ABL KI, including nilotinib and dasatinib. More recent data suggest that several Aurora kinase (AuK) inhibitors block the kinase activity of BCR/ABL T315I. We have examined the growth-inhibitory effects of the AuK/ABL inhibitor R763/AS703569 (Merck-Serono, Darmstadt, Germany) on primary CML cells (chronic phase, n=12), the CML cell line K562, and Ba/F3 cells transfected with various imatinib-resistant mutants of BCR/ABL. As assessed by 3H-thymidine-uptake, R763/AS703569 was found to inhibit proliferation in imatinib-sensitive and imatinib-resistant primary CML cells in all donors tested, in imatinib-resistant and imatinib-responsive K562 cells, and in Ba/F3 cells harbouring various mutants of BCR/ABL (E255K, Y253F, H396P, T315I). The effects of R763/AS703569 on BCR/ABL-transformed cells were dose-dependent with IC50 values ranging between 0.001–0.1 μ M in K562 cells, <0.001-1 μ M in primary CML cells, and 0.001–0.1 μ M in BCR/ABL+ Ba/F3 cells. In all three patients in whom an imatinib-resistant BCR/ABL mutation was detected (one with V379I, one with F359V, and one with T315), R763/AS703569 was found to inhibit growth of primary CML cells at pharmacologic concentrations (IC50: 0.5, 0.005, and 0.05 μ M, respectively). As assessed by flow cytometry, the growth-inhibitory effects of R763/AS703569 were accompanied by DNA endoreduplication and consecutive apoptosis. Western blot experiments using anti-pCrkL antibody were performed and confirmed that R763/AS703569 blocks BCR/ABL activity at 1 μ M in K562 cells and BCR/ABL-transformed Ba/F3 cells. In addition, R763/AS703569 was found to block Aurora kinase A and Lyn phosphorylation in CML cells. By contrast, no effect of R763/AS703569 on phosphorylation or expression of Polo-like kinase-1 (Plk-1) was seen. In a next step, we explored the effects of drug combinations on growth of K562 cells and BCR/ABL-transformed Ba/F3 cells. In these experiments, R763/AS703569 was found to synergize with the ABL/multi-kinase inhibitors nilotinib (Novartis, Basel Switzerland) and dasatinib (Bristol-Myers Squibb, Princeton, NJ) in producing growth inhibition in CML cells (figure). Moreover, R763/AS703569 and the Plk-1 inhibitor BI 2536 (Boehringer Ingelheim, Vienna, Austria) were found to produce synergistic growth-inhibitory effects on CML cells in all samples tested. Synergistic effects of the KI applied were also seen in Ba/F3 cells exhibiting BCR/ABL T315I. In summary, our data show that the novel AuK/ABL inhibitor R763/AS703569 produces growth inhibition and apoptosis in BCR/ABL-transformed cells including those harbouring BCR/ABL T315I or other imatinib-resistant BCR/ABL mutants. Moreover, our data show that R763/AS703569 synergize with other multi-kinase/ABL inhibitors as well as with the Plk-1 inhibitor BI 2536 in producing growth inhibition in imatinib-resistant CML cells. Whether R763/AS703569, applied alone or in combination with other targeted drugs, can produces anti-leukemic effects in patients with advanced CML remains to be determined in clinical trials. [Display omitted] [Display omitted] Synergistic growth-inhibitory effects of R763/AS703569 and nilotinib in BCR/ABL T315I+ Ba/F3 cells (left), and R763/AS703569 and dasatinib in K562 cells (right). Disclosures:Valent:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.

The Dark Side of NRF2: Upregulation of NRF2 as a Mechanism for Resistance to Imatinib In CML.

AbstractAbstract 3401Chronic myeloid leukemia (CML) is a myeloproliferative disorder that results from a balanced translocation between chromosomes 9 and 22, creating the Philadelphia chromosome, and giving rise to a 210 kd (p210) fusion protein between Bcr and Abl. In addition to conferring anti-apoptotic properties, proliferative advantage and growth factor independence, BCR-ABL is associated with increased reactive oxygen species (ROS), DNA damage and altered repair that are proposed to play a role in genomic instability and disease progression. Imatinib mesylate, and other tyrosine kinase inhibitors (TKI)s, induce a substantial clinical response in patients with BCR-ABL-positive CML. However, resistance to TKIs through acquisition of mutations in BCR-ABL or BCR-ABL independent mechanisms is an increasing clinical problem. Thus, delineating mechanisms for disease resistance may allow the development of novel therapies for TKI-refractory disease.Cells deal with high levels of intracellular ROS by transcriptionally activating a number of anti-oxidant pathways. In particular, acute increases in ROS levels activate the Nrf2 pathway. Since NRF2 is known to transcriptionally activate drug resistance genes, and some anti-oxidant genes are transcriptionally increased in imatinib-resistant cells, we characterized the role of NRF2 in imatinib resistance in CML. We studied two imatinib-resistant (IR) BCR-ABL-positive cell lines, P210Mo7eIR with no detectable mutations in BCR-ABL and Baf3P210IR with BCR-ABL mutation T315I, derived from isogenic imatinib-sensitive (IS; P210Mo7e and Baf3P210) cells and parental controls (Mo7e and Baf3), and demonstrated that one of the IR cell lines, P210Mo7eIR, had very high steady state levels of Nrf2 (>10-fold) compared with isogenic controls. In addition, real-time PCR studies of NRF2 anti-oxidant target transcripts including, NQO1, NQO2, HMO1, GCLM and GCLC in P210Mo7eIR cells showed that they significantly increased compared with control cells. Importantly, to determine if NRF2 plays a role in the sensitivity of P210Mo7eIR cells to imatinib, we performed siRNA knockdown of NRF2 followed by growth of these cells in the presence of imatinib, and find that downregulation of NRF2 resensitizes P210Mo7eIR cells to imatinib, dramatically increasing cell death, compared with that of siRNA scrambled controls. Furthermore, inhibition of NQO1/2 leads to sensitivity of P210Mo7eIR cells to imatinib. Conversely, overexpression of NRF2 cDNA constructs in imatinib sensitive P210Mo7e renders these cells resistant to imatinib, compared with mutated NRF2 and vehicle controls. To determine whether this mechanism is operative in primary samples from IR cells, we examined 5 IS and 5 IR CML BM samples for steady state levels of NRF2 protein and transcript levels of NRF2 target genes. One of 5 IR CML samples with no BCR-ABL mutations and no IS CML samples had significantly increased NRF2 levels and target anti-oxidant activity. These data suggest that significant upregulation of Nrf2 acute stress pathways may be an alternative mechanism for TKI resistance that does not involves mutations in BCR-ABL. Thus, inhibition of NRF2 itself or its downstream targets may be important in therapeutic strategies in this sub group of TKI resistant CML. Disclosures:No relevant conflicts of interest to declare.

Human Blast Crisis Leukemia Stem Cell Inhibition with a Novel Smoothened Antagonist.

AbstractAbstract 1223Sonic hedgehog (Shh) pathway activators, particularly smoothened (SMO), are implicated in driving mouse leukemia progression by promoting leukemia stem cell maintenance. Thus, we investigated the role of the Shh signaling pathway in a humanized mouse model of blast crisis (BC) CML as well as the effects of a novel SMO antagonist on leukemia stem cell (LSC) quiescence, survival and self-renewal both in vitro and in vivo. Methods:Human BC CML progenitor cells (n=3 patients) were treated for 7 days in vitro with vehicle (DMSO) or a clinical grade SMO antagonist (Pfizer; PF-04449913) and harvested for Shh target gene Q-PCR analysis. FACS-purified BC CML LSCs (CD34+CD38+Lin-PI-) were transplanted intrahepatically into newborn RAG2-/-gc-/- mice. When human progenitors were detectable in peripheral blood (8 weeks), mice were treated for 14 days daily by oral gavage with 200μl of drug or vehicle (50% PEG in HBSS). The treatment included dasatinib alone (50 mg/kg), PF-04449913 (100 mg/kg) with or without dasatinib (50mg/kg). After treatment the percentage of LSC (CD34+CD38+CD45RA+CD123+Lin-PI-) was determined by FACS analysis of the bone marrow, peripheral blood, spleen and liver and the number of myeloid sarcomas was evaluated. In addition, cell cycle analysis was also performed on the same hematopoietic tissues of vehicle compared with treated mice. To determine if self-renewal potential was abrogated by treatment, human leukemic progenitors were serially transplanted and myeloid sarcoma formation was assessed in secondary recipients after 8 weeks. Results:BC CML progenitors (n=3 patient samples) harbored increased expression of Gli and downregulation of Ptch1 transcripts compared to normal cord blood. Notably, Gli expression decreased following in vitro treatment with PF-04449913. Treatment with PF-04449913 alone in human BC CML LSC transplanted mice (n=12 exp., n=46 mice) significantly reduced spleen size (p ≤ 0.05). However, SMO inhibition alone did not significantly inhibit bone marrow engraftment compared with vehicle (n=12 exp., n= 47 mice). Although dasatinib alone decreases myeloid sarcoma formation (p ≤ 0.05), like the SMO antagonist alone it did not eradicate the LSC in the liver or bone marrow (n=12 exp., n=36 mice). Combination treatment (n=12 exp., n=34 mice) with PF-04449913 and dasatinib revealed a significant decrease in LSC hepatic engraftment (P≤0.05) compared with the PF-04449913 or dasatinib alone. Moreover, combination treatment completely eradicated the capacity of LSC to form myeloid sarcomas formation (0.0) in secondary transplant recipients (n=5) compared with dasatinib that had an average of 1.8 tumors (n=6), PF-04449913 that had an average of 4.9 (n=7) and vehicle group that had 3.8 tumors (n=16).Hematopoietic progenitor assays performed with normal cord blood CD34+ progenitors (n=3 samples) showed no reduction in colony survival following treatment with PF-04449913 (1μM) compared with vehicle. Furthermore, there was no significant inhibition of normal CD34+ colony replating following PF-04449913 compared with vehicle treatment. Cell cycle analysis demonstrated a reduction in quiescent human leukemic cells in the peripheral blood and marrow following SMO inhibitor treatment. Conclusions:Combination therapy with a potent BCR-ABL inhibitor, dasatinib, and a selective SMO inhibitor abrogates the self-renewal capacity of human imatinib resistant blast crisis leukemia stem cells. A phase I clinical trial with PF-04449913 is currently underway and a Phase 1b trial involving dasatinib and PF-04449913 for patients with advanced phase, imatinib resistant CML will begin in the near future. Disclosures:Jamieson:Pfizer: Research Funding; CIRM: Research Funding.

Treatment with TKIs Overcomes Imatinib Resistance through the PLCgamma-1 Signaling Pathway In Imatinib Resistant Human CML Cell Lines

AbstractAbstract 4468Suppression of BCR-ABL1 catalytic activity by the tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) has dramatically improved the natural history of Chronic Myeloid Leukemia (CML) and - to date - represents the first and most successful example of effective anti-cancer targeted therapy {Druker, 2009}. Despite the success, patients can become resistant. Since IM-resistance in CML patients occurs despite adequate suppression of BCR-ABL activity, it is likely due to the activation of other pathways, and for this reason we need to discover novel Bcr-Abl independent pathways than can become the targets of resistant cells.Therefore we used immortalized CML human cell lines K562 and LAM84, both sensitive and resistant to Imatinib, to study the signaling in sensitive/resistant cell lines following treatment with 4 different compounds. Reverse-Phase protein microarrays were used to quantitatively map 35 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. Cells were incubated with IM 1uM, Dasatinib 1uM, Nilotinib 1uM or LY-29400210 uM and after 2 or 12 hours were placed in a preservative that suppresses fluctuations in kinase pathway proteins (Espina, 2008).5/35 protein endpoints were linked together and suppressed by Dasatinib, even in the resistant cell line: PLC-y-1-Tyr783, and its upstream (ShCTyr317 SrcTyr416) and downstream targets (mTORSer2448, STAT5Tyr694, ERKThr202/Tyr204) without interfering with AKT activation status on Ser473 compared to Imatinib (p=0.0031 for K562, p= 0.042 for LAM84), Nilotinib (p=0.0034 for K562, p= 0.043 for LAM84), LY-294002 (p=0.0009 for K562, p= 0.015 for LAM84).In Imatinib-sensitive cell line LAM84 there were no differences between Dasatinib and Imatinib in the modulation of the pathway, compared to IM-sensitive K562 cell line (p=0.005), thus confirming the different profile among these two CML models.Dasatinib showed a greater suppression of the PLC-y-1 pathway compared to LY-294002 in both resistant cell lines (K562, p=0.0009 and LAM84, p=0.015).These data confirm and extend the conclusions of Markova et al. (Oncogene 2010) showing PLC-y-1 as a mechanism of death in sensitive cells. Our data showed the mechanism by which second generation TKIs (dasatinib) can overcome Imatinib resistance by suppressing the PLC-y-1 pathway. Disclosures:No relevant conflicts of interest to declare.

Comparasion Between Nilotinib and Dasatinib as Second-Line Therapy for Patients with Chronic Myeloid Leukemia: A Single Center Retrospective Study.

AbstractAbstract 3440Nilotinib and dasatinib are second-generation tyrosine kinase inhibitors (TKI) used in patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib. There are no randomized clinical trials comparing these drugs in this context. The aim of this study was to compare, retrospectively, the hematological, cytogenetic and molecular response in patients submitted to these second-generation TKI at Hemorio, a public brazilian institution. A total of 114 patients were analyzed, 63 received nilotinib and 51 dasatinib as second-line therapy (55.3% and 44.7%, respectively). The following variables were equally distributed between these two groups (nilotinib vs. dasatinib, respectively): male sex (54% vs. 60.8%, p=0.46), median age at diagnosis (46 vs. 45 years, p=0.76), median time in months using imatinib before the switch (45.2 vs. 44.1, p=0.96), resistance to imatinib (98.4% vs. 98%, p=0.88), presence of the mutation T315I (3.2% vs. 3.9%, p=0.09), patients in chronic phase before the switch (85.7% vs. 86.3%, p=0.93). Use of another second generation TKI, as a third-line therapy, was necessary in 30 out of the 114 patients analyzed (26.1%) because of lack of response. This modification was slightly more frequent in the group initially submitted to nilotinib (31.7% vs. 19.6%, p=0.21). Patients who used a third-line therapy were excluded from response and survival analyzes. Response rates after the second-generation TKI were similar between these two groups (nilotinib vs. dasatinib): complete hematological response until three months (77.8% vs. 87.3%, p=0.24), complete cytogenetic response until six months (21.6% vs. 22.2%, p=0.95) and 12 months (32.4% vs. 33.3%, p=0.94) and major molecular response reached before 12 months (32.7% vs. 21.6%, p=0.25). Two-year overall survival (OS) and progression free-survival (PFS) were similar between these two groups (nilotinib vs. dasatinib, respectively): 92.2% vs. 87.8% (p=0.38) for OS and 87.8% vs. 83.7% (p=0.14) for PFS. Although not statistically significant, two-year OS was inferior in the group of patients who needed a third-line therapy (70.5% vs. 95.6%, p=0.70). Our results suggest that the response and survival rates are similar between nilotinib and dasatinib as second-line therapy for patients with imatinib resistant or intolerant CML. Also, they suggest an inferior prognosis for patients who need a third-line therapy. In this way, the choice between these two TKI for second-line therapy should be guided by the clinical characteristics and the mutation status of the patient. Disclosures:Lobo:NOVARTIS: Research Funding.

Impact of Prior Second-Generation Tyrosine Kinase Inhibitors on the Outcome of Hematopoietic Stem Cell Transplantation for Chronic Myeloid Leukemia

AbstractAbstract 2267Prior to the introduction of the tyrosine kinase inhibitors (TKI) into clinical practice, the only curative therapy for chronic myeloid leukaemia (CML) was allogeneic hematopoietic stem cell transplantation (HSCT). HSCT is nowadays recommended for patients in accelerated phase, blastic phase or with the T315I mutation and for patients who experience second-line TKIs (TKI2) failure or intolerance. The impact of TKI2 for CML on the results of subsequent HSCT has not yet been clearly established, although preliminary data does not suggest an increase of non relapse mortality (NRM) in patients previously treated with these agents. To assess whether exposure to TKI2s before HSCT adversely affects outcome, we retrospectively analyzed 31 patients with CML reported to the French registry for HSCT between January 2001 and December 2008 who received a first HSCT for imatinib resistant or intolerant CML subsequently treated with either nilotinib or dasatinib or both.The median age at diagnosis was 39.8 years (range, 19–61). At the start of TKI, 15 (48%) patients were in chronic phase, 12 (39%) patients were in accelerated phase and 4 (13%) patients were in blast crisis. For chronic phase patients, 6 (40%) patients were classified as low Sokal risk, 7 (46%) as intermediate risk, and 2 (14%) as high risk. After imatinib failure (among the 27 patients in chronic or accelerated phase at diagnosis), 21 and 6 patients received dasatinib and nilotinib, respectively. Among these patients, 9 patients received a third line therapy, including 5 sequential therapies with both drugs. The best response to the second and third-line treatments with TKI2 was a complete molecular response in 2 patients, a major molecular response in 2 patients, a complete cytogenetic response (CCR) in 7 patients, a complete hematologic response in 14 patients and no response in 2 patients. 14 patients eventually failed TKI2 treatment because of resistance, whereas 8 were considered as intolerant. Of 14 patients who developed resistance to TKI2, 2 had a mutation identified (T315I in both cases).Median interval from diagnosis to HSCT was 19 months (range, 3–151). At time of transplant, 21 patients were in chronic phase, 10 in accelerated phase and none in blast crisis. 19 patients received a graft from an unrelated donor whereas 12 a match related donor. Stem cell source was peripheral blood, bone marrow or cord blood in 20, 8 and 3 patients, respectively. The conditioning regimen was myeloablative in 21 patients combining either TBI and cyclophosphamide (9 patients) or high dose IV busulfan and cyclophosphamide (12 patients), and a RIC for 10 patients. All patients engrafted successfully: median time to neutrophil and platelet recovery was 18 days and 21 days respectively. Grade 2–4 acute graft-versus-host disease (aGVHD) was observed in 11 (37.9%) patients; grade 3–4 aGVHD occurred in 6 (20.6%) patients. Chronic GVHD was observed in 15 (60%) of 25 patients alive after day 100. The median follow-up after HSCT is 27 months (range, 1.2–50.2). At time of analysis, 11 (35%) patients died, 7 (22.5%) from NRM and 4 (12.5%) from progression of disease. NRM was due to infection (3 patients), GVHD (2 patients), post transplant lymphoma disease (1 patient) and unknown cause (1 patient). The 1-year overall survival (OS) was 79.2% (95% CI, 64,3-94,1%) and the estimated 2-year OS was 55,5% (95% CI, 35,0-75,9%). One-year relapse and NRM rates were respectively 10,3% (95% CI, 2.5–24.6%) and 19.1% (95% CI, 6.7–36.2%). In univariate analysis, no variable had a significant impact on outcome among Sokal score, disease phase at diagnosis, Grathwol score, age at HSCT, time from diagnosis to HSCT, or quality of response before HSCT. In a multivariate analysis, only quality of response (at least better than CCR) was significantly associated with a better outcome in terms of OS (p=0.0459, HR=0.17, 95% CI=0.03-0.97).In conclusion, TKI2 prior to HSCT did not result in an increased risk of NRM. However, our observation that patients in cytogenetic or molecular response at time of HSCT have a significant better outcome underscores the importance of a stringent prospective molecular monitoring under TKI2 therapy and the need for prognostic factors of response under TKI2. Such elements could help to better define after initiating TKI2 treatment patients that could really benefit from HSCT and the best timing of the procedure. Disclosures:No relevant conflicts of interest to declare.

Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

AbstractAbstract 4469The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures:No relevant conflicts of interest to declare.

Prior Treatment with Dasatinib Followed by Stem Cell Transplantation In Patients with CML In Advanced Phases with BCR-ABL Mutation.

AbstractAbstract 4485 Background:The prognosis for patients with chronic myeloid leukemia (CML) in advanced phases remains dismal, especially for those who develop ABL mutation. Administration of second-generation tyrosine kinase inhibitors (TKI) might provide an effective approach for patients with BCL-ABL mutation, but it only offers short-tem benefit in most cases. Allogeneic stem cell transplantation (allo-SCT) seems the only curative therapy for advanced CML, and limited data exist on the effectiveness of allo-SCT following prior treatment with new TKI dasatinib. Objective and Methods:To evaluate the efficacy of this combination therapy, patients with advanced disease harboring BCL-ABL mutation were enrolled between May 2009 and May 2010, and given dasatinib for a predetermined period, and then referred to myeloablative allo-SCT. All the patients were conditioned with busulfan/cyclophosphamide based regimens. ATG was used for those who received HLA-haploidentical transplantation. Cyclosporin, mycophenolate mofetil and short-term methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. Results:(1) Five patients with accelerated phase (n=1) and blast crisis (n=4) were enrolled. The median age was 37 years (range, 11–42 years). All the patients had a history of chronic phase (CP) before enrollment. Three patients progressed to advanced phases on imatinib. The other two patients didn't receive any TKIs during the CP. When progressing to blast crisis, imatinib was administered but showed no response. BCR-ABL mutations were detected at the beginning of enrollment. The mutations were F359C, D296G, E459G, Y253H, Y253F. (2) After enrollment, patients were treated with dasatinib for 1–3months (median, 2 months). At the time of transplant, all patients achieved complete hematological response and returned to CP, and two patients achieved partial cytogenetic response. Dasatinib was well-tolerated expect one patient who developed pleural effusion. (3) Donors were HLA-haploidentical related in two cases,HLA-identical related in one case, and HLA-identical unrelated in two cases, respectively. All patients engrafted successfully, with a median time to neutrophil (>500/ul) and platelet (>30000/ul) recovery of 15 and 17 days respectively. Two patients experienced grade III-IV acute GVHD). Chronic GVHD (cGVHD) was observed in three patients, including two with extensive cGVHD. The relapse rate after transplantation was 0. Only one patient died from pulmonary fungal infection while in complete molecular remission. After a median follow-up of 6.5 months, four patients were alive with complete cytogenetic remission, including three in complete molecular remission. Conclusion:Dasatinib given before allo-HSCT do not negatively affect transplant engraftment and response rate. Myeloablative transplantation for imatinib-resistant CML may have a satisfactory outcome when combined with dasatinib, which could provide a good quality of remission prior to transplantation and a curable opportunity after transplantation. Disclosures:No relevant conflicts of interest to declare.

Nilotinib and Dasatinib Produce Synergistic Growth-Inhibitory Effects In Imatinib-Resistant CML Cells, Including Subclones Bearing the Multi-Resistant BCR/ABL Mutant T315I

AbstractAbstract 2280In most patients with chronic myeloid leukemia (CML), complete cytogenetic remission can be achieved with the BCR/ABL tyrosine kinase inhibitor (TKI) imatinib. However, not all patients are long-term responders. A major cause of acquired resistance against imatinib is the development of BCR/ABL mutations in subclones. In most of these patients, a second generation TKI is prescribed. However, the T315I mutant of BCR/ABL introduces resistance against most TKI, including nilotinib and dasatinib. One approach to overcome drug resistance in BCR/ABL T315I+ CML cells may be to apply drug combinations. Recent data suggest that the mechanisms through which dasatinib and nilotinib act on BCR/ABL differ from each other and that both drugs act on multiple additional targets in CML cells. Here, we show that dasatinib and nilotinib cooperate with each other in producing growth inhibition in imatinib-sensitive and imatinib-resistant CML cells, including subclones bearing BCR/ABL T315I. The drug combination was tested on leukemic cells obtained from 9 patients with chronic phase (CP) CML and 3 with blast phase (PB) of CML. Samples were assessed from 4 patients at the time of diagnosis, and against cells from 8 patients (CP, n=5; BP, n=3) who had developed resistance against one or more BCR/ABL TKI. In all 3 patients in PB, the T315I mutant was detectable. As expected, nilotinib and dasatinib failed to inhibit proliferation of cells harbouring BCR/ABL T315I when applied as single agents. However, the combination xnilotinib+dasatinibx produced synergistic effects in most samples, including primary CML cells and Ba/F3 cells harbouring BCR/ABL T315I. Interestingly, in all 3 patients with BP (BCR/ABL T315I+), strong cooperative or even synergistic growth-inhibitory effects were observed in primary CML cells, resulting in substantial anti-leukemic effects seen at reasonable (pharmacologic) drug concentrations (< 1 μ M) (figure). Based on these results, we treated one patient with TKI-resistant CML in hematologic relapse in whom 2 BCR/ABL mutant-bearing subclones, one clinically resistant against nilotinib (F359V) and one clinically resistant against dasatinib (F317L) had been detected, with a combination of nilotinb (800 mg p.o. daily) and dasatinib (50 mg/day p.o., days 1–5 every third week). A transient hematologic response was obtained in this patient, and except for mild bone pain, no side effects were recorded. Moreover, we were able to show that during treatment with xnilotinib+dasatinibx, the number of CD34+/CD38-/CD33+ CML stem cells decreased from clearly measurable levels (0.005%) to nearly undetectable levels (0.0002%). Finally, ex vivo analyses of leukemic blood cells confirmed, that the combination xnilotinib+dasatinibx produced strong cooperative growth-inhibitory effects in both disease-components, i.e. the F359V-bearing subclone and the F317L-bearing subclone. In summary, our data show that the combination of dasatinib and nilotinib can override acquired TKI resistance in CML, and can suppress growth of various imatinib-resistant subclones including cells that bear BCR/ABL T315I or other BCR/ABL mutants. Whether this combination can suppress imatinib-resistant subclones in CML for prolonged time periods or even can eradicate neoplastic stem cells remains in CML patients to be determined.Synergistic effects of nilotinib and dasatinib on primary leukemic cells obtained from a patient with a BCR/ABL T315I+ blast phase of CML [Display omitted] Disclosures:Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Molecular Mechanism Regulating Foxo In Leukemia Initiating Cells of Chronic Myeloid Leukemia.

AbstractAbstract 3391Chronic myeloid leukemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. Although the development of imatinib, a small molecule inhibitor of ABL, represents a breakthrough in the treatment of CML, major part of patients treated in chronic phase CML are not off therapy due to resistance or intolerance. Recent studies have suggested that imatinib is a potent inhibitor against differentiated leukemia cells, but does not deplete leukemia-initiating cells (LICs) responsible for recurrence of CML. To date, therapeutics that can eradicate CML LICs, however, have remained under investigation. To overcome these clinical problems, here we studied the molecular mechanisms regulating maintenance of imatinib-resistant CML LICs by forkhead transcription factor Foxo3a.We first generated a mouse CML model by using retroviral induction of BCR-ABL-ires-GFP gene into mouse immature hematopoietic cells, and the cells were subsequently transplanted into irradiated recipient mice. These experiments showed that CML LICs were highly enriched in c-Kit+Lin−Sca-1+ (KLS+) population in BCR-ABL+ CML cells. Serial transplantation experiments for CML LICs originated from Foxo3a-deficient mice and littermate wild-type mice indicated that Foxo3a-deficiency reduced lethality of recipient mice at third transplantation. Although recipients that transplanted with wild-type LICs developed CML and acute lymphocytic leukemia (ALL) at third transplantation, we did not observe development of ALL or CML in recipients of Foxo3a deficient LICs after 45 days post-third transplantation, suggesting that the Foxo3a deficient LICs lose their potential to generate malignancies. In addition, a combination of Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo, indicating that Foxo3a plays an essential role for the maintenance of imatinib-resistant CML LICs (Naka et al., Nature 463, 676–680, 2010).Interestingly, when we examined sub-cellular localization of Foxo3a transcription factor in the CML LICs, we found two CML LIC populations; one population was the cells with nuclear localization of Foxo3a (Foxo3a transcription factor is active) and the other population was the cells with cytoplasmic localization of Foxo3a (Foxo3a is inactive). To understand the molecular mechanisms regulating Foxo3a in CML LICs, we next evaluated the activity of upstream BCR-ABL, PI3K, PDK1, and Akt signaling pathway by fluorescence immunohistochemistry. BCR-ABL activity that was determined by phosphorylation levels of CrkL, a down-stream target of BCR-ABL, was detected in almost all of the CML LICs. However, unexpectedly, phosphorylation levels of Akt in the CML LICs with nuclear localization of Foxo3a appeared to be lower than that in the CML LICs with cytoplasmic localization of Foxo3a, despite it is widely believed that BCR-ABL induces activation of Akt signal. Consistent with Akt phosphorylation status, we detected low levels phosphorylation of PDK1 and PI3K p85, upstream regulators for Akt, in the CML LICs with nuclear localization of Foxo3a. Interestingly, expression levels of the cell proliferation antigen Ki67 were lower in the CML LICs with nuclear Foxo3a than that in the CML LICs with cytoplasmic Foxo3a. These results suggest that Foxo3a responsible for maintenance of imatinib-resistant CML LICs may be regulated by molecular mechanisms that are involved in dormancy in CML LICs. Disclosures:No relevant conflicts of interest to declare.

A Computational Approach to Analyze the Mechanism of Action of the Kinase Inhibitor Bafetinib

Prediction of drug action in human cells is a major challenge in biomedical research. Additionally, there is strong interest in finding new applications for approved drugs and identifying potential side effects. We present a computational strategy to predict mechanisms, risks and potential new domains of drug treatment on the basis of target profiles acquired through chemical proteomics. Functional protein-protein interaction networks that share one biological function are constructed and their crosstalk with the drug is scored regarding function disruption. We apply this procedure to the target profile of the second-generation BCR-ABL inhibitor bafetinib which is in development for the treatment of imatinib-resistant chronic myeloid leukemia. Beside the well known effect on apoptosis, we propose potential treatment of lung cancer and IGF1R expressing blast crisis.

Carboxyamidotriazole inhibits cell growth of imatinib-resistant chronic myeloid leukaemia cells including T315I Bcr–Abl mutant by a redox-mediated mechanism

Mutation of the Bcr–Abl oncoprotein is one of most frequent mechanisms by which chronic myelogenous leukemia (CML) cells become resistant to imatinib. Here, we show that treatment of cell lines harbouring wild type or mutant BCR–ABL with carboxyamidotriazole (CAI), a calcium influx and signal transduction inhibitor, inhibits cell growth, the expression of Bcr–Abl and its downstream signalling, and induces apoptosis. Moreover, we show that CAI acts by increasing intracellular ROS. Clinically significant, CAI has also inhibitory effects on T315I Bcr–Abl mutant, a mutation that causes CML cells to become insensitive to imatinib and second generation abl kinase inhibitors.

Extensive pleural and pericardial effusion in chronic myeloid leukemia during treatment with dasatinib at 100 mg or 50 mg daily

Dasatinib is considered an effective drug in imatinib-resistant chronic myeloid leukemia. Although reported to be well-tolerated, severe events such as pleural or pericardial effusion have been reported at 140 mg daily. We examined our chronic myeloid leukemia patients treated with dasatinib at 100 mg or 50 mg daily and identified 4 of 13 patients who developed marked effusion formation. In 2 patients, grade III/IV pleural and/or pericardial effusions were recorded. All 4 patients had received previous anti-leukemia therapy but none had pre-existing cardiac or pulmonary diseases. In 3 patients, dasatinib had to be discontinued despite treatment with diuretics and glucocorticosteroids. In conclusion, dasatinib-treated chronic myeloid leukemia patients are at risk for the development of pleural and pericardial effusions even when the drug is administered at 100 mg or 50 mg daily. Therefore, all patients should be examined for pre-existing comorbidity and risk factors before starting dasatinib and all should have repeated chest X-rays during long-term dasatinib therapy.

Efficacy of dasatinib for the treatment of intractable chronic myeloid leukemia

Dasatinib (DAS) is a well tolerated oral dual SRC inhibitor with remarkable activity against all phases of imatinib-resistant chronic myeloid leukemia (CML). This paper focuses on the activity of DAS in intractable CML, and reviews outcomes of patients enrolled on DAS clinical trials. Safety and tolerability as well as practical tips for management of side-effects, and drug interactions are included.

Activity of the potent dual Abl/Src tyrosine kinase inhibitor FB2 against Bcr–Abl positive cell lines in vitro and in vivo

We have previously shown the inhibition of the small-molecule inhibitor FB2 on imatinib-sensitive and resistance CML cell lines with the wild-type Bcr–Abl fusion gene. Here we report the potent and selective antiproliferation on FB2 on transfected Ba/F3 p210 cell lines expressing various isoforms of Bcr–Abl (wild-type, Y253F, T315I). FB2 which orients Bcr–Abl and Src kinase activities, is shown to override imatinib-resistance CML involving Y253F mutation in the Abl kinase domain of the fusion protein except T315I in vivo and in vitro. Thus, we present FB2 that displays potency toward Bcr–Abl and Src as the molecular target, and which could potentially be used to override drug resistance in CML.

Characteristics of BCR–ABL kinase domain point mutations in Chinese imatinib-resistant chronic myeloid leukemia patients

To explore the characteristics of BCR-ABL kinase domain point mutations in Chinese chronic myeloid leukemia (CML) patients, a cohort of 127 patients with hematologic or cytogenetic resistance to imatinib are screened by direct sequencing. Mutations are found in 74 patients (58%). More patients with hematologic resistance show mutations compared to patients with cytogenetic resistance (70% vs 44%, p = 0.002), and more patients with acquired resistance present mutations compared to patients with primary resistance (68% vs 48%, p = 0.031). Frequencies of mutations were similar in early chronic phase (ECP), late chronic phase (LCP), accelerated phase, and blast phase (BP) patients (56%, 58%, 50%, and 69%, respectively; p > 0.05). Overall, 25 mutants are found in 21 amino acid sites, and four of them (I418V, E450A, E453L, and E455K) are first reported here. All patients with these four mutants either progress to or reenter the BP. The most frequent mutant is M244V, followed by Y253H, F359C/V/I, G250E, E255K, and T315I. Only seven patients (9%) have T315I mutants, and all showed hematologic resistance. Three of them were in the ECP and three in the LCP. Look-back studies show that mutants were detected 0-20 (median 7) months ahead of the appearance of clinical resistance in 15 tested patients with acquired resistance. ABL mutations are common in Chinese imatinib-resistant CML patients and are associated with clinical resistance. Chinese patients also seem to have unique profiles in the types and frequencies of some mutants, the disease phases of patients with T315I mutation, and the frequency of mutations in CP patients.

Abstract A4: Withaferin A induces apoptosis in imatinib-resistant CML cells by repressing the Akt signaling pathway

Abstract Although imatinib therapy of chronic myelogenous leukemia is effective, the resistance to imatinib challenges the treatment of this disease. Therefore, search of novel drugs to overcome imatinib resistance is a critical issue in clinic. Withaferin A (WA), an extract of Withania somniferia, exhibits anticancer activity on a number of solid tumors. In this study, we investigate the effect of WA on imatinib-sensitive and -resistant CML cells. WA at low concentrations induces autophagy in imatinib- sensitive K562 cells. Co-treatment of chloroquine suppresses autophagy and switches WA-treated K562 cells to apoptosis. This data indicates that autophagy protects K562 cells from apoptosis induced by WA. However, we find that WA triggers caspase activation and apoptosis in imatinib-resistant T315I-positive cells and this effect is associated with downregulation of Akt activity. Treatment of the AKT inhibitor LY294002 also causes apoptosis in imatinib-resistant T315I- positive cells. Ectopic expression of constitutively active Akt reverses WA-induced apoptosis and caspase activation in imatinib-resistant T315I-positive cells. Molecular study demonstrates that WA represses the Akt signaling pathway by decreasing Akt expression. We find that WA causes Akt degradation through the ubiquitin- and proteasome-dependent pathway. More importantly, WA also induces AKT downregulation and apoptosis in primary CML cells. Taken together, our results suggest that imatinib-resistant T315I-positive cells are more addicted to Akt-dependent survival pathway and are more sensitive to WA. Therefore, WA can be useful for the treatment of imatinib-resistant CML. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A4.