Overexpression Of ABCG2 Research Articles

Mobocertinib antagonizes multidrug resistance in ABCB1- and ABCG2-overexpressing cancer cells: In vitro and in vivo studies.

Overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1 and ABCG2, strongly correlates with multidrug resistance (MDR), rendering cancer chemotherapy ineffective. Exploration and identification of novel inhibitors targeting ABCB1 and ABCG2 are necessary to overcome the related MDR. Mobocertinib is an approved EGFR/HER2 inhibitor for non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations. This study demonstrates that mobocertinib can potentially reverse ABCB1- and ABCG2-mediated MDR. Our findings indicate a strong interaction between mobocertinib and these two proteins, supported by its high binding affinity with ABCB1 and ABCG2 models. Through inhibiting the drug efflux function of ABCB1 and ABCG2, mobocertinib facilitates substrate drugs accumulation, thereby re-sensitizing substrate drugs in drug-resistant cancer cells. Additionally, mobocertinib inhibited the ATPase activity of ABCB1 and ABCG2 without changing the expression levels or subcellular localization. In the tumor-bearing mouse model, mobocertinib boosted the antitumor effect of paclitaxel and topotecan, resulting in tumor regression. In summary, our study uncovers a novel potential for repurposing mobocertinib as a dual inhibitor of ABCB1 and ABCG2, and suggests the combination of mobocertinib with substrate drugs as a strategy to counteract MDR.

Impact of ABCB1 and ABCG2 Transporter in Outcome of Gallbladder Cancer

BackgroundGallbladder cancer (GBC) is a biologically aggressive malignancy requiring appropriate biomarkers to improve its outcome. Role of ABC transporters (ABCB1 and ABCG2) has been linked to cancer aggressiveness, tumorigenesis and multidrug resistance. Herein, we studied the prognostic implication of ABCB1 and ABCG2 in GBC. MethodsFresh tissue (tumour & normal) samples collected from 54 patients who underwent R0 resection, were analysed for mRNA and protein expression of ABCB1 and ABCG2 by quantitative Real-Time PCR and western blotting respectively in this prospective study. The molecular findings were correlated with clinical-pathological parameters using χ2 and fisher exact test. The molecular changes in ABCB1 and ABCG2 was analysed for predicting overall survival (OS), disease free survival (DFS) and response to chemotherapy using Kaplan Meier log rank test and Cox regression multivariate analysis. ResultsThe mean age of the cohort was 50 ±13.2 with 26 (48.1%) patients had early stage gallbladder cancer (GBC). Overexpression of ABCB1 and ABCG2 was noted in 32/54(59%) and 40/54 (74%) cases respectively. The protein expression of ABCB1(P-glycoprotein) and ABCG2 (BCRP) was higher in 27/54 (50%) and 37/54 (59%) cases respectively. The mean OS and DFS was 20.7 ±11.5 and 19.3 ± 12.2 months at median follow up 24 months. The TNM stage, lymph node metastasis, and presence of gallstone were significant factors for predicting OS and DFS on multivariate analysis. Both ABCB1 and ABCG2 did not show any significant correlation with OS and DFS with similar incidence of late death and recurrence among over-expression and down-expression. Sub- group comparison suggests that change in expression pattern of ABCB1 and ABCG2 may not affect response to chemotherapy in GBC. ConclusionAltered expression of ABCB1 and ABCG2 may not be a useful prognostic marker for survival or response to chemotherapy in GBC. Presently, histo-pathological characteristics and associated gallstones are the important predictors for survival and recurrence in GBC.

Abstract LB260: Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly polymerase inhibitor

Abstract Aims: The overexpression of ABC transporters on cancer cell membranes is one of the most common causes of multidrug resistance (MDR). This study investigates the impact of ABCC1 and ABCG2 on the resistance to talazoparib (BMN-673), a potent poly (ADP-ribose) polymerase (PARP) inhibitor, in ovarian cancer treatment. Methods: The cell viability test was used to indicate the effect of talazoparib in different cell lines. Computational molecular docking analysis was conducted to simulate the interaction between talazoparib and ABCC1 or ABCG2. The mechanism of talazoparib resistance was investigated by constructing talazoparib-resistant subline A2780/T4 from A2780 through drug selection with gradually increasing talazoparib concentration. Results: Talazoparib cytotoxicity decreased in drug-selected or gene-transfected cell lines overexpressing ABCC1 or ABCG2 but can be restored by ABCC1 or ABCG2 inhibitors. Talazoparib competitively inhibited substrate drug efflux activity of ABCC1 or ABCG2. Upregulated ABCC1 and ABCG2 protein expression on the plasma membrane of A2780/T4 cells enhances resistance to other substrate drugs, which could be overcome by the knockout of either gene. In vivo experiments confirmed the retention of drug-resistant characteristics in tumor xenograft mouse models. Conclusions: The therapeutic efficacy of talazoparib in cancer may be compromised by its susceptibility to MDR, which is attributed to its interactions with the ABCC1 or ABCG2 transporters. The overexpression of these transporters can potentially diminish the therapeutic impact of talazoparib in cancer treatment. Citation Format: Shuo Fang, Xiaona Fang. Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly polymerase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB260.

Overexpression of ABCC1 and ABCG2 confers resistance to talazoparib, a poly (ADP-Ribose) polymerase inhibitor

AimsThe overexpression of ABC transporters on cancer cell membranes is one of the most common causes of multidrug resistance (MDR). This study investigates the impact of ABCC1 and ABCG2 on the resistance to talazoparib (BMN-673), a potent poly (ADP-ribose) polymerase (PARP) inhibitor, in ovarian cancer treatment. MethodsThe cell viability test was used to indicate the effect of talazoparib in different cell lines. Computational molecular docking analysis was conducted to simulate the interaction between talazoparib and ABCC1 or ABCG2. The mechanism of talazoparib resistance was investigated by constructing talazoparib-resistant subline A2780/T4 from A2780 through drug selection with gradually increasing talazoparib concentration. ResultsTalazoparib cytotoxicity decreased in drug-selected or gene-transfected cell lines overexpressing ABCC1 or ABCG2 but can be restored by ABCC1 or ABCG2 inhibitors. Talazoparib competitively inhibited substrate drug efflux activity of ABCC1 or ABCG2. Upregulated ABCC1 and ABCG2 protein expression on the plasma membrane of A2780/T4 cells enhances resistance to other substrate drugs, which could be overcome by the knockout of either gene. In vivo experiments confirmed the retention of drug-resistant characteristics in tumor xenograft mouse models. ConclusionsThe therapeutic efficacy of talazoparib in cancer may be compromised by its susceptibility to MDR, which is attributed to its interactions with the ABCC1 or ABCG2 transporters. The overexpression of these transporters can potentially diminish the therapeutic impact of talazoparib in cancer treatment.

MiR-548c-3p through suppressing Tyms and Abcg2 increases the sensitivity of colorectal cancer cells to 5-fluorouracil

BackgroundColorectal cancer, is one of most prevalent the cancer in the world. 5-Fluorouracil is a standard chemotherapeutic drug while the acquisition of resistance to 5-Fluorouracil is one of the problems during treatment. In this study, we aimed to find the miRNAs that modulate the expression of Tyms and Abcg2 as resistance-inducing genes in the resistant cell lines to 5-Fluorouracil. Methods5-Fluorouracil-resistant HCT116 and SW480 cell lines were generated by consecutive treatment of cells with 5-Fluorouracil. This resistance induction was validated by MTT assays. The expression of the Tyms and Abcg2 gene and miR-548c-3p were studied by quantitative real-time PCR in the cell lines. ResultsWe hypothesized that miR-548c-3p is targeting Tyms and Abcg2 simultaneously. Increased expression Tyms gene in the two most resistant cell lines derived from HCT116 and all resistant cell lines derived from SW480 except one were seen. Increased expression of Abcg2 was observed in the most resistant HCT116-derived cell line and all resistant cell lines, derived from SW480. In all resistant cell lines, the expression of miR-548c-3p was decreased. ConclusionIt can be concluded downregulation of miR548c-3p is in line with Tyms and Abcg2 overexpression in resistant cell lines to 5-Fluorouracil.

Neuropilin-2 Inhibits Drug Resistance and Progression of Melanoma Involving the MiR-331-3p Regulated Cascade.

MicroRNAs (miRs) are small noncoding RNAs that are crucial in the development and progression of tumours. Melanoma is an aggressive form of skin cancer and is resistant to most of the chemotherapeutic agents. However, the role of miRs in melanoma remains poorly studied. The work aimed to demonstrate that miR-331-3p is downregulated in melanoma against the benign melanocytic nevi. RT-PCR analysis was performed for the expression of proteins; cell proliferation and wound healing assays were carried out. Flow cytometry study was conducted for cell cycle analysis; colony formation assay was performed by soft agar method. For developing a tumour xenograft model, nu/nu mice were selected. Up-regulation of miR-331-3p in melanoma cells decreased cell proliferation, cell migration, and also drug resistance. Over-expression of miR-331-3p resulted in suppression of NRP2 and up-regulation of E-cadherin levels. Moreover, the levels of MDR1, ABCG-2, and ABCG-5 were decreased. However, the knockdown of NRP2 demonstrated similar effects as that of miR- 331-3p overexpression in tumour cells. Overexpression of miR-331-3p caused significant inhibition of tumour growth and its metastasis in mice model of melanoma, which was associated with depletion of NRP2 protein and increased expression of E-cadherin. However, the effects of miR- 331-3p on the migration, cell proliferation, and self-renewal were overturned by the upregulation of NRP2, which also resulted in the inhibition of E-cadherin and overexpression of MDR-1, ABCG-2, and ABCG-5. The findings point out the key role of miR-331-3p in the progression and drug resistance of melanoma involving NRP2.

The acquisition order of leukemic drug resistance mutations is directed by the selective fitness associated with each resistance mechanism

In Chronic Myeloid Leukemia, the transition from drug sensitive to drug resistant disease is poorly understood. Here, we used exploratory sequencing of gene transcripts to determine the mechanisms of drug resistance in a dasatinib resistant cell line model. Importantly, cell samples were collected sequentially during drug exposure and dose escalation, revealing several resistance mechanisms which fluctuated over time. BCR::ABL1 overexpression, BCR::ABL1 kinase domain mutation, and overexpression of the small molecule transporter ABCG2, were identified as dasatinib resistance mechanisms. The acquisition of mutations followed an order corresponding with the increase in selective fitness associated with each resistance mechanism. Additionally, it was demonstrated that ABCG2 overexpression confers partial ponatinib resistance. The results of this study have broad applicability and help direct effective therapeutic drug usage and dosing regimens and may be useful for clinicians to select the most efficacious therapy at the most beneficial time.

The AKT inhibitor, MK-2206, attenuates ABCG2-mediated drug resistance in lung and colon cancer cells.

Introduction: The overexpression of ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, are two of the major mediators of multidrug resistance (MDR) in cancers. Although multiple ABCB1 and ABCG2 inhibitors have been developed and some have undergone evaluation in clinical trials, none have been clinically approved. The compound, MK-2206, an inhibitor of the protein kinases AKT1/2/3, is undergoing evaluation in multiple clinical trials for the treatment of certain types of cancers, including those resistant to erlotinib. In this in vitro study, we conducted in vitro experiments to determine if MK-2206 attenuates multidrug resistance in cancer cells overexpressing the ABCB1 or ABCG2 transporter. Methodology: The efficacy of MK-2206 (0.03-1μM), in combination with the ABCB1 transporter sub-strates doxorubicin and paclitaxel, and ABCG2 transporter substrates mitoxantrone, SN-38 and topotecan, were determined in the cancer cell lines, KB-C2 and SW620/Ad300, which overexpress the ABCB1 transporter or H460/MX20 and S1-M1-80, which overexpress the ABCG2 transporter, respectively. The expression level and the localization of ABCG2 transporter on the cancer cells membranes were determined using western blot and immunofluorescence assays, respectively, following the incubation of cells with MK-2206. Finally, the interaction between MK-2206 and human ABCG2 transporter was predicted using computer-aided molecular modeling. Results: MK-2206 significantly increased the efficacy of anticancer compounds that were substrates for the ABCG2 but not the ABCB1 transporter. MK-2206 alone (0.03-1μM) did not significantly alter the viability of H460/MX20 and S1-M1-80 cancer cells, which overexpress the ABCG2 transporter, compared to cells incubated with vehicle. However, MK-2206 (0.3 and 1μM) significantly increased the anticancer efficacy of mitoxantrone, SN-38 and topotecan, in H460/MX20 and S1-M1-80 cancer cells, as indicated by a significant decrease in their IC50 values, compared to cells incubated with vehicle. MK-2206 significantly increased the basal activity of the ABCG2 ATPase (EC50 = 0.46μM) but did not significantly alter its expression level and sub-localization in the membrane. The molecular modeling results suggested that MK-2206 binds to the active pocket of the ABCG2 transporter, by a hydrogen bond, hydrophobic interactions and π-π stacking. Conclusion: These in vitro data indicated that MK-2206 surmounts resistance to mitoxantrone, SN-38 and topotecan in cancer cells overexpressing the ABCG2 transporter. If these results can be translated to humans, it is possible that MK-2206 could be used to surmount MDR in cancer cells overexpressing the ABCG2 transporter.

Monoacylglycerol Lipase Inhibitor JJKK048 Ameliorates ABCG2 Transporter-Mediated Regorafenib Resistance Induced by Hypoxia in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression.The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl2) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays.Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC50) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy.In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

ABCG2 plays a central role in the dysregulation of 25-hydrovitamin D in Crohn's disease

Vitamin D (VD) deficiency, as indicated by the main circulating form of VD metabolite 25-hydrovitamin D3 (25(OH)D3), in patients with Crohn's disease (CD) has been well documented, but the reasons for this remain unclear. In this study, 367 patients with CD and 57 healthy individuals who were enrolled, and the association between 25 (OH)D3 level and clinical biochemical characteristics including hepatic and renal functions, inflammatory response was analyzed with binary logistic regression models. VD metabolic enzymes and transporters were screened with bioinformatical analysis and identified with qRT-PCR and western blot. Compared to the healthy controls, serum 25(OH)D3 was significantly reduced in patients with CD, but the protein level of adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) was evidently increased in the ileum and colon. Meanwhile, in lipopolysaccharide (LPS)-treated CaCO2 cells, the mRNA and protein levels of ABCG2 were significantly increased, and the overexpression of ABCG2 obviously promoted 25(OH)D3 efflux, but, Ko143, an ABCG2 inhibitors, substantially prevented the efflux of 25(OH)D3. In addition, in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD model mice, the ABCG2 protein levels were significantly increased in the ileum, colon, kidney and liver, and administration of Ko143 significantly inhibited the efflux of 25 (OH) D3in vivo. All of these findings suggest that VD deficiency in patients with CD may be associated with an abnormal increase in ABCG2 expression, but not directly implicated in hepatic and renal function, and inflammatory response in patients with CD.

ABCB1 and ABCG2 Overexpression Mediates Resistance to the Phosphatidylinositol 3-Kinase Inhibitor HS-173 in Cancer Cell Lines.

Constitutive activation of the phosphoinositide-3-kinase (PI3K)/Akt signaling pathway is crucial for tumor growth and progression. As such, this pathway has been an enticing target for drug discovery. Although HS-173 is a potent PI3K inhibitor that halts cancer cell proliferation via G2/M cell cycle arrest, the resistance mechanisms to HS-173 have not been investigated. In this study, we investigated the susceptibility of HS-173 to efflux mediated by the multidrug efflux transporters ABCB1 and ABCG2, which are two of the most well-known ATP-binding cassette (ABC) transporters associated with the development of cancer multidrug resistance (MDR). We found that the overexpression of ABCB1 or ABCG2 significantly reduced the efficacy of HS-173 in human cancer cells. Our data show that the intracellular accumulation of HS-173 was substantially reduced by ABCB1 and ABCG2, affecting G2/M arrest and apoptosis induced by HS-173. More importantly, the efficacy of HS-173 in multidrug-resistant cancer cells could be recovered by inhibiting the drug-efflux function of ABCB1 and ABCG2. Taken together, our study has demonstrated that HS-173 is a substrate for both ABCB1 and ABCG2, resulting in decreased intracellular concentration of this drug, which may have implications for its clinical use.

IFIT2 Depletion Promotes Cancer Stem Cell-like Phenotypes in Oral Cancer

(1) Background: Cancer stem cells (CSCs) are a small cell population associated with chemoresistance, metastasis and increased mortality rate in oral cancer. Interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) depletion results in epithelial to mesenchymal transition, invasion, metastasis, and chemoresistance in oral cancer. To date, no study has demonstrated the effect of IFIT2 depletion on the CSC-like phenotype in oral cancer cells. (2) Methods: Q-PCR, sphere formation, Hoechst 33,342 dye exclusion, immunofluorescence staining, and flow cytometry assays were performed to evaluate the expression of the CSC markers in IFIT2-depleted cells. A tumorigenicity assay was adopted to assess the tumor formation ability. Immunohistochemical staining was used to examine the protein levels of IFIT2 and CD24 in oral cancer patients. (3) Results: The cultured IFIT2 knockdown cells exhibited an overexpression of ABCG2 and CD44 and a downregulation of CD24 and gave rise to CSC-like phenotypes. Clinically, there was a positive correlation between IFIT2 and CD24 in the patients. IFIT2high/CD24high/CD44low expression profiles predicted a better prognosis in HNC, including oral cancer. The TNF-α blockade abolished the IFIT2 depletion-induced sphere formation, indicating that TNF-α may be involved in the CSC-like phenotypes in oral cancer. (4) Conclusions: The present study demonstrates that IFIT2 depletion promotes CSC-like phenotypes in oral cancer.

Bixin and Fuxoxanthin Alone and in Combination with Cisplatin Regulate ABCC1 and ABCC2 Transcription in A549 Lung Cancer Cells.

The ATP-Binding Cassette (ABC) transporter has long been studied to confer drug resistance in human tumors and play important role in metabolic processes and cellular signaling. The overexpression of ABCB1, ABCC1, ABCC2, ABCC3, and ABCG2 leads to decreased sensitivity of lung cancer to cisplatin. At the transcription level, the expression of ABC transporters is highly regulated and requires the complex interplay of factors involved in differentiation and development, cell survival and apoptosis upon intrinsic and environmental stress. The p53 regulation of drug-resistance genes is also complex yet not well understood. Previously, we demonstrated the synergistic interaction between bixin or fucoxanthin with cisplatin in A549 lung cancer cells. Current study aims to identify whether carotenoids enhancing therapeutic effect of Cisplatin due to the ability to reverse drug resistance associated proteins, such as ABC transporter and regulating the tumor suppressor corresponding gene, p53. Real-Time Quantitative-Polymerase Chain Reaction (RT-qPCR) was performed to estimate the expression level of ABCC1 and ABCC2, and p53 of A549 cell lines in response to carotenoids alone and in combination with cisplatin. The administration of bixin or fucoxanthin decreases the expression of ABCC1 and ABCC2. Both carotenoids, either alone or in combination with cisplatin, upregulated p53 gene expression indicating the mechanism of proliferation inhibition and apoptosis occurs via the p53 caspase-independent pathway.

Abstract 1095: Establishment and characterization of a topotecan resistant lung cancer NCI H460/TPT10 cell line and a tumor xenograft model

Abstract While topotecan (TPT) is a chemotherapeutic drug in treating lung cancer, the development of TPT resistance in tumors reserves as a major obstacle to chemotherapeutic success. Therefore, a better understanding of the mechanisms of TPT resistance is critical. In this study, the first topotecan-resistant human non-small cell lung cancer (NSCLC) cell line, termed NCI-H460/TPT10, was established from the parental NCI-H460 cell line. NCI-H460/TPT10 cells exhibited a 394.7-fold resistance to TPT, and cross-resistance to SN-38, mitoxantrone, and doxorubicin, compared to parental NCI-H460 cells. Overexpression of ABCG2 localized on the cell membrane, but not ABCB1 or ABCC1, was found in NCI-H460/TPT10 cells, indicating that ABCG2 was likely to be involved in TPT-resistance. This was confirmed by the abolishment of drug resistance in NCI-H460/TPT10 cells after ABCG2 knockout. Moreover, the involvement of functional ABCG2 as a drug efflux pump conferring multidrug resistance (MDR) was indicated by low intracellular accumulation of TPT in NCI-H460/TPT10 cells, and the reversal effects by ABCG2 inhibitor Ko143 and cabozantinib. The NCI-H460/TPT10 and its parental cell line were further used to establish in vivo tumor xenograft mouse models, which verified their capability to serve as clinically relevant models for drug screening and the development of targeted strategies to overcome ABCG2-mediated MDR in NSCLC. Citation Format: Qiu-Xu Teng, Zi-Ning Lei, Wei Zhang, Ying-Fang Fan, Jing-Quan Wang, Chao-Yun Cai, Dong-Hua Yang, John Wurpel, Zhe-Sheng Chen. Establishment and characterization of a topotecan resistant lung cancer NCI H460/TPT10 cell line and a tumor xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1095.

The multi-targeted tyrosine kinase inhibitor SKLB610 resensitizes ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic drugs

The overexpression of ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein) or ABCG2 (BCRP/MXR/ABCP) in cancer cells is frequently associated with the development of multidrug resistance (MDR) in cancer patients, which remains a major obstacle to effective cancer treatment. By utilizing energy derived from ATP hydrolysis, both transporters have been shown to reduce the chemosensitivity of cancer cells by actively effluxing cytotoxic anticancer drugs out of cancer cells. Knowing that there are presently no approved drugs or other therapeutics for the treatment of multidrug-resistant cancers, in recent years, studies have investigated the repurposing of tyrosine kinase inhibitors (TKIs) to act as agents against MDR mediated by ABCB1 and/or ABCG2. SKLB610 is a multi-targeted TKI with potent activity against vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor 2 (FGFR2). In this study, we investigate the interaction of SKLB610 with ABCB1 and ABCG2. We discovered that neither ABCB1 nor ABCG2 confers resistance to SKLB610, but SKLB610 selectively sensitizes ABCG2-overexpressing multidrug-resistant cancer cells to cytotoxic anticancer agents in a concentration-dependent manner. Our data indicate that SKLB610 reverses ABCG2-mediated MDR by attenuating the drug-efflux function of ABCG2 without affecting its total cell expression. These findings are further supported by results of SKLB610-stimulated ABCG2 ATPase activity and in silico docking of SKLB610 in the drug-binding pocket of ABCG2. In summary, we reveal the potential of SKLB610 to overcome resistance to cytotoxic anticancer drugs, which offers an additional treatment option for patients with multidrug-resistant cancers and warrants further investigation.

Lazertinib improves the efficacy of chemotherapeutic drugs in ABCB1 or ABCG2 overexpression cancer cells in vitro, in vivo, and ex vivo

Multidrug resistance (MDR) is the major cause of chemotherapy failure, which is usually caused by the overexpression of ATP-binding cassette (ABC) transporters such as ABCB1 and ABCG2. To date, no MDR modulator has been clinically approved. Here, we found that lazertinib (YH25448; a novel third-generation tyrosine kinase inhibitor [TKI]) could enhance the anticancer efficacy of MDR transporter substrate anticancer drugs in vitro，in vivo, and ex vivo. Mechanistically, lazertinib was shown to inhibit the drug efflux activities of ABCB1 and ABCG2 and thus increase the intracellular accumulation of the transporter substrate anticancer drug. Moreover, lazertinib was found to stimulate the ATPase activity of ABCB1/ABCG2 and inhibit the photolabeling of the transporters by 125I-iodoarylazidoprazosin (IAAP). However, lazertinib neither changed the expression or locolization of ABCB1 and ABCG2 nor blocked the signal pathway of Akt or Erk1/2 at a drug concentration effective for MDR reversal. Overall, our results demonstrate that lazertinib effectively reverses ABCB1- or ABCG2-mediated MDR by competitively binding to the ATP-binding site and inhibiting drug efflux function. This is the first report demonstrating the novel combined use of lazertinib and conventional chemotherapeutical drugs to overcome MDR in ABCB1/ABCG2-overexpressing cancer cells.

Tepotinib Inhibits Several Drug Efflux Transporters and Biotransformation Enzymes: The Role in Drug-Drug Interactions and Targeting Cytostatic Resistance In Vitro and Ex Vivo.

Tepotinib is a novel tyrosine kinase inhibitor recently approved for the treatment of non-small cell lung cancer (NSCLC). In this study, we evaluated the tepotinib’s potential to perpetrate pharmacokinetic drug interactions and modulate multidrug resistance (MDR). Accumulation studies showed that tepotinib potently inhibits ABCB1 and ABCG2 efflux transporters, which was confirmed by molecular docking. In addition, tepotinib inhibited several recombinant cytochrome P450 (CYP) isoforms with varying potency. In subsequent drug combination experiments, tepotinib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCB1 and ABCG2 overexpression, respectively. Remarkably, MDR-modulatory properties were confirmed in ex vivo explants derived from NSCLC patients. Furthermore, we demonstrated that anticancer effect of tepotinib is not influenced by the presence of ABC transporters associated with MDR, although monolayer transport assays designated it as ABCB1 substrate. Finally, tested drug was observed to have negligible effect on the expression of clinically relevant drug efflux transporters and CYP enzymes. In conclusion, our findings provide complex overview on the tepotinib’s drug interaction profile and suggest a promising novel therapeutic strategy for future clinical investigations.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB131.

ABCG2 but Not ABCB1 RNA Levels at Diagnosis and during Treatment Correlates with Response to Imatinib and 2G-Tkis Front-Line Therapy in Chronic Phase CML Patients

Introduction. Overexpression of multidrug resistance proteins ABCB1 and ABCG2 confers resistance to anticancer drugs, including tyrosine kinase inhibitors (TKIs). However, little is known about the significance of ABCB1 and ABCG2 expression during TKI treatment for chronic myeloid leukemia (CML) and possible correlation with response to therapy.Aims. To investigate the role of ABCB1 and ABCG2 expression during the first 12 months of treatment with imatinib and 2G-TKIs for chronic phase CML patients and correlation with molecular response.Methods. We analysed expression of ABCB1 and ABCG2 RNA at diagnosis and after 3, 6 and 12 months of TKI therapy in 20 patients with CML, divided in two groups according to response to treatment. Group A (n=10) comprised patients treated with imatinib that displayed optimal response at 3 months (defined as BCR/ABL <10%), 6 months (BCR/ABL < 1%) and at 12 months (BCR/ABL < 0.1%); group B (n=10) included patients treated with imatinib (n=5) or 2G-TKIs (nilotinib in 3 and dasatinib in 2 cases, respectively) who did not meet the criteria for optimal molecular response at all three timepoint or that switched to alternative treatment after at least the 3rd month. RNA levels were expressed using the 2-ΔΔCt method proposed by Livak and Schmittgen, using ABL as normalizing gene and the expression level of the K562 cell line as calibrator, in order to standardize the results.Results. ABCB1 expression at diagnosis was similar in group A (mean±SD = 0.21±0.22) and in group B (0.15±0.20; p=0.48); similarly, expression during treatment was similar in responding and non-responding patients at 3 (0.82±0.32 vs 0.83±0.41; p=0.66) 6 (0.85±0.22 vs 0.90±0.50; p=0.65) and 12 (1.05±0.24 vs 0.77±0.42; p=0.21) months. Conversely, ABCG2 expression at diagnosis was lower in group A (0.85±0.71) compared to group B (2.83±2.84; p=0.06). RNA levels increased in both groups during treatment, but were always lower in patients with optimal response compared to cases without response or treatment failure: at 3 months (5.12±3.31 vs 49.44±82.70; p=0.13), at 6 months (8.23±8.55 vs 12.71±10.04; p=0.32) and at 12 months (6.05±4.87 vs 13.00±9.93; p=0.05).Considering response to therapy, all patients in group A are still in imatinib with sustained optimal response; all patients in group B receiving imatinib switched to alternative therapy (nilotinib = 2, dasatinib = 1, allogeneic SCT for evolution to blast phase = 1) or died for non-CML causes while not in complete cytogenetic response (n=1), while 3/5 patients receiving 2G-TKI changed therapy (nilotinib to dasatinib = 1, dasatinib to ponatinib = 1, nilotinib to allogeneic SCT for aplasia = 1) and 2/5 patients continued the same drug achieving MMR beyond the 12th month.Discussion. With the limits of a small number of patients, our data suggest that high ABCG2 levels at diagnosis seems to be associated with non-optimal response to imatinib and 2G-TKIs, and an increase in ABCG2 during the first months of treatment is a hallmark of poor response. DisclosuresTiribelli:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria.

Cabozantinib Reverses Topotecan Resistance in Human Non-Small Cell Lung Cancer NCI-H460/TPT10 Cell Line and Tumor Xenograft Model

Cabozantinib (CBZ) is a small molecule tyrosine kinase receptor inhibitor, which could also inhibit the ABCG2 transporter function. Therefore, CBZ could re-sensitize cancer cells that are resistant to ABCG2 substrate drugs including topotecan (TPT). However, its reversal effect against TPT resistance has not been tested in a TPT-induced resistant cancer model. In this study, a new TPT selected human non-small cell lung cancer (NSCLC)-resistant cell model NCI-H460/TPT10 with ABCG2 overexpression and its parental NCI-H460 cells were utilized to investigate the role of CBZ in drug resistance. The in vitro study showed that CBZ, at a non-toxic concentration, could re-sensitize NCI-H460/TPT10 cells to TPT by restoring intracellular TPT accumulation via inhibiting ABCG2 function. In addition, the increased cytotoxicity by co-administration of CBZ and TPT may be contributed by the synergistic effect on downregulating ABCG2 expression in NCI-H460/TPT10 cells. To further verify the applicability of the NCI-H460/TPT10 cell line to test multidrug resistance (MDR) reversal agents in vivo and to evaluate the in vivo efficacy of CBZ on reversing TPT resistance, a tumor xenograft mouse model was established by implanting NCI-H460 and NCI-H460/TPT10 into nude mice. The NCI-H460/TPT10 xenograft tumors treated with the combination of TPT and CBZ dramatically reduced in size compared to tumors treated with TPT or CBZ alone. The TPT-resistant phenotype of NCI-H460/TPT10 cell line and the reversal capability of CBZ in NCI-H460/TPT10 cells could be extended from in vitro cell model to in vivo xenograft model. Collectively, CBZ is considered to be a potential approach in overcoming ABCG2-mediated MDR in NSCLC. The established NCI-H460/TPT10 xenograft model could be a sound clinically relevant resource for future drug screening to eradicate ABCG2-mediated MDR in NSCLC.