Downregulation of lncRNA CCDC26 contributes to imatinib resistance in human gastrointestinal stromal tumors through IGF-1R upregulation

Imatinib is the first line of therapy for patients with metastatic or gastrointestinal stromal tumors (GIST). However, drug resistance limits the long-term effect of imatinib. Long non-coding RNAs (lncRNAs) are emerging as key players in regulating drug resistance in cancer. In this study, we investigated the association between lncRNA CCDC26 and IGF-1R in GIST and their involvement in drug resistance. Considering the key role of lncRNAs in drug resistance in cancer, we hypothesized that IGF-1R is regulated by lncRNAs. The expression of a series of reported drug resistance-related lncRNAs, including CCDC26, ARF, H19, NBR2, NEAT1, and HOTAIR, in GIST cells treated with imatinib H19 was examined at various time-points by qRT-PCR. Based on our results and published literature, CCDC26, a strongly down-regulated lncRNA following imatinib treatment, was chosen as our research target. GIST cells with high expression of CCDC26 were sensitive to imatinib treatment while knockdown of CCDC26 significantly increased the resistance to imatinib. Furthermore, we found that CCDC26 interacted with c-KIT by RNA pull down, and that CCDC26 knockdown up-regulated the expression of IGF-1R. Moreover, IGF-1R inhibition reversed CCDC26 knockdown-mediated imatinib resistance in GIST. These results indicated that treatments targeting CCDC26-IGF-1R axis would be useful in increasing sensitivity to imatinib in GIST.

Detección de mutaciones en el dominio tirosina quinasa de BCR-ABL1 en pacientes colombianos con leucemia mieloide crónica LMC, resistentes al imatinib / Detection of BCR-ABL1 tyrosine kinase domain mutations in chronic myeloid leukaemia patients with Imatinib resistance in Colombia

Resumen Introducción: Las mutaciones en el dominio BCR-ABL1, tirosina quinasa (TK) son mecanismos importantes de resistencia de los inhibidores de la tirosina quinasa (ITK) en pacientes con leucemia mieloide crónica (LMC). Objetivo: Determinar el tipo y la frecuencia de las mutaciones en el dominio tirosina quinasa del gen BCR-ABL1, asociadas con falla en la respuesta al tratamiento con imatinib en pacientes con LMC y correlacionar el perfil de mutaciones con los hallazgos clínicos, demográficos, respuesta citogenética y respuesta molecular. Materiales y métodos: Se realizó un estudio descriptivo de tipo prospectivo en pacientes con LMC en tratamiento con IMATINIB a quienes se les realizó cariotipo y análisis de mutaciones del dominio BCR-ABL1 mediante la técnica de PCR anidada. Resultados: De los 23 pacientes estudiados en cuatro se encontraron mutaciones: dos presentaron la mutación E255K, uno presentó la mutación H396P y otro presentó doble mutación L387L y T389P. Las mutaciones E255K que se ubican en la región P-loop y H396P en A-loop se asocian con mal pronóstico. La mutación T389P localizada en la región A-loop no está informada en algunas bases de datos. Conclusiones: En este estudio encontramos cuatro mutaciones en el dominio tirosina quinasa (E255K, H396P, L387L y T389P) que podrían aportar información valiosa y guiar las decisiones de tratamiento. Es importante destacar que esta investigación de análisis mutacional del dominio BCR-ABL es la primera que se realiza en el país con la particularidad adicional de cubrir una población triétnica.

Abstract Mutations in the BCR-ABL1 tyrosine kinase domain mutations, are one of the principal mechanisms associated with tyrosine kinase inhibitors (TKI) resistance in patients with chronic myeloid leukaemia (CML). Objectives: To determine the type and frequency of mutations in the tyrosine kinase domain of the BCR-ABL1 gene associated with failure to respond to treatment with Imatinib and Imatinib in patients with CMK, and to correlate the mutation profile with the clinical and demographic variables, as well as the cytogenetic and molecular response. Materials and methods: A descriptive prospective study was carried out on patients with CML treated with Imatinib. Karyotyping and analysis of the BCR-ABL1 domain mutations were performed on the patients using nested PCR. Results: Four types of mutations were found in the 23 patients studied, of which two of them were the E225 mutation, one with the H396P mutation, another with a double mutation L387L and T389P. Both the E255K mutation located in the P-loop region, and H396P mutation in the A-loop region, are associated with a poor prognosis. The T389P mutation located within A-loop region has not been reported in any of the databases. Conclusions: Four mutations were found in the tyrosine kinase domain (E255K, H396P, L387L and T389P) were found in this study. These findings provide valuable information and as a guideline to help make treatment decisions. It is important to point out that this analytical study on mutations of the BCR-ABL domain is the first one carried out in the country and, specifically, in a tri-ethnic population.

The mechanism of STAT5-ROS signal pathway to mediated imatinib resistance of K562 cells / 安徽医科大学学报

Objective To investigate the mechanism of STAT5-ROS pathway to mediate IM resistance of K562 cells.K562 cells were cultured with imatinib at gradually increased concentrations to generate resistance cell line.Methods CCK-8 assay was used to clarify the resistance ratio.The STAT5A and STAT5B mRNA levels were detected by RT-PCR.Flow cytometry assay was used to detect the level of ROS and cell apoptosis.The expression of STAT5 protein was detected by Western blot.Results Imatinib resistance cell line K562/G was successfully induced by gradually increasing concentrations of IM.The IC50 of K562/G was eighty times higher than K562 by CCK-8.Cell growth curve showed that K562/G was not inhibited in 20 μmol/L imatinib, whereas the K562 cell was significantly inhibited by up to 0.1 μmol/L imatinib.Intracellular level of ROS in K562/G was obviously higher than that of K562 cells(P=0.000 1).The apoptosis ratio of K562 was lower than that of K562/G when the same concentration of IM react on the two cell lines for the same time(P<0.05).The expression of STAT5A and STAT5B mRNA in K562/G was higher than in K562(P=0.000 1,P=0.017 0).Then,the level of STAT5 proteins in K562/G cells was significantly increased (P=0.009 0).Conclusion The STAT5 is highly expressed in CML.STAT5-ROS is closely related to the formation of imatinib resistance of chronic myeloid leukemia.

Curcumin derivative C085 inhibits proliferation of K562/G01 cells and activity of Bcr-Abl kinase in vitro / 中国药理学通报

Aim To find new kinase inhibitors that o-vercome the imatinib resistance in the treatment of chronic myeloid leukemia ( CML ) by synthesizing C085, a novel derivative of curcumin , and testing its activities against wild-type( WT) and imatinib-resistant mutant Abl kinases , as well as in imatinib-resistant CML cells in vitro.Methods Cell proliferation and apoptosis were examined using MTT assay and flow cy-tometry, respectively;Kinase activity was measured u-sing Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant ( Q252H, Y253F, and T315I) Abl kinases.The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting .Colony forming units ( CFU ) growth was used to test the effects of C 085 on human leukemia progenitor/stem cells.Results C085 suppressed the growth of imatinib-resistant K562/G01 cells and po-tently inhibited both WT and mutant ( Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP com-petitive manner with the values of IC 50 at low nanomole levels.C085 dose-dependently down-regulated Bcr-Abl kinase activity in K562/G01 cells as judged by auto-phosphorylation and Stat 5 , Crkl phosphorylation , and inhibited the phosphorylation of downstream targets Raf,Mek and Erk with protein content reducing .C085 could directly impact mitochondrial PT hole and make it open, which prevents the activation of caspase cas-cade reaction and induces the apoptosis .Furthermore, C085 significantly suppressed CFU growth , implicating that C085 could inhibit human leukemia progenitor/stem cells.Conclusion C085 may inhibit K562/G01 cells through inhibiting Bcr-Abl kinase activity and down-regulating the downstream signal proteins .Di-rectly acting on mitochondrial PT hole and then activa-ting apoptosis-associated proteins are also involved in the pro-apoptotic effect of C085.C085 is a promising compound for the treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib re-sistance .

Sunitinib in patients with imatinib‑resistant gastrointestinal stromal tumor: A single center experience.

AIM: The outcome of patients with advanced gastrointestinal stromal tumor (GIST) has improved with the use of imatinib. Despite high response rates with this drug resistance eventually develops in nearly all patients. We present an analysis of prospectively collected data on sunitinib efficacy and safety in patients with imatinib‑resistant GIST. SUBJECTS AND METHODS: Between November 2006 and October 2007, patients with GIST were accrued in an approved sunitinib patient access protocol. Key eligibility criteria included tumor resistance to imatinib and/or patient intolerance to this drug. Patients received sunitinib at a starting dose of 50 mg once daily for 4 weeks in a 6 week cycle, with standardized dose modification titrated to toxicity. Patients were continued on sunitinib until disease progression or unacceptable toxicity. The endpoints were safety, overall survival (OS) and objective response rate (ORR). RESULTS: Fifteen patients, all of whom had imatinib resistance and none intolerance, with median age of 48 (26–69) years, were treated on the protocol. The most common sites of primary disease were small intestine (40%), stomach (26.7%) and retroperitoneal (26.7%). A median of 10 (1–47) cycles of sunitinib were delivered, 9 (60%) patients required dose reductions due to toxicity whereas dose delay of > 2 weeks was required in only one (6.7%) patient. There were no toxicity‑related drug discontinuations. Hypothyroidism (n = 4; 26.7%) and hand‑foot syndrome (n = 3; 20%) were the most common toxicities. There were no complete and 4 (26.7%) partial responses while prolonged disease stability was seen in 8 (53.3%) patients. At a median follow‑up of 81 months in surviving patients, the median progression‑free and overall survivals were 15.5 and 18.7 months, respectively. CONCLUSIONS: Sunitinib appears to be an effective and well‑tolerated treatment for Indian patients with imatinib‑resistant GIST with outcomes similar to that reported previously. Adverse effects can be reasonably well managed using a dose modification strategy.

Imatinib resistance in chronic myelogenous leukemia: an emerging challenge.

Resistance to imatinib is a significant clinical issue, and the underlying mechanism of this resistance is multifactorial. The efficacy of imatinib in chronic myeloid leukemia (CML) in achieving a high remission rate and improving prognosis has seriously been challenged by the development of mutants of BCR-ABL gene, which resist the action of imatinib, which is a tyrosine kinase inhibitor. We present here a case of a 35-year-old male, a known case of CML on imatinib therapy, the patient eventually landed in blast crisis and succumbed to the disease and secondary infections.

Inhibition of c-Myc by 10058-F4 overcomes imatinib resistance in chronic myeloid leukemia cells / 中国病理生理杂志

AIM: To investigate the effect of c-Myc inhibitor 10058-F4 on human chronic myeloid leukemia ( CML) K562 cells and imatinib-resistant K562/G cells.METHODS: The protein expression of c-Myc was detected by Western blotting .Cell proliferation was evaluated by MTT assay and colony formation assay .PI staining was used to deter-mine the cell cycle distribution .Annexin V-PI staining was applied for apoptosis detection .RESULTS:Imatinib-resistant K562/G cells displayed lower sensitivity to imatinib than K 562 cells with high expression of c-Myc.Treatment with specific c-Myc inhibitor 10058-F4 inhibited the cell proliferation in a dose-and time-dependent manner , and K562/G displayed more sensitivity to 10058-F4 than K562 cells.10058-F4 also induced cell cycle arrest in G 0/G1 phase and induced apoptot-ic cell death in the 2 cells.Importantly, 10058-F4 suppressed the colony formation ability in K 562 and K562/G cells. CONCLUSION:c-Myc is a novel target to overcome imatinib-induced drug resistance , and c-Myc inhibitor provides a new approach in CML therapy .

Leucemia Mielóide Crônica: novas drogas em desenvolvimento / Chronic Myeloid Leukemia: development of new drugs

A LMC é um modelo de investigação biológica e clinica que deve ser seguido nesta nova fase da oncologia moderna. A resposta terapêutica ao uso do imatinibe como droga de primeira linha mudou os conceitos e paradigmas e criou uma expectativa que drogas mais potentes possam ser desenvolvidas no futuro. Infelizmente nem todos conseguem atingir essa situação ideal. Por esta razão, Baccarani M sugeriu que a falência de resposta subótima, precaução ou alerta fossem estudadas no sentido de serem desenvolvidas intervenções terapêuticas diferenciadas mais precoces. A resistência ao imatinibe existe e depende de vários mecanismos. Tanto mais tardia a introdução do imatinibe e mais avançada for a fase evolutiva da doença maior a freqüência de resistência. Do ponto de vista biológico, a superexpressão do BCR-ABL, os defeitos genéticos adicionais e as mutações que podem atingir várias regiões da molécula - a alça de fosfato, a alça de ativação, o domínio da quinase são os mais importantes fatores associados à resistência ao imatinibe. Por esta razão, são necessárias outras opções terapêuticas e hoje há o desenvolvimento de um grande número de drogas para um número maior de alvos. Inicialmente temos o dasatinibe, já aprovado nos EUA, na Europa e também no Brasil; o nilotinibe, em fase avançada de estudos clínicos (inclusive de fase III), e também já aprovado para uso nos EUA; o bosutinibe, o INNO - 406 bem como outras drogas que atuam em alvos como as aurora-quinases ou inibidores de histona-deacetilases.

Chronic Myeloid Leukemia (CML) is a model of clinical and biological investigation that may be useful for other neoplastic diseases. The therapeutic response to imatinib as the front line therapy has changed concepts and procedures in CML and has created hope concerning new more potent drugs for this and other oncological diseases that have a similar mechanism of action. However, not all patients achieve this ideal situation. Thus, Baccarani et al. suggested that cases of failure of suboptimal response, as a precaution or in warning situations should be studied in order to develop differentiated new therapies earlier. Resistance to imatinib exists and may depend of several mechanisms. Delay in its use or the advanced phase of the disease are more frequently associated with imatinib resistance. In respect to the biological point of view, over expression of the BCR-ABL gene, additional genetics abnormalities and mutations that involve other regions of the molecule (P-Loop, A-Loop or kinase dominion) are the most important factors associated to imatinib resistance. Hence, new therapeutic options are necessary in order to overcome resistance and to act on other target cells. Currently, Dasatinib was approved in the US, Europe and Brazil. Nilotinib is in advanced phase III studies and was recently approved in the US. Both Dasatinib and Nilotinib have been approved for intolerant and refractory imatinib patients. Bosutinib, INNO - 406 as well as other TK, aurora kinase and histone deacetylase inhibitors are in clinical studies and probably will be available in the future.

Erythroleukemic Blast Crisis of Chronic Myeloid Leukemia / 대한진단검사의학회지

Erythroleukemic blast crisis of chronic myeloid leukemia (CML) is very rare. We report two cases of erythroleukemic blast crisis of CML resistant to imatinib treatment. Both patients made a rapid progression to blast crisis 6 and 4 months after diagnosis while being treated with imatinib 400 mg/day. Bone marrow aspiration revealed predominant erythroid precursors with 65.4% and 54.8% each. There were significant proportions (more than 20%) of myeloblasts among non-erythroid cells. Immunophenotyping revealed expression of glycophorin A confirming erythroleukemic blast crisis. The karyotyping result of patient 1 was 46,XX,t(9;22)(q34;q11.2)[3]/52,idem,+8,+12,+18,+21,+22,+der(22)t(9;22)[17] and that of patient 2 was 46,XX,inv(3)(q21q26.2),t(9;22)(q34;q11.2)[20]. Patient 1 showed no response to imatinib and BMS-354825 in the following bone marrow study. She died of septic shock as a complication of an infection after 69 days of blast crisis. Patient 2 received allogeneic bone marrow transplantation (BMT) in the cytogenetically no response state, but she also died of graft-versus-host disease 9 weeks after BMT. The poor prognosis and rapid progression of disease in both cases were correspondent to most of the reported cases. During the course of the disease of the two patients, we monitored the BCR-ABL chimeric mRNA with real-time quantitative polymerase chain reaction (RT-PCR), and it was found useful in predicting the imatinib response and progression to blast crisis of CML. Although both of our cases showed the typical bad prognosis and findings of erythroleukemic blast crisis of CML, the karyotypes were different from the expected type of t(3;21)(q26;q22). But the relationship between additional changes of EVI1 on chromosome 3q26 shown in case 2, and progression to the erythroleukemic blast crisis need further investigation.

Clinical Resistance to the Tyrosine Kinase Inhibitor Imatinib (STI571) and Detection of BCR-ABL Gene Mutations in Korean Patients with Chronic Myeloid Leukemia

BACKGROUND: Imatinib mesylate, the tyrosine kinase activity of the BCR-ABL fusion gene, induces a remarkable remission in chronic myeloid leukemia (CML) patients. However, resistance to imatinib has been observed in a significant proportion of subjects, with the point mutations of the BCR-ABL kinase domain clinically identified as a possible mechanism. The aim of this study was to investigate clinical resistance to imatinib in Korean CML patients, and search for the point mutation of the BCR-ABL gene. METHODS: The clinical data and cytogenetic results of thirty two CML patients, who were treated with imatinib, between Jan. 2002 and Aug. 2003, were evaluated. Mutational analyses for the point mutations of the BCR-ABL kinase domain in clinically resistant patients were tested using RT-PCR and direct sequencing methods. RESULTS: Complete hematological remission was obtained in all CML patients with a chronic phase and in 4 of 6 CML with accelerated or blast crisis. However, 4 patients (2 in the chronic phase and 2 with blast crisis) relapsed to blast crisis following continued treatment. A major cytogenetic response was observed in 67% of the chronic phase patients, but in 2, the Philadelphia chromosomes reemerged in a follow-up chromosome study. Mutational analyses showed point mutations in the 351st amino acid of the BCR-ABL kinase domain in 2 patients: M351T, which has previously been reported in many studies, and a novel substitution, M351L. CONCLUSION: The frequency of imatinib resistance in Koreans was similar to that found in well-controlled western studies. Point mutations of the BCR-ABL kinase domain were detected in two patients. Further studies, with more sensitive methods and a greater number of patients will help reveal other mechanisms of imatinib resistance and establish more effective treatment plans.