Imatinib-sensitive CML Research Articles

Assessment of azithromycin as an anticancer agent for treatment of imatinib sensitive and resistant CML cells

IntroductionChronic Myeloid Leukemia (CML) is a hematological disease which is characterized by the presence of BCR-ABL fusion protein. Imatinib (IMA), a tyrosine kinase inhibitor of BCR-ABL, is used as a frontline treatment.Although IMA aids in killing a majority of leukemia cells, it may not kill CML stem cells which are the primary roots of disease and therapy resistance. Recently, antimicrobial drugs have been gaining attention because of their selective targeting of cancer cells. Therefore, we now ask if combinational therapy of IMA with a targeted antimicrobial drug Azithromycin (AZT) can enhance the treatment efficiency in IMA resistant CML. MethodsK562S (IMA sensitive) and K562R (IMA resistant) cells were treated with increasing concentrations of AZT to determine its effects on cell proliferation and apoptosis. Cell viability, apoptosis, caspase3/7 activity and P-glycoprotein (Pgp) function were investigated with spectrophotometric MTT assay and flow cytometric Annexin V staining, caspase 3/7 activity, and Rhodamine123 staining assays respectively. The expression levels of pro-apoptotic (BAX, BAD and BIM), anti- apoptotic (BCL-XL and BCL-2) and drug transporter (MDR-1 and MRP-1) genes were assessed with qRT-PCR. ResultsAZT treatment alone inhibited cell viability, induced apoptosis and enhanced caspase 3/7 activity in both K562S and high MDR-1 (Pgp) expressing K562R cells. Moreover, combination of AZT/IMA suppressed cell viability, induced apoptosis and caspase3/7 activity more effectively and significantly compared to K562R cells treated with only IMA or AZT. Furthermore, AZT and AZT/IMA combination decreased Pgp function in K562R cells in comparison with their controls. Based on qRT-PCR data, single AZT and combined AZT/IMA treatment also induced BAX/BCL-2 ratio significantly in both K562S and K562R cells. ConclusionSingle AZT and AZT/IMA combinational treatment can be proposed as a promising and effective treatment strategy for CML. One of the mechanisms underlying the potent anticancer effect of combined AZT/IMA could be its ability to inhibit Pgp function and increase intracellular accumulation of IMA which leads to the induction of apoptosis in K562R cells.

Abstract 366: Association of MLL2/KMT2D and MLL3/KMT2C with chronic myeloid leukemia

Abstract Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm linked to the Philadelphia chromosome presence that generates the BCR-ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR-ABL tyrosine kinase. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the MLL2/KMT2D and MLL3/KMT2C genes of histone methyltrasnferases. Here we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression. Both genes were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. Our results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance. Citation Format: Doralina do Amaral Rabello Ramos, Vivian D'Afonseca da Silva Ferreira, Maria Gabriela Berzoti-Coelho, Sandra Mara Burin, Cíntia Leticia Magro, Maira da Costa Cacemiro, Belinda Pinto Simões, Felipe Saldanha-Araujo, Fabíola Attié Castro, Fábio Pittella-Silva. Association of MLL2/KMT2D and MLL3/KMT2C with chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 366.

MLL2/KMT2D and MLL3/KMT2C expression correlates with disease progression and response to imatinib mesylate in chronic myeloid leukemia

BackgroundChronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm whose pathogenesis is linked to the Philadelphia chromosome presence that generates the BCR–ABL1 fusion oncogene. Tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM) dramatically improved the treatment efficiency and survival of CML patients by targeting BCR–ABL tyrosine kinase. The disease shows three distinct clinical-laboratory stages: chronic phase, accelerated phase and blast crisis. Although patients in the chronic phase respond well to treatment, patients in the accelerated phase or blast crisis usually show therapy resistance and CML relapse. It is crucial, therefore, to identify biomarkers to predict CML genetic evolution and resistance to TKI therapy, considering not only the effects of genetic aberrations but also the role of epigenetic alterations during the disease. Although dysregulations in epigenetic modulators such as histone methyltrasnferases have already been described for some hematologic malignancies, to date very limited data is available for CML, especially when considering the lysine methyltransferase MLL2/KMT2D and MLL3/KMT2C.MethodsHere we investigated the expression profile of both genes in CML patients in different stages of the disease, in patients showing different responses to therapy with IM and in non-neoplastic control samples. Imatinib sensitive and resistant CML cell lines were also used to investigate whether treatment with other tyrosine kinase inhibitors interfered in their expression.ResultsIn patients, both methyltransferases were either upregulated or with basal expression level during the chronic phase compared to controls. Interestingly, MLL3/KMT2C and specially MLL2/KMT2D levels decreased during disease progression correlating with distinct clinical stages. Furthermore, MLL2/KMT2D was decreased in patients resistant to IM treatment. A rescue in the expression of both MLL genes was observed in KCL22S, a CML cell line sensitive to IM, after treatment with dasatinib or nilotinib which was associated with a higher rate of apoptosis, an enhanced expression of p21 (CDKN1A) and a concomitant decrease in the expression of CDK2, CDK4 and Cyclin B1 (CCNB1) in comparison to untreated KCL22S control or IM resistant KCL22R cell line, which suggests involvement of p53 regulated pathway.ConclusionOur results established a new association between MLL2/KMT2D and MLL3/KMT2C genes with CML and suggest that MLL2/KMT2D is associated with disease evolution and may be a potential marker to predict the development of therapy resistance.

The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

BIRC6 Is Up‐Regulated Via Transcriptional and Post‐Transcriptional Mechanisms in Imatinib Resistant Chronic Myeloid Leukemia

BIRC6 is a recently discovered anti‐apoptotic protein and increased expression is correlated with adverse outcomes in childhood leukemia and other cancers. The expression of BIRC6 was examined in cell models of imatinib sensitive and resistant CML (MYL/MYL‐R). A significant increase in BIRC6 mRNA and protein expression was observed in the MYL‐R cells compared to the drug sensitive MYL cells. Knockdown of BIRC6 in MYL‐R cells increased imatinib sensitivity confirming the importance of this protein in drug resistance. In this study we investigated the mechanisms responsible for the up‐regulation of BIRC6 in MYL‐R cells. Using a novel CDK9 inhibitor (HY‐16462), we were able to identify CDK9 as a required kinase for BIRC6 mRNA transcription. In comparison with MCL‐1, BIRC6 protein levels remained stable despite inhibition of BIRC6 mRNA transcription by the CDK9 inhibitor. This data showed that the regulation of BIRC6 protein levels was post‐transcriptionally determined. A time course treatment with cyclohexamide showed a difference in BIRC6 degradation between MYL and MYL‐R cells. The half‐life of BIRC6 protein in MYL cells was around 3.5 hours compared to 6 hours, nearly double, in MYL‐R cells. Mass spectrometry experiments demonstrated that BIRC6 was differentially phosphorylated in MYL‐R cells. In summary, these studies identify BIRC6 as a key component of cell survival in drug‐resistant leukemia and show that its regulation lies with both mRNA transcription and protein stability. These studies suggest that compounds targeting BIRC6 or BIRC6 stability could be developed to treat cancers resistant to traditional therapies.

Dual Inhibition of Bcr-Abl and Hsp90 By C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells

Purpose: Although such tyrosine kinase inhibitors (TKIs) as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of CML patients, TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition.Experimental Design: Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation and immunoblotting were applied to examine Hsp90 function; Colony-forming unit (CFU), long-term culture-initiating cells (LTC-ICs), and flow cytometry were used to test CML progenitor and stem cells.Results: Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, inhibited the growth of both imatinib-sensitive and resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34+CD38+ and CD34+CD38- cells in vitro. Moreover, C086 could decreased the number of CD45+, CD45+CD34+CD38+ and CD45+CD34+CD38- cells in CML NOD-SCID mice.Conclusions: Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs. DisclosuresNo relevant conflicts of interest to declare.

Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance. Here we investigated derivatives of apoptin, a chicken anemia viral protein with selective toxicity towards cancer cells, which can be directed towards inhibiting multiple hyperactive kinases including BCR-ABL1. Our earlier studies revealed that a proline-rich segment of apoptin interacts with the SH3 domain of fusion protein BCR-ABL1 (p210) and acts as a negative regulator of BCR-ABL1 kinase and its downstream targets. In this study we show for the first time, the therapeutic potential of apoptin-derived decapeptide for the treatment of CML by establishing the minimal region of apoptin interaction domain with BCR-ABL1. We further show that the apoptin decapeptide is able to inhibit BCR-ABL1 down stream target c-Myc with a comparable efficacy to full-length apoptin and Imatinib. The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples. The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

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.

Beneficial effects of combining a type II ATP competitive inhibitor with an allosteric competitive inhibitor of BCR-ABL for the treatment of imatinib-sensitive and imatinib-resistant CML

Beneficial effects of combining a type II ATP competitive inhibitor with an allosteric competitive inhibitor of BCR-ABL for the treatment of imatinib-sensitive and imatinib-resistant CML

Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

BackgroundChronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB) transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I.ResultsIn this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I) and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5) or imatinib-resistant (KBM5-T315I) CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways.ConclusionTo our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for further drug development to overcome imatinib resistance in CML patients.

A Novel β-catenin Inhibitor, AV65 Suppresses the Growth of CML Cell Lines Which Acquire Imatinib-Resistance Because of Abl Kinase Domain Mutations Including T315I and Hypoxia-Adaptation.

Imatinib and the second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib have dramatically changed the management of CML. However, these agents are effective neither for CML cells harboring T315I mutation nor for CML stem cells. Genetic analysis of blasts from CML patients in blast crisis has identified numerous members of the Wnt/b-catenin pathway as being activated (Radich et al, PNAS 2006) and loss of b-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007). Thus, b-catenin signaling inhibition may be useful for CML treatment. We herein describe the effects of a novel b-catenin inhibitor, AV65 on various imatinib-sensitive and –resistant CML cell lines (Table 1). Eight imatinib-sensitive human CML cell lines and one normal cell line derived from hepatocyte were exposed to AV65 for 72 hours and its anti-proliferative effects were examined by MTT assay. AV65 induced apoptosis in these CML cell lines time- and does-dependent manners and inhibited the growth of all eight CML cell lines with IC50 at ranging from 9.8 to 33.1nM. While that for hepatocyte was 204.8nM. Interestingly, AV65 induced polyploidies in K562 but not in BV173. In addition, AV65 augmented the anti-proliferative effects of imatinib additively against K562 and BV173. These findings suggested that AV65 alone or the combination with imatinib was effective for imatinib-sensitive CML cells. To investigated the effects of AV65 against imatinib-resistant CML cells, four imatinib-resistant CML cell lines such as K562/IMR (bcr-abl amplification), MYL-R1 (LYN overexpression), KBM5/ STIR (harboring T315I) and K562/D1-9 (P-glycoprotein overexpression), and BaF3 cells expressing the wild type BCR-ABL or its ten different mutant BCR-ABL forms including T315I, were used. There was little difference on the induction of apoptosis and anti-proliferative effects of AV65 except K562/D1-9 between each parental cell line and its resistant clone and AV65 inhibited the growth of all examined BaF3 cells harboring various mutations including T315I with IC50 at ranging from 21.6 to 46.5nM. IC50 values of AV65 for K562 and K562/D1-9 were 11.0 and 60.1nM, respectively. These findings suggested that the effects of AV65 were independent either to BCR-ABL expression level, ABL mutations or LYN overexpression, but might be affected by P-glycoprotein. Next, the effects of AV65 against the hypoxia-adapted CML cell lines such as K562/ HA and KCL22/HA were also investigated. These cell lines were resistant to imatinib, dasatinib, INNO-406 (another second-generation ABL TKI; Kimura et al, Blood 2005) and alkylating agents via the up-regulation of glyoxalase-I, a detoxification enzyme for the cytotoxic byproducts of glycolysis. Intriguingly, AV65 inhibited the growth of hypoxiaadapted CML cell lines at almost same concentration compared with their parental cell lines. Although CML stem cell niche has not been definitely identified, it might located in hypoxia because the inoculated human leukemia cells to immunodeficient mice preferably localized on the surface of osteoblasts in the epiphysis (Ninomiya et al, Leukemia 2007) where could be hypoxic. Taken together, AV65 may be effective for CML stem cells in hypoxia. In conclusion, AV65 inhibited the growth of CML cell lines which acquire imatinib-resistance because of Abl kinase domain mutations including T315I and hypoxiaadaptation. Therefore, AV65 may become a promising agent for CML treatment including both imatinib and the second-generation Abl TKIs-resistant patients. [Display omitted]

Evaluation of the Role of PlGF and the Therapeutic Potential of Anti-PlGF in BCR/ABL+ Leukemia.

Introduction of imatinib and second-generation BCR/ABL inhibitors has revolutionized treatment of patients with Philadelphia chromosome positive (Ph+) leukemia, but leukemia cells persist even in successfully treated patients, and some patients develop resistance and ultimately relapse. The reasons for these drawbacks are not entirely resolved, but besides BCR/ABL the host stroma potentially plays an important (independent) role. Placental Growth Factor (PlGF), a homologue of VEGF, was already proven to be abundantly secreted by stromal cells in solid tumors. Therefore, we investigated the role of PlGF, and the therapeutic potential of αPlGF, a monoclonal antibody against PlGF, which we recently reported to have a broad anti-tumoral potential in pre-clinical models of solid tumors (Fischer et al., Cell, 2007), in BCR/ABL+ lymphoid and myeloid leukemia. First, we studied expression of PlGF by 5 different human and murine BCR/ABL+ leukemia cell lines (Bv-173, BaF3, 32D, K562, KCL22) in vitro and found that neither of the cell lines secreted PlGF protein, but expressed its target receptor VEGFR-1. In contrast, primary murine adherent bone marrow stromal cells (BMDSC) expressed abundant amounts of PlGF protein (up to 105 pg/ml/105 cells), indicating a potential stroma-related function of PlGF. Second, we analyzed whether PlGF could induce proliferation and thereby revealed dose-dependent induction of proliferation by recombinant PlGF in all analyzed leukemia cell lines. This pro-proliferative effect of PlGF was nearly completely abrogated by both αPlGF and an extracellular anti-VEGFR-1 antibody, thus indicating that it is mediated primarily by VEGFR-1. Third, we studied potential paracrine interactions between BMDSCs and leukemia cells by performing co-culture experiments. Remarkably, coculture of BMDSC with leukemia cells significantly induced proliferation of both cell types. We hypothesized, that this induction of proliferation might be mediated by PlGF and indeed found its nearly complete abrogation upon addition of αPlGF to the co-cultures. Furthermore, BMDSCs significantly upregulated PlGF secretion (2.1 fold; N=3; P=0.005) when cultured in presence of leukemia cells. Thus, we conclude, that stromal derived PlGF promotes proliferation of leukemia cells in a paracrine fashion and at the same time acts as autocrine pro-proliferative signal for stromal cells. To test this hypothesis in vivo, we established 3 different murine models of BCR/ABL+ myeloid and lymphoid leukemia. Subsequently, we analyzed PlGF protein as present in blood and bone marrow of diseased mice in comparison to healthy mice, and detected no PlGF protein in the peripheral blood of healthy mice and low amounts of PlGF protein in their bone marrow. In contrast, leukemic mice showed PlGF protein (76.5 ± 18.4 pg / ml plasma; N=7) in their circulation at levels comparable to mice bearing solid tumors, and, interestingly more than 8.9 fold (N=7; P<0.0001) elevated PlGF levels in their bone marrow, compared to healthy mice, again indicating that PlGF represents a stroma derived, novel pathogenetic factor in Ph+ leukemia. In order to investigate the therapeutic potential of PlGF inhibition in murine BCR/ABL+ leukemia, we treated mice bearing leukemia induced by syngeneic BCR/ABL+ BaF3 cells with αPlGF, and found a significant prolongation of median survival by 18 days (N=9; P=0.015), compared to control antibodies. Encouraged by these positive results, we established models of imatinib-sensitive and imatinib-resistant (T315I mutant) CML by transducing primary bone marrow cells and subsequent transplantation into lethally irradiated recipient mice. Interestingly, also in these aggressive models, we found a significant prolongation of survival of diseased mice induced by blockade of PlGF compared to controls (median survival prolongation in wt BCR/ABL induced leukemia 5 days; N=11; P=0.002; in T315I mutant 4 days; N=12; P=0.039). Bone marrow histology and phenotypic analysis by FACS revealed decreased infiltration of spleen and bone marrow with leukemia cells (reduction in the bone marrow by 38% and in the spleen by 24%). In conclusion, these data indicate that PlGF represents a stromal derived factor promoting the progression of Ph+ leukemia, independent of BCR/ABL mutational status, and might represent a novel target produced by the leukemic stroma, potentially useful adjunct to BCR/ABL kinase inhibitors.

Outcome of Relapsed Philadelphia Chromosome Positive Chronic Myeloid Leukemia (Ph+ CML) after Allogeneic Hematopoietic Cell Transplantation (HCT).

In the imatinib era, HCT is mainly offered to patients (pts) with advanced disease or imatinib resistance. In this analysis, relapse incidence (RI) and the long-term efficacy of imatinib and immunomodulation for relapse after HCT for Ph+ CML were evaluated.Patients and Methods: From January 2000 to May 2006, 65 pts (38 m, 27 f; median age 41 (range 20–68) years) underwent HCT at the University of Leipzig. Median interval between diagnosis and HCT was 22 (2–264) months. Prior to HCT, 50 pts (77%) received imatinib for a median duration of 9 (range 1–54) months. Disease phase prior to imatinib was chronic phase (CP),n=25 (50%), accelerated phase (AP),n=18 (36%), blast phase (BP),n=7 (14%). HCT was performed because of resistance to imatinib, n=26 (BCR-ABL kinase domain (KD) mutations were found in 6 pts prior to HCT: F359V, G250E, Y253H, M315T, E255K, L248V. One patient had a K247R polymorphism), AP/BP prior to imatinib,n=14, or imatinib-intolerance/pts preference,n=10. HCT was performed in 15 (23%) pts (CP,n=12, AP,n=3) without prior imatinib. Conditioning consisted of 12 Gy TBI/cyclophosphamid 120 mg/kg in 44 (68%), and fludarabin 30 mg/m2/day for 3 days/2 Gy TBI in 21 (32%) pts. Donors were MRD, n=20; and MUD, n=45. After HCT, blood and marrow were examined at 3 months interval. A molecular relapse was diagnosed if two samples at least four weeks apart were BCR-ABL positive by reverse-transcriptase polymerase chain reaction (RT-PCR; sensitivity, approximately 1/106). In case of relapse, cyclosporine was tapered and withdrawn. Additionally, imatinib 400 mg/d was started. If a complete molecular remission (CMR) was achieved, imatinib was continued for another 12 months and then stopped.Results: Engraftment occurred in 61 (94%) pts. OS and TRM at 5 years were 70%, and 23% respectively. At a median time of 7 months after HCT, 31/48 (65%) relapsed [molecular,n=18 (58%), cytogenetic,n=4 (13%), hematological,n=9 (29%)]. Type of conditioning, and donor type had no influence on RI which tended to be higher in pts with imatinib prior to HCT (p=0.03). Incidence of chronic GvHD in pts without and those with relapse was 65% and 13% respectively (p<0.001). RI tended to be higher in pts with imatinib resistance prior to HCT (77%) compared to imatinib sensitive CML (54%) (p=0.08). 6/7 pts with KD mutations relapsed after HCT. In 4 pts the same mutations were detected. All suffered from refractory relapse. Overall, CMR was achieved in 22/31 (71%) pts after a median treatment time of 5 months. 9/17 (53%) of imatinib resistant pts prior to HCT including two pts with KD mutations achieved CMR. After imatinib withdrawal, molecular relapse recurred in 11/22 (50%) pts after a median time of 3 months. Donor lymphocyte infusion was given and imatinib restarted. All pts achieved a sustained CMR. Overall, if chronic GvHD occurred at any time point (n=8), CMR was sustained even after imatinib withdrawal. In the remaining pts, imatinib therapy had to be continued indefinitely.Conclusions: HCT can be curative even in pts with advanced CML or imatinib resistance. Some KD mutations could survive HCT and could be associated with refractory relapse. Nevertheless, relapse could be treated with imatinib. In the absence of GvHD, CMR after relapse under imatinib does not seem to be durable after imatinib withdrawal in most pts. RI was significantly less in pts with chronic GvHD which was also associated with sustained CMR after relapse even after imatinib withdrawal.

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.

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.