ABCB1 Expression Levels Research Articles

Clinically relevant interactions of anti-apoptotic Bcl-2 protein inhibitors with ABC transporters.

Antiproliferative effects of ABT-263 and ABT-199 might be reduced by ABCB1 or ABCG2, however, this effect depends on transporter expression levels. Since the expression levels of ABCB1 and ABCG2 are rarely high in clinical samples, their contribution to the overall resistance to ABT-263 or ABT-199 is probably low. Inhibition study revealed that ABT-199, but not ABT-263, fully inhibited low expression level of ABCG2. Our data suggest that ABT-199 should be evaluated beyond its original application as an inhibitor of the ABCG2 transporter in clinical settings.

Reversal of ABCB1 mediated efflux by imatinib and nilotinib in cells expressing various transporter levels

Recently, it has been suggested that imatinib (IM) and nilotinib (NIL) could be studied beyond their original application, as inhibitors of the drug efflux pump ABCB1 (P-glycoprotein, MDR1). Since the reversal of ABCB1-mediated resistance has never been successfully demonstrated in the clinic, we addressed the question of whether IM and NIL may actually serve as efficient inhibitors of ABCB1. Here we define an efficient inhibitor as a compound that achieves full (90–100%) reversal of drug efflux at a concentration that does not exhibit significant off-target toxicity in vitro. In this study, human leukemia K562 cells expressing various levels of ABCB1 were used. We observed that cells expressing higher ABCB1 levels required higher concentrations of IM and NIL to achieve full reversal of drug efflux. Among the well-known ABCB1 inhibitors, a similar effect was found for cyclosporin A (CsA) but not for zosuquidar. IM was efficient only in cells with the low and moderate ABCB1 expression at high concentrations that were cytotoxic in the absence of Bcr-Abl. In contrast, NIL was as efficient an inhibitor of ABCB1 as CsA. Low and moderate expression levels of ABCB1 could be efficiently inhibited by NIL concentrations without cytotoxic effects in the absence of Bcr-Abl. However, high expression levels of ABCB1 required higher NIL concentrations with off-target cytotoxic effects. In conclusion, application of NIL, but not of IM, in clinics is promising, however, only in cells with low ABCB1 expression levels. We hypothesize that some patients may benefit from an inhibitor exhibiting an ABCB1 expression-dependent effect.

Genetic variability in the regulation of the expression cluster of MDR genes in patients with breast cancer.

We aimed to investigate the association between the polymorphism and expression patterns of multiple drug resistance genes (MDR) in breast cancer (BC). The MDR gene expression levels were measured in tumor tissues of 106 breast cancer patients using quantitative real-time PCR. Affymetrix CytoScan™ HD Array chips were used to assess genotypes. Pairwise correlation analysis for ABCB1, ABCC1, ABCC2 and ABCG2 gene expression levels was carried out to reveal co-expression clusters. Associations between SNPs of MDR genes and their preoperative expression levels were assessed using analysis of covariance adjusting for covariates. The SNPs associated with the expression of the ABCB1, ABCC1, ABCC2 and ABCG2 genes before NAC were detected. In addition, 21 SNPs associated with the expression of four ABC-transporter genes and involved in the expression regulation were identified. Validation in an independent sample confirmed the association between the MDR cluster genes and 11 SNPs. Four MDR genes: ABCB1, ABCC1, ABCC2 and ABCG2 were shown to form the functional expression cluster in breast tumor. Further studies are required to discover precise mechanisms of the cluster regulation, thereby providing new approaches and targets to combat the development of the MDR phenotype during chemotherapy.

Mebendazole, an antiparasitic drug, inhibits drug transporters expression in preclinical model of gastric peritoneal carcinomatosis

The present study aimed to investigate whether MBZ down-regulates drug transporter expression (ABCB1, ABCC1, SLC47A1). mRNA expression level of ABCB1, ABCC1 and SLC47A1 was evaluated by qPCR and protein expression levels MDR-1 was performed by western blotting in malignant ascites cells (AGP-01) treated with MBZ for 24h. The mRNA expression level of ABCB1 and ABCC1 significantly decreased at a 1.0μM of MBZ compared to negative control, while SLC47A1 extremely decreased at all tested concentrations of MBZ. Protein expression levels MDR-1 significantly decreased at a 1.0μM of MBZ compared to negative control. Therefore, our results showed MBZ may play an important role in inhibiting MDR gene expression in malignant ascites cells.

Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters.

Multidrug resistance is a major problem in colon cancer treatment. However, its molecular mechanisms remain unclear. Recently, the epithelial-mesenchymal transition (EMT) in anticancer drug resistance has attracted increasing attention. This study investigated whether vincristine treatment induces EMT and promotes multidrug resistance in colon cancer. The result showed that vincristine treatment increases the expression of several ATP-binding cassette transporters in invasive human colon adenocarcinoma cell line (HCT-8). Vincristine-resistant HCT-8 cells (HCT-8/V) acquire a mesenchymal phenotype, and thus its migratory and invasive ability are increased both in vitro and in vivo. The master transcriptional factors of EMT, especially Twist1, were significantly increased in the HCT-8/V cell line. Moreover, the ectopic expression of Twist1 increased the chemoresistance of HCT-8 cells to vincristine and increased the expression levels and promoter activities of ABCB1 and ABCC1. Furthermore, Twist1 silencing reverses the EMT phenotype, enhances the chemosensitivity of HCT-8/ V cells to anticancer agents in vitro and in vivo, and downregulates the expression of ABCB1 and ABCC1. Twist1-mediated promotion of ABCB1 and ABCC1 expression levels plays an important role in the drug resistance of colon cancer cells.

ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling

BackgroundLung cancer (LC) is still the most common cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all LC cases but is not a single entity. It is now accepted that, apart from the characteristic driver mutations, the unique molecular signatures of adeno- (AC) and squamous cell carcinomas (SCC), the two most common NSCLC subtypes should be taken into consideration for their management. Therapeutic interventions, however, frequently lead to chemotherapy resistance highlighting the need for in-depth analysis of regulatory mechanisms of multidrug resistance to increase therapeutic efficiency.MethodsNon-canonical Wnt5a and canonical Wnt7b and ABC transporter expressions were tested in primary human LC (n = 90) resections of AC and SCC. To investigate drug transporter activity, a three dimensional (3D) human lung aggregate tissue model was set up using differentiated primary human lung cell types. Following modification of the canonical, beta-catenin dependent Wnt pathway or treatment with cisplatin, drug transporter analysis was performed at mRNA, protein and functional level using qRT-PCR, immunohistochemistry, immune-fluorescent staining and transport function analysis.ResultsNon-canonical Wnt5a is significantly up-regulated in SCC samples making the microenvironment different from AC, where the beta-catenin dependent Wnt7b is more prominent. In primary cancer tissues ABCB1 and ABCG2 expression levels were different in the two NSCLC subtypes. Non-canonical rhWnt5a induced down-regulation of both ABCB1 and ABCG2 transporters in the primary human lung aggregate tissue model recreating the SCC-like transporter pattern. Inhibition of the beta-catenin or canonical Wnt pathway resulted in similar down-regulation of both ABC transporter expression and function. In contrast, cisplatin, the frequently used adjuvant chemotherapeutic agent, activated beta-catenin dependent signaling that lead to up-regulation of both ABCB1 and ABCG2 transporter expression and activity.ConclusionsThe difference in the Wnt microenvironment in AC and SCC leads to variations in ABC transporter expression. Cisplatin via induction of canonical Wnt signaling up-regulates ABCB1 and ABCG2 drug transporters that are not transporters for cisplatin itself but are transporters for drugs that are frequently used in combination therapy with cisplatin modulating drug response.

Regorafenib overcomes chemotherapeutic multidrug resistance mediated by ABCB1 transporter in colorectal cancer: In vitro and in vivo study

Chemotherapeutic multidrug resistance (MDR) is a significant challenge to overcome in clinic practice. Several mechanisms contribute to MDR, one of which is the augmented drug efflux induced by the upregulation of ABCB1 in cancer cells. Regorafenib, a multikinase inhibitor targeting the RAS/RAF/MEK/ERK pathway, was approved by the FDA to treat metastatic colorectal cancer and gastrointestinal stromal tumors. We investigated whether and how regorafenib overcame MDR mediated by ABCB1. The results showed that regorafenib reversed the ABCB1-mediated MDR and increased the accumulation of [3H]-paclitaxel in ABCB1-overexpressing cells by suppressing efflux activity of ABCB1, but not altering expression level and localization of ABCB1. Regorafenib inhibited ATPase activity of ABCB1. In mice bearing resistant colorectal tumors, regorafenib raised the intratumoral concentration of paclitaxel and suppressed the growth of resistant colorectal tumors. But regorafenib did not induce cardiotoxicity/myelosuppression of paclitaxel in mice. Strategy to reposition one FDA-approved anticancer drug regorafenib to overcome the resistance of another FDA-approved, widely used chemotherapeutic paclitaxel, may be a promising direction for the field of adjuvant chemotherapy. This study provides clinical rationale for combination of conventional chemotherapy and targeted anticancer agents.

Drug resistance of cancer cells is crucially affected by expression levels of ABC-transporters

Dasatinib (DAS), a second generation of tyrosine kinase inhibitor (TKI), represents excellent choice for the treatment of chronic myeloid leukemia resistant to imatinib. Unfortunately, recent laboratory studies suggested that antiproliferative effect of DAS might be significantly reduced due to the overexpression of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2. However, whether these drug transporters might compromise therapeutic effect of DAS in clinic is unclear. We believe that the drug transporter expression level is a crucial factor that affects the results and its consideration may help to explain the existing controversy. In addition, clinically relevant concentrations of drug must be used. In our study, human leukemia K562 cells with high and low expression levels of ABCB1 or ABCG2 were used. DAS was applied at nanomolar concentrations. We observed that K562 cells expressing high levels of ABCB1 and ABCG2 contained significantly reduced intracellular levels of DAS and these cells exhibited significantly increased resistance to this drug. Importantly, cells with the low expression of ABCB1 or ABCG2 effluxed DAS less efficiently, however, still significantly. Accordingly, the observed resistance was lower but significant. Conclusions: The antiproliferative effects of DAS might be reduced by ABCB1 or ABCG2. However, the actual effect of these ABC transporters on DAS efficiency depends on their expression levels. The lower expression levels of ABC transporters mediate lower resistance. Considering the fact that expression levels of ABCB1 and ABCG2 transporters are usually low in clinical samples, their contribution to the overall resistance to DAS is probably low but significant.

Thiazole-valine peptidomimetic (TTT-28) antagonizes multidrug resistance in vitro and in vivo by selectively inhibiting the efflux activity of ABCB1

Multidrug resistance (MDR) attenuates the chemotherapy efficacy and increases the probability of cancer recurrence. The accelerated drug efflux mediated by ATP-binding cassette (ABC) transporters is one of the major MDR mechanisms. This study investigated if TTT-28, a newly synthesized thiazole-valine peptidomimetic, could reverse ABCB1-mediated MDR in vitro and in vivo. TTT-28 reversed the ABCB1-mediated MDR and increased the accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells by selectively blocking the efflux function of ABCB1, but not interfering with the expression level and localization of ABCB1. Animal study revealed that TTT-28 enhanced the intratumoral concentration of paclitaxel and promoted apoptosis, thereby potently inhibiting the growth of ABCB1 overexpressing tumors. But TTT-28 did not induce the toxicity (cardiotoxicity/myelosuppression) of paclitaxel in mice. In this study, we synthesized and evaluated a novel selective inhibitor of ABCB1 (TTT-28) with high efficacy and low toxicity. The identification and characterization of this new thiazole-valine peptidomimetic will facilitate design and synthesis of a new generation of ABCB1 inhibitors, leading to further research on multidrug resistance and combination chemotherapy. Furthermore, the strategy that co-administer MDR-ABCB1 inhibitor to overcome the resistance of one FDA approved, widely used chemotherapeutic paclitaxel, may be promising direction for the field of adjuvant chemotherapy.

Clinical significance of the ABCB1 and ABCG2 gene expression levels in acute lymphoblastic leukemia

ABSTRACTObjectives: Acute lymphoblastic leukemia (ALL) is a clonal disease that accounts for 20% of acute leukemias in adults. A high percentage of adult patients (ranging from 70 to 80%) reach complete remission; however, the 5-year survival rate is only 20–40%. One of the main obstacles to treatment success is the drug resistance of leukemic cells. Therefore, our research group analyzed the ABCB1 and ABCG2 gene expression levels in 61 patients diagnosed with ALL and assessed whether the levels affected the clinical parameters and 40-month survival rate.Methods: The ABCB1 and ABCG2 gene expression levels were analyzed using real-time polymerase chain reaction in 61 patients diagnosed with ALL and 99 healthy donors as controls. The association between ABCB1 and ABCG2 gene expression levels and clinical variables was determined using the Chi-square test and Fisher’s exact test. Overall survival (OS) was determined using the Kaplan–Meier method.Results: The results showed high ABCB1 and ABCG2 gene levels, which were 4.5 and 2.3 times the levels of healthy donors, respectively. A total of 52% of the study patients expressed high ABCB1 levels and were significantly associated with the high-risk patient group and a decreased 40-month survival rate of 78%. Only 49% of the patients expressed high ABCG2 gene levels. No association was found between the clinical parameters and the ABCG2 gene expression levels.Conclusions: Early detection of ABCB1 gene expression levels could be important for the diagnosis and monitoring of ALL patients.

Effect of the Adition of Metformin on the expressión of Multidrug-Resistance Genes (ABCB1, ABCG2) in Acute Lymphoblastic Leukemia: In Vitro and In Vivo Study

INTRODUCTION. The over expresión of the multidrug resistance genes (MDR) specifficaly ABCB1 and ABCG2 correlate with a poor prognosis in patients with ALL. Different drugs specially the chomotherapeutic agents are they substrates. This genes encode a family of different membrane transports that Works with energy specifically the ATP. Metformin is a biguanide whose mechanism of action is the activiation of AMPK and depletes the levels of ATP.PRIMARY OBJECTIVE: Evaluate the efficacy of the use of Metformin on the levels of expression of the MDR genes (ABCB1 y ABCG2) by RQ-PCR in ALL cell lines and patients with ALL during a preinduction treatment with prednisone.RESULTS.In vitro study. The ALL cell lines RS4, ReH, MOLT4 and SUB15 were elavluated. After the addition of Metformin (10uM) the expression level of ABCB1, ABCG2 started to decrease after the 24hrs of culture (mean expression level of 0.3823 ABCB1 and 0.4265 of ABCB2) with a máximum effect at 48 hours (0.2415 ABCB1 and 0.3452 ABCB2) The difference of levels were statystically significant (p= 0.0045).In vivo study. About 108 patients were evaluated, 44 (40.7%) in the arm of prednisone and 64 (59.3%) in the arm of prednisone plus metformin. Fort eh subanalysis of the MDR genes by RQ-PCR we select 25 patients (14 in the prednisone arm and 11 in the metformin arm). The mean levels of the genes was 0.1906 for the ABCB1 gene and 0.3371 of the ABDG2. At the moment of diagnosis about 32% were considerer with high level of expression (n=8), 28% (n=7) of low expression and 40% (n=10) do not express any of the both genes. After the treatment with Metformin 4 patients with high expression at diagnosis change the patter for low expression. Although there is no difference in means of the expression of genes in both arms (p = 0.391 , p = 0. 828) in the group of metformin the prognosis in the high expression group was better than in the prednisone arm (p=0.043). Fort he entire cohort the complete remission rate was higher in the Metformin cohort comparing with prednisone (86.4% versus 67.2%) and the refractoriness was higher in the group of prednisone (n=16, 25%) p=0.002. About the relapse, the addition of Metformin reduce the relapse rate ( 6 vs 21 patients, p = 0.027).Conclusion. The addition of metformin in a controlled enviroment reduce the expression of the MDR genes, but in vivo the impact is primarily on the refractory leukemias and the relapse rate DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-595 sensitizes ovarian cancer cells to cisplatin by targeting ABCB1.

Ovarian cancer is among the leading cause of cancer-related deaths in females. In this study, we demonstrated that miR-595 expression was downregulated in the ovarian cancer tissues and cell lines. miR-595 expression was lower in the lymph node metastases tissues than in the primary ovarian cancer tissues and normal tissues. Furthermore, miR-595 overexpression suppressed the ovarian cancer cell proliferation, colony formation and invasion and promoted the sensitivity of ovarian cancer cell to cisplatin. We identified ABCB1 as a direct target gene of miR-595 in the ovarian cancer cell. ABCB1 expression was upregulated in the ovarian cancer tissues and cell lines. Morevoer, the expression level of ABCB1 was inversely correlated with miR-595 in the ovarian cancer tissues. In addition, overexpression of ABCB1 decreased the miR-595-overexpressing HO8910PM and SKOV-3 cell sensitivity to cisplatin. Ectopic expression of ABCB1 promoted the miR-595-overexpressing HO8910PM and SKOV-3 cell proliferation, colony formation and invasion. These data suggested that miR-595 acted a tumor suppressor role in ovarian cancer development and increased the sensitivity of ovarian cancer to cisplatin.

ABCB1 2677G>T/A variant enhances chemosensitivity to anti-cancer agents acting on microtubule dynamics through LAMP1 inhibition

Overexpression of ABCB1 associated with single nucleotide variants in cancers was reported to encode a protein responsible for drug resistance. We studied chemosensitivity-related genes associated with ABCB1 2677G>T/A variant. The associated genes were identified based on the results of the significance analysis of microarray, and then prediction accuracy was evaluated using the prediction analysis of microarray. Functional assay of the selected gene was performed by using siRNA and drug accumulation study.A higher frequency of chemoresistance to microtubule-modulating agents was found in cell lines with wild-type ABCB1 compared to cell lines with 2677G>T/A ABCB1 variant. Based on the pharmacogenetic association study with 2677 variant, we identified seven genes that could predict chemosensitivity to microtubule dynamics modulators. The classification accuracy with these seven genes was 90.0%, and the predicted probability was 0.73. LAMP1 was the only gene that was commonly related to chemosensitivity. LAMP1 expression levels were relatively higher in chemoresistant ABCB1 wild-type compared to chemosensitive polymorphic cells. But, there was no difference in ABCB1 expression levels between the two groups. Following LAMP1 siRNA, chemosensitivity was restored due to increased intracellular drug accumulation in wild type cell line. In conclusion, ABCB1 2677G>T/A variant enhances chemosensitivity on microtubule dynamics through LAMP1 inhibition.

Targeting CXCR4 and FAK reverses doxorubicin resistance and suppresses invasion in non-small cell lung carcinoma.

Current high lung cancer mortality rates are mainly due to the occurrence of metastases and therapeutic resistance. Therefore, simultaneous targeting of these processes may be a valid approach for the treatment of this type of cancer. Here, we assessed relationships between CXC chemokine receptor type 4 (CXCR4) and focal adhesion kinase (FAK) gene expression levels and expression levels of the drug resistance-related genes ABCB1 and ABCC1, and tested the potential of CXCR4 and FAK inhibitors to reverse doxorubicin (DOX) resistance and to decrease the invasive capacity of non-small cell lung carcinoma (NSCLC) cells. qRT-PCR was used for gene expression analyses in primary lung tissue samples obtained from 30 NSCLC patients and the human NSCLC-derived cell lines NCI-H460, NCI-H460/R and COR-L23. MTT, flow cytometry, cell death and β-galactosidase activity assays were used to assess the in vitro impact of CXCR4 and FAK inhibitors on DOX sensitivity. In addition, invasion and gelatin degradation assays were used to assess the in vitro impact of the respective inhibitors on metastasis-related processes in combination with DOX treatment. We found that ABCB1 over-expression was significantly associated with CXCR4 and FAK over-expression, whereas ABCC1 over-expression was associated with increased FAK expression. We also found that CXCR4 and FAK inhibitors strongly synergized with DOX in reducing cell viability, arresting the cell cycle in the S or G2/M phases and inducing senescence. Additionally, we found that DOX enhanced the anti-invasive potential of CXCR4 and FAK inhibitors by reducing gelatin degradation and invasion. From our data we conclude that targeting of CXCR4 and FAK may overcome ABCB1 and ABCC1-dependent DOX resistance in NSCLC cells and that simultaneous treatment of these cells with DOX may potentiate the anti-invasive effects of CXCR4 and FAK inhibitors.

Effect of abemaciclib (LY2835219) on enhancement of chemotherapeutic agents in ABCB1 and ABCG2 overexpressing cells in vitro and in vivo

Multidrug resistance (MDR) is the major obstacle of the success in cancer chemotherapy. The overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1 and ABCG2, play a significant role in mediating MDR by pumping anticancer drugs out of cancer cells. Abemaciclib (LY2835219) is an orally bioavailable CDK4/6 inhibitor under phase III clinical trials. Here, we found that LY2835219 remarkably enhanced the efficacy of chemotherapeutic drugs in ABCB1 or ABCG2 over-expressing cancer cells in vitro and in vivo. Furthermore, LY2835219 significantly increased the intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) by inhibiting ABCB1 or ABCG2-mediated drug efflux in the transporters-overexpressing cells. Mechanistically, LY2835219 is likely a competitive inhibitor of ABCB1 and ABCG2 for its competition with [125I]-iodoarylazidoprazosin for photo affinity labeling of the transporters. On the other hand, at the transporters-inhibiting concentrations, LY2835219 did not alter the expression level of ABCB1 and ABCG2, and the phosphorylation status of retinoblastoma (Rb) pathway in both parental and their resistant cells. In conclusion, these findings revealed a novel role of LY2835219 in reversing ABCB1 or ABCG2-mediated MDR, which may be benefit to the patients with MDR cancer for combinational therapy.

Potential pharmacokinetic effect of rifampicin on enrofloxacin in broilers: Roles of P-glycoprotein and BCRP induction by rifampicin

P-glycoprotein ( P-GP: , encoding gene Abcb1) and Breast Cancer Resistance Protein ( BCRP: , encoding gene Abcg2) are transport proteins that play a major role in modulating the bioavailability of oral drugs in humans and rodents. It has been shown that rifampicin is the typical inducer of P-gp in rodents by activating the nuclear receptor. However, its effect on Abcb1, Abcg2, CYP3A, and chicken xenobiotic-sensing orphan nuclear receptor ( CXR: ) mRNA expression in broilers is poorly understood. This study explored the effect of rifampicin on mRNA expression of Abcb1, Abcg2, CYP3A37, CXR as well as its effect on the pharmacokinetics of enrofloxacin in broilers. The mRNA levels of Abcb1, Abcg2, CYP3A37, and CXR were significantly increased in the liver (except Abcg2), kidney, jejunum, and ileum (P < 0.05) but not significantly changed in the duodenum (P > 0.05) after treated with rifampicin. Further analysis revealed that the variation tendencies of Abcb1, Abcg2, and CYP3A37 expression levels were significantly correlated with CXR mRNA expression levels in liver, kidney, jejunum, and ileum. Coadministration of rifampicin significantly changed the pharmacokinetic behavior of enrofloxacin orally administered by showing clearly lower AUC0-∞, AUC0-t, and Cmax as well as longer Tmax. The bioavailability of orally administered enrofloxacin was decreased from 72.5% to 24.8% by rifampicin. However, rifampicin did not significantly change the pharmacokinetics of enrofloxacin following intravenous administration. Our study shows that rifampicin up-regulated the small intestinal level of P-gp and BCRP and suggests that P-gp and BCRP are key factors that affected pharmacokinetic behavior of orally administered enrofloxacin by limiting its absorption from the intestine in broilers.

ABCB1 Overexpression Is a Key Initiator of Resistance to Tyrosine Kinase Inhibitors in CML Cell Lines.

The tyrosine kinase inhibitor (TKI) imatinib has resulted in excellent responses in the majority of Chronic Myeloid Leukaemia (CML) patients; however, resistance is observed in 20–30% of patients. More recently, resistance to the second generation TKIs, nilotinib and dasatinib, has also been observed albeit at a lower incidence. ABCB1 has previously been implicated in TKI export and its overexpression linked to TKI resistance. In this study the dynamics of nilotinib resistance was studied in CML cell lines with particular focus on ABCB1 expression levels during development of resistance. Results revealed ABCB1 overexpression is likely an important initiator of nilotinib resistance in vitro. ABCB1 overexpression was also observed in cell lines as an intermediate step during development of resistance to imatinib and dasatinib in vitro. We conclude that ABCB1 overexpression may provide an initial platform to facilitate development of additional mechanisms for resistance to TKIs. This provides a rationale for investigating this phenomenon in patients undergoing TKI therapy.

Osimertinib (AZD9291) Attenuates the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCB1 in Vitro.

The effectiveness of cancer chemotherapy is often circumvented by multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in human cancer cells. Furthermore, some TKIs are transported by ABCB1, which results in low oral bioavailability, reduced distribution, and the development of acquired resistance to these TKIs. In this study, we investigated the interaction between ABCB1 and osimertinib, a novel selective, irreversible third-generation EGFR TKI that has recently been approved by the U.S. Food and Drug Administration. We also evaluated the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells, which can present a therapeutic challenge to clinicians in the future. We revealed that although osimertinib stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer resistance to osimertinib. Our results suggest that it is unlikely that the overexpression of ABCB1 can be a major contributor to the development of osimertinib resistance in cancer patients. More significantly, we revealed an additional action of osimertinib that directly inhibits the function of ABCB1 without affecting the expression level of ABCB1, enhances drug-induced apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. Considering that osimertinib is a clinically approved third-generation EGFR TKI, our findings suggest that a combination therapy with osimertinib and conventional anticancer drugs may be beneficial to patients with MDR tumors.

The Allosteric Inhibitor ABL001 Is Susceptible to Resistance in Vitro Mediated By Overexpression of the Drug Efflux Transporters ABCB1 and ABCG2

Tyrosine Kinase Inhibitors (TKIs) result in excellent responses in Chronic Myeloid Leukaemia (CML) patients. However, secondary resistance is observed in ~35% of patients, mostly due to Bcr-Abl kinase domain (KD) mutations. Overexpression of the efflux transporter ABCB1 is a known mediator of primary resistance to imatinib (IM) and nilotinib (NIL). ABL001, a potent allosteric inhibitor, binds to the myristate pocket of the Bcr-Abl KD. In this study, we modelled ABL001 resistance in vitro with particular focus on ABCB1 and ABCG2.ABL001 sensitivity was evaluated in BCR -ABL1 + cell lines K562, K562-Dox (ABCB1 overexpressing) and K562-ABCG2 (transfected with ABCG2 overexpression vector). Also, K562, K562-Dox and KU812 cells were made resistant to ABL001 by culturing long term in increasing concentrations of ABL001 (increased once >80% survival in culture for >10 days). Parental ABL001 naive cells were maintained in parallel culture. Initial onset of resistance was characterised by significantly increased IC50ABL001 based on AnnexinV/7-AAD cytotoxicity assays performed at each ABL001 dose escalation. Resistance mechanisms were interrogated during escalation and once 10 μM was reached (based on clinically achievable doses). Protein expression levels of efflux transporters ABCB1/ABCG2 were examined by flow cytometry; KD mutation sequencing and Bcr-Abl protein quantitation were performed.K562-Dox and K562-ABCG2 cells demonstrated significantly increased IC50ABL001 compared with parental K562 control cells: 256 and 299 nM respectively vs 23 nM, p<0.001 suggesting both ABCB1 and ABCG2 are important in ABL001 transport. Furthermore, resistance was reversible through use of specific inhibitors cyclosporin (ABCB1) and Ko143 (ABCG2). IC50ABL001 +inhibitors was comparable to that of control K562 cells: K562-Dox +cyclosporin=11 nM; K562-ABCG2 +Ko143=15 nM.A prior study identified ABCB1 overexpression as an initiator of resistance to IM and NIL (expression increased up to 7- and 5-fold in IM and NIL resistant cells compared with respective control cells p<0.002). In this study, expression levels of ABCB1 and ABCG2 were interrogated in 3 ABL001 resistant cell lines; results indicated overexpression of both transporters was integral in development of resistance. Up to 17- and 60-fold greater levels of ABCB1 and ABCG2 respectively was observed in resistant vs control cells (Table 1). No KD mutations were detected in early resistance intermediates; however, a myristate pocket mutation was detected in later stage KU812 ABL001 resistant cells (percentage mutation correlated with IC50ABL001).Table 1ABL001 resistance characteristics in selected resistant intermediatesIC50ABL001 (nM)Fold change (MFI)Bcr-Abl over expressionKD mutationABCB1ABCG2K562 Control23NNK562 500 nM ABL001>2500 p <0.001-1.4 p =0.00348.3 p =0.007YNK562 10 μM ABL00127, 800p <0.001-2.3 p =0.53560.3 p <0.001YNK562 10 μM ABL001 +Ko14389 p <0.001K562-Dox Control256NNK562-Dox 500 nM ABL0012393 p <0.0012.3 p <0.001-2.9 p <0.001NNK562-Dox 500 nM ABL001 +cyclosporin17 p <0.001K562-Dox 10 μM ABL001>50, 000 p <0.001-1.6 p <0.001-0.1 p <0.001NNKU812 Control2.7NNKU812 5 nM ABL0016.4 p <0.0012.2 p =0.0084.0 p =0.002YNKU812 10 μM ABL00133, 300 p <0.0018.1 p =0.00311.0 p =0.010YY (90%)p-value: resistant vs respective control; n>3; N=No; Y=YesWhile further in vitro and in vivo studies will determine the clinical relevance of efflux-mediated resistance to ABL001 mono- and combination therapy, our preclinical data provide evidence that kinase domain mutations may not be the predominant cause of ABL001 resistance; drug transporters likely play an important role as well. Susceptibility to ABCB1 overexpression is well recognised for IM and NIL resistance and we now show it is also relevant for ABL001. ABCG2-mediated resistance has not been observed with NIL or IM but is clearly important for ABL001. ABL001 treatment induced ABCG2 overexpression in two cell lines; both have negligible basal expression. ABCG2 overexpression preceded Bcr-Abl overexpression and mutation emergence. Importantly, ABL001 resistance was completely reversible in the presence of the ABCG2 inhibitor Ko143 (K562 10 μM ABL001 cells). Given the lack of strong evidence for ABCG2-mediated transport of NIL or IM at clinically relevant concentrations, our data provide a strong rationale for the use of ABL001 in combination with either of these TKIs. DisclosuresBranford:Ariad: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract B165: Optimizing selective CDK7 inhibition in MYCN-driven neuroblastoma

Abstract Amplification of the MYCN gene defines approximately 50% of high-risk neuroblastomas (NB), and is associated with aggressive disease and a poor clinical outcome. We recently demonstrated that MYCN-amplified NBs are dependent on MYCN-driven global transcriptional amplification, and that they can be selectively targeted using a first-in-class covalent cyclin-dependent kinase (CDK) 7/12 inhibitor, THZ1. CDK7, CDK9 and CDK12 directly phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAPII) to regulate gene transcription. NB cells expressing high levels of MYCN were 10 times more sensitive to THZ1 than normal cells or NB cells not driven by amplified MYCN. THZ1 caused tumor growth inhibition in murine models of human NB, without general toxicity indicating the clinical potential of this therapeutic strategy. To identify mechanisms of resistance to THZ1, we generated THZ1-resistant NB cells (THZ1-R) by exposing sensitive cells to escalating sub-lethal doses of THZ1. THZ1-R cells exhibited no changes MYCN levels or RNAPII activity, eliminating alterations in CDK7 itself and/or emergence of compensatory pathways to account for the resistance. A survey of common multidrug resistance mechanisms revealed that extended THZ1 exposure exclusively enhanced the transcript levels of ATP-Binding Cassette sub-family member B1 (ABCB1, also MDR1) by 500-fold, which was translated to the protein level as well. The importance of ABCB1 in THZ1-resistance was demonstrated by rescue of THZ1 activity using the selective ABCB1 inhibitor tariquidar and shRNA-mediated gene knockdown. Subsequent screening of CDK inhibitors of the SBI-E class in these ABCB1-overexpressing NB cells afforded the identification of a covalent CDK9/12 inhibitor (SBI-E-9) devoid of efflux-substrate characteristics. Regardless of the expression levels of ABCB1, SBI-E-9 selectively suppressed MYCN expression and MYCN-associated transcriptional activity. In addition, we evaluated the in vitro efficacy of a series of covalent CDK7 inhibitors with distinctive scaffolds from THZ1. Although these compounds do not escape ABCB1-mediated drug efflux, their specific targeting of CDK7 over CDK12 correlated with greater differential selectivity in MYCN-amplified vs. MYCN non-amplified NB cells (30-fold versus 10-fold with THZ1). In conclusion, we report, for the first time, ABCB1-regulated drug efflux as a major mechanism of resistance to the first-generation covalent CDK7/12 inhibitor, THZ1, which can be overcome by a newly identified, covalent CDK9/12 inhibitor, SBI-E-9. Additionally, we have identified second generation covalent CDK7 inhibitors that display a higher selectivity against MYCN-driven NB through augmented CDK7 activity. Hence, these covalent CDK inhibitors represent attractive therapeutic options for MYCN-driven NB, and perhaps other MYC-dependent cancers. Citation Format: Yang Gao, Yanke Liang, Tinghu Zhang, Nathanael S. Gray, Rani E. George. Optimizing selective CDK7 inhibition in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B165.