Synergistic Effects Of Imatinib Research Articles

Synergistic Effect of Imatinib and Ruxolitinib in a Patient with JAK2V617F positive Myelofibrosis and Concomitant BCR-ABL1 positive Chronic Myeloid Leukemia

Introduction:JAK2V617Fmutation is present in majority of polycythemia vera (PV) and in 50-60% of patients with primary myelofibrosis (MF). JAK1/2 inhibitor ruxolitinib (RUX) is approved for treatment of both PV and intermediate/high risk MF. Two independent randomized studies showed the benefit of RUX in MF with significant reduction in spleen volume and improvement in constitutional symptoms compared to best supportive care. Clonal evolution may occur in PV and MF upon acquisition of new mutations and transformation to acute myeloid leukemia. Rarely, patients with JAK2V617Fpositive PV or MF have also concomitant BCR-ABL1 fusion gene with phenotype of chronic myelogenous leukemia (CML). Imatinib, a BCR-ABL1 inhibitor, induces long-term cytogenetic and molecular remissions in CML. Single agent imatinib has shown no clinical benefit in MF patients despite in vitro efficacy (Gaikwad et al Exp. Hematol. 2007). We describe here a patient with JAK2V617F positive post-PV MF who later developed concomitant BCR-ABL+ CML. Splenomegaly was initially resolved by RUX but returned coincidently with the diagnosis of CML and was again normalized by imatinib alone. Massive splenomegaly reappeared along with elevated hematocrit on imatinib and was resolved by the addition of low dose RUX.Case report: A 71-year old woman initially presented in September 2011 with isolated erythrocytosis and splenomegaly. A diagnosis of PV was established based on the presence of the JAK2V617Fmutation and hypercellular marrow. Bone marrow karyotype was normal (Table 1). The patient was randomized to pegylated interferon arm in the MPD Consortium study. A complete hematologic response was achieved after nearly 2 years on maximal dose 180 mcg weekly. In early 2015, she developed constitutional symptoms, and progressive splenomegaly. Progression to post-PV MF was confirmed on bone marrow evaluation. The patient was taken off study and started on RUX 20 mg BID. She developed severe anemia 4 months later requiring dose reduction to 10 mg BID. The anemia markedly improved, while JAK2V617F allele burden remained high at 88% with normal cytogenetics. In December 2017, the patient developed anemia, fatigue and rapid regrowth of spleen. Marrow karyotype showed Philadelphia (Ph) chromosome in 25% of the cells analyzed, PCR was positive for BCR-ABL1 p210, and JAK2V617F allele burden was reduced to 56.4%. In January 2018, she started imatinib 400 mg daily and RUX was discontinued due to toxicity concerns. In April 2018, her hematocrit increased and massive splenomegaly returned. Restart of initial RUX 20 mg BID was followed by severe pancytopenia which normalized when RUX was reduced to 5 mg BID with continuing imatinib regimen. After 8 weeks, splenomegaly completely resolved (Figure 1). A complete cytogenetic response and major molecular response were achieved after 3 months and 6 months of combination therapy respectively.Methods: Mononuclear cells from peripheral blood collected in June 2018 were plated in methylcellulose without erythropoietin or other cytokines. After 14 days, colonies were plucked, with ½ of each colony used for JAK2V617F allele-specific PCR and ½ prepared as cytospins for BCR-ABL fluorescent in situ hybridization (FISH) with the Vysis LSI BCR/ABL Dual Color, Dual Fusion Translocation Probe from Abbott Laboratories.Results: Analysis of 3-6 EPO-independent colonies of each CFU-E, G, M, GM, GEMM, BFU-E showed no BCR-ABL1 positive cells, while a single CFU-GEMM colony was positive and also homozygous for JAK2V617F. Frozen marrow cells from the time of the original diagnosis are being analyzed.Conclusion: The recurrence of splenomegaly in this patient following single agent RUX indicates emergence of novel BCR-ABL1+ clone. While single agent imatinib temporarily normalized the splenomegaly and leukocytosis for several months, MF phenotype reoccurred. Splenomegaly resolved completely in 8 weeks with the addition of very low dose RUX to standard dose imatinib, providing the first clinical observation of in vivo synergism of RUX and imatinib. We are investigating serial samples during her course, which will be presented at the meeting. The analysis of a single clone demonstrate that BCR-ABL1 mutation did not occur in previously normal dormant stem cell but in JAK2V617F positive progenitors and represent in this pts a novel subclone of previous PV/MF stem cell. DisclosuresDeininger:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Safe dose of intravitreal imatinib and its effect on laser-induced choroidal neovascularization: a rat-model experiment.

BackgroundThis two-phase experimental study was conducted to determine the maximum safe dose of intravitreal imatinib (IVI) and its inhibitory effect on a rat model of choroidal neovascularization (CNV).MethodsIn phase I, 60 rats were divided into six groups (A to F); five of which received IVI with concentrations of 330 (A), 250 (B), 165 (C), 80 (D), and 40 (E) µg/5 µl, and the control group (F) received balanced salt solution (BSS). In addition to electroretinography (ERG), routine histopathological analysis and immunohistochemistry for glial fibrillary acidic protein were performed. In phase II, CNV was induced by laser photocoagulation in 25 rats and the animals were divided into two groups. One group received the maximum safe dose of IVI, determined in phase I, and the other received intravitreal BSS. After 4 weeks, the groups were compared in terms of mean scores of fluorescein leakage in fluorescein angiography and the mean CNV areas in histopathological sections.ResultsIn phase I, ERG and the histopathological findings revealed retinal toxicity in groups A to D and A to C, respectively; therefore, a dose of 40 µg/5 µl imatinib was specified as the maximum safe dose for phase II. In phase II, late phase fluorescein leakage and the CNV areas were not significantly different between the imatinib-treated eyes and the controls (p = 0.62 and p = 0.5, respectively).ConclusionsDespite the safety of IVI with a dose of 40 µg/5 µl, no inhibitory effect on laser-induced CNV was observed. Further studies are required to investigate the possible synergistic effects of Imatinib with conventional anti-CNV drugs.

Synergistic effects of imatinib and carboplatin on VEGF, PDGF and PDGF-Rα/ß expression in squamous cell carcinoma of the head and neck in vitro

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. The development of new treatment modalities in order to improve long-term survival of patients with HNSCC is imperative. Numerous studies have demonstrated that carcinogenesis and tumor cell dissemination is influenced by the tumor microenvironment. The protein-kinase-receptors (PTKs) are essential elements of the intracellular signal transduction pathway and regulate cell growth, development and apoptosis. Cell proliferation, migration, induction of tumor vascularization and carcinogenesis, invasion is regulated by a variety of angiogenic factors, such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor) and their respective tyrosine kinase receptors (PDGF-R and VEGF-R). They present promising targets for anti-cancer therapy through abrogation of impaired signaling pathways. Indeed, imatinib, a small molecule drug targeting these protein kinases, has antiproliferative effects in several cancer types. The purpose of this study was to investigate the potential synergism of imatinib and carboplatin on the expression of PDGF, PDGF-R α/ß and VEGF in different HNSCC cell lines. Several tumor cell lines were subjected to increasing concentrations of carboplatin (3 and 7.5 µmol/l) and imatinib (18 and 30 µmol/l) and ELISA, immunohistochemical methods and RQ-PRC after 48, 72, 120 and 240 h were used to assess their expression levels. While PDGF-Rα/ß expression was unimpaired at lower imatinib concentrations (18 µmol/l), PDGF-Rα/ß expression was suppressed at 30 µmol/l, and suppression was enhanced by the presence of carboplatin. By RQ-PCR, a significant reduction of PDGF-Rα/ß expression was detected (p<0.5). We observed explicit significant reduction in VEGF levels with increasing concentrations of imatinib and with the combination of the two chemotherapeutic drugs (p<0.5). We report for the first time evidence of synergism of imatinib and carboplatin in suppressing VEGF, PDGF and PDGF-Rα/ß expression in HNSCC.

A case of myeloid neoplasm associated with eosinophilia and KIAA1509-PDGFRβ responsive to combination treatment with imatinib mesylate and prednisolone

A 41-year-old woman presented with dyspnoea, persistent leucocytosis and eosinophilia for 8 months. High-resolution computed tomography scan and pathology of bronchoalveolar lavage confirmed the diagnosis of hypereosinophilic pneumonitis. The patient was treated with prednisolone (0·5-1 mg/kg/day) for more than 20 weeks under the impression of hypereosinophilic syndrome, but without improvement of leucocytosis and eosinophilia. The bone marrow aspiration smear disclosed hypercellular marrow with myeloid hyperplasia and eosinophilia. The fusion gene detection was positive for KIAA1509-PDGFRβ. Myeloid neoplasm associated with eosinophilia and abnormality of PDGFRβ was then diagnosed (Tefferi A, Vardiman JW, Leukemia, 22, 2008, 14). The tyrosine kinase inhibitor, imatinib mesylate (Glivec; 200 mg/day), was administered along with prednisolone (0·25-1 mg/kg/day). White blood cell (WBC) count decreased from 49,500/μL to 17,200/μL, and eosinophil count decreased from 1932/μL to 35/μL, which represent percentage dropped from 7·7%> to 0·2%. Withdrawal of prednisolone was done to avoid adverse events. However, absolute eosinophil count increased progressively despite the continue administration of imatinib and negative detection PDGFRβ fusion gene. The patient then received combination therapy of imatinib and prednisolone again. WBC and absolute eosinophil were normalized subsequently. We had discontinued the prednisolone one more time, and rebound of eosinophilia was seen again. The phenomenon of rebounding of eosinophilia was observed in two subsequent withdrawals of prednisolone. Either steroid or imatinib mesylate alone failed to achieve complete haematological response. A synergistic effect of imatinib and steroid is postulated.

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

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

Synergistic effects of imatinib (STI 571) in combination with chemotherapeutic drugs in head and neck cancer

The tyrosine kinase inhibitor imatinib (STI 571; glivec) is a potent inhibitor of bcr-abl, c-kit and platelet-derived growth factor receptors. Imatinib was evaluated both alone and in combination with established chemotherapeutic agents in adenoid cystic carcinoma (ACC) primary cultures and established cell lines representing squamous cell carcinoma of the head and neck (HNSCC). Over 90% of ACC tumors are c-kit-positive, and these primary cultures proved to be of short-term usefulness in assessing chemosensitivity. Interaction was determined over a wide range of drug combinations using a statistical three-dimensional analysis model. Both ACC short-term cultures and HNSCC cell lines were demonstrated to have a response ranging from additive to synergistic when imatinib and cisplatin were combined. The interaction of imatinib on cisplatin-induced DNA cross-linking was further investigated using the comet-X assay. In contrast, significant antagonism was observed when imatinib and gemcitabine were combined. Since gemcitabine is activated by deoxycytidine kinase (dCK), the effect of imatinib on this enzyme was investigated. A dose-dependent inhibition of dCK was observed, highlighting this kinase as a possible additional secondary molecular target for imatinib. This work demonstrates a synergistic interaction between cisplatin and imatinib, which may prove to be clinically relevant in the future management of both ACC and HNSCC.

Identification of Hypusination Inhibitors as a Novel Therapuetical Option in BCR-ABL Positive Leukemias Using a Proteomics Approach.

Introduction: BCR-ABL has been shown to be both necessary and sufficient for the induction of chronic myeloid leukemia (CML) in experimental models. The selective tyrosine kinase inhibitor imatinib has been shown to induce major cytogenetic remissions in >80% of CML patients in chronic phase. However, resistance to imatinib develops frequently especially in later stages of the disease. Therefore, investigations on potential synergistic treatment strategies are required in order to be able to overcome clinical resistance.Materials and Methods: Protein expression analysis of K562 leukemic cells was performed using 2-dimensional gel electrophoresis after treatment with imatinib (4 μM) or DMSO (as a solvent control) for 24 hours and 48 hours. Resulting differentially expressed prominent proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Differential expression was confirmed by western blot analysis for some candidate proteins. Cytotoxicity was measured in K562 cells and primary CD34+ cells from patients with CML at diagnosis using MTT-assay, the fraction of apoptotic cells was measured by flow cytometry using propydium iodide (PI).Results: Using 2-dimensional gel electrophoresis, thirteen candidate proteins were identified to be down-regulated whereas 6 proteins were up-regulated by cell treatment with imatinib. Interestingly, we detected the down-regulation of the eukaryotic translation initiation factor 5A (eif5A), which is essential for cell proliferation. Eif5a represents the only known eukaryotic protein activated by posttranslational hypusination. Formation of hypusine is one of the most specific polyamine-dependent biochemical reactions and its biosynthesis involves two enzymatic steps which can be inhibited with the compounds ciclopirox and GC-7. To assess the effects of the hypusination inhibitors as single agent and in combination with imatinib on Bcr-Abl positive or negative cells, we performed cytotoxicity- and apoptosis-assays. Hypusination inhibition resulted in substantial and dose-dependent cytotoxicity and in an increasing fraction of apoptotic cells in K562, HL-60 and primary Bcr-Abl positiv CD34+ cells. Interestingly, synergistic effects of imatinib and ciclopirox or GC-7 could be detected on cellular cytotoxicity and apoptosis in both Bcr-Abl positive K562 cells and Bcr-Abl positive primary CD34+ cells from four newly diagnosed CML patients.Discussion: We could detect significant differences in protein expression levels in the BCR-ABL-dependent cell line K562 upon treatment with imatinib. Furthermore, we observed down-regulation of eif5a which so far has not been shown to be involved in proliferation control of Bcr-Abl positive leukemias. Eif5a suggests to be a promising target for imatinib-based synergistic treatment strategies. Considering that a number of hypusination inhibitors are clinically approved drugs with acceptable toxicity profiles, our results might have important implications for the design of novel synergistic treatment strategies in patients with Bcr-Abl-positive leukemias and other imatinib-responsive diseases.