ATP-binding Cassette Drug Transporters Research Articles

ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells.

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

Epidermal Growth Factor Receptor Inhibitor Mobocertinib Resensitizes Multidrug-Resistant Cancer Cells by Attenuating the Human ATP-Binding Cassette Subfamily B Member 1 and Subfamily G Member 2.

ATP-binding cassette (ABC) transporters, notably ABCB1 (P-glycoprotein) and ABCG2, play a crucial role in the development of multidrug resistance (MDR) during the administration of chemotherapy for cancer patients. With a lack of approved treatments for addressing multidrug-resistant cancers, MDR remains a substantial challenge to the effective management of cancer. Rather than focusing on developing novel synthetic inhibitors, a promising approach to combat MDR involves repurposing approved therapeutic agents to enhance the sensitivity to cytotoxic antiproliferative drugs of multidrug-resistant cancer cells with high expression of ABCB1 or ABCG2. In this investigation, we observed a substantial reversal of MDR conferred by ABCB1 and ABCG2 in multidrug-resistant cancer cells through the use of mobocertinib, an approved third-generation inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. Mobocertinib demonstrated the ability to hinder drug transport function without causing changes in protein expression. The interactions between mobocertinib and ABCB1, as well as ABCG2, were validated through ATPase assays. Furthermore, in silico docking simulations were utilized to substantiate the binding of mobocertinib within the drug-binding pockets of both ABCB1 and ABCG2. We conclude that further testing of mobocertinib in combination therapy is warranted for patients with tumors expressing elevated levels of the ABC drug transporters ABCB1 and ABCG2.

Imperatorin Restores Chemosensitivity of Multidrug-Resistant Cancer Cells by Antagonizing ABCG2-Mediated Drug Transport.

The high expression of the ATP-binding cassette (ABC) drug transporter ABCG2 in cancer cells contributes to the emergence of multidrug resistance (MDR) in individuals afflicted with either solid tumors or blood cancers. MDR poses a major impediment in the realm of clinical cancer chemotherapy. Recently, substantial endeavors have been dedicated to identifying bioactive compounds isolated from nature capable of counteracting ABCG2-mediated MDR in cancer cells. Imperatorin, a natural coumarin derivative renowned for its diverse pharmacological properties, has not previously been explored for its impact on cancer drug resistance. This study investigates the chemosensitizing potential of imperatorin in ABCG2-overexpressing cancer cells. Experimental results reveal that at sub-toxic concentrations, imperatorin significantly antagonizes the activity of ABCG2 and reverses ABCG2-mediated MDR in a concentration-dependent manner. Furthermore, biochemical data and in silico analysis of imperatorin docking to the inward-open conformation of human ABCG2 indicate that imperatorin directly interacts with multiple residues situated within the transmembrane substrate-binding pocket of ABCG2. Taken together, these results furnish substantiation that imperatorin holds promise for further evaluation as a potent inhibitor of ABCG2, warranting exploration in combination drug therapy to enhance the effectiveness of therapeutic agents for patients afflicted with tumors that exhibit high levels of ABCG2.

Perspectives on drug repurposing to overcome cancer multidrug resistance mediated by ABCB1 and ABCG2

The overexpression of the human ATP-binding cassette (ABC) transporters in cancer cells is a common mechanism involved in developing multidrug resistance (MDR). Unfortunately, there are currently no approved drugs specifically designed to treat multidrug-resistant cancers, making MDR a significant obstacle to successful chemotherapy. Despite over two decades of research, developing transporter-specific inhibitors for clinical use has proven to be a challenging endeavor. As an alternative approach, drug repurposing has gained traction as a more practical method to discover clinically effective modulators of drug transporters. This involves exploring new indications for already-approved drugs, bypassing the lengthy process of developing novel synthetic inhibitors. In this context, we will discuss the mechanisms of ABC drug transporters ABCB1 and ABCG2, their roles in cancer MDR, and the inhibitors that have been evaluated for their potential to reverse MDR mediated by these drug transporters. Our focus will be on providing an up-to-date report on approved drugs tested for their inhibitory activities against these drug efflux pumps. Lastly, we will explore the challenges and prospects of repurposing already approved medications for clinical use to overcome chemoresistance in patients with high tumor expression of ABCB1 and/or ABCG2.

Single-nucleotide polymorphisms in ABC drug transporters alter expression and circulating tenofovir in healthy South African women exposed to pre-exposure prophylaxis.

Aim: We investigated if single-nucleotide polymorphisms (SNPs) in ATP-binding cassette (ABC)drug transporters alter gene expression and tenofovir disposition in South African women taking Truvada® for HIV prevention. Materials & methods: In 393 women, real-time PCR was used to determine the associations between six SNPs in ABC transporter genes, mRNA expression and circulating-tenofovir. Results: Univariable and multivariable analyses showed that CT and TT relative to CC genotypes for theABCC4(3463C/T) SNPhad significantly higher tenofovir levels. In contrast, the AA genotype for the ABCC4(4976A/G) SNP showed significantly less tenofovir, while mRNA expression was increased. Conclusion: SNPs in the ABCC4 gene may differentially affect gene expression and circulating tenofovir. Their impact may inform on low pre-exposure prophylaxis efficacy and discern effective drugs in clinical trials of African women enriched for certain genotypes.

Drug transporter expression and activity in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice with humanized livers

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.

MicroRNAs as Biomarkers of Surgical Outcome in Mesial Temporal Lobe Epilepsy: A Systematic Review

Mesial temporal lobe epilepsy is the most common type of epilepsy. For most patients suffering from TLE, the only treatment option is surgery. However, there is a high possibility of relapse. Invasive EEG as a method for predicting the outcome of surgical treatment is a very complex and invasive manipulation, so the search for outcome biomarkers is an urgent task. MicroRNAs as potential biomarkers of surgical outcome are the subject of this study. For this study, a systematic search for publications in databases such as PubMed, Springer, Web of Science, Scopus, ScienceDirect, and MDPI was carried out. The following keywords were used: temporal lobe epilepsy, microRNA, biomarkers, surgery, and outcome. Three microRNAs were studied as prognostic biomarkers of surgical outcome: miR-27a-3p, miR-328-3p, and miR-654-3p. According to the results of the study, only miR-654-3p showed a good ability to discriminate between patients with poor and good surgical outcomes. MiR-654-3p is involved in the following biological pathways: ATP-binding cassette drug transporters, glutamate transporter SLC7A11, and TP53. A specific target for miR-654-3p is GLRA2, the glycine receptor subunit. MicroRNAs, which are diagnostic biomarkers of TLE, and epileptogenesis, miR-134-5p, MiR-30a, miRs-143, etc., can be considered as potential biomarkers of surgical outcome, as they can be indicators of early and late relapses. These microRNAs are involved in the processes characteristic of epilepsy: oxidative stress and apoptosis. The study of miRNAs as potential predictive biomarkers of surgical outcome is an urgent task and should be continued. However, when studying miRNA expression profiles, it is important to take into account and note a number of factors, such as the type of sample under study, the time of sampling for the study, the type and duration of the disease, and the type of antiepileptic treatment. Without taking into account all these factors, it is impossible to assess the influence and involvement of miRNAs in epileptic processes.

Hydroxygenkwanin Improves the Efficacy of Cytotoxic Drugs in ABCG2-Overexpressing Multidrug-Resistant Cancer Cells.

Hydroxygenkwanin, a flavonoid isolated from the leaves of the Daphne genkwa plant, is known to have pharmacological properties; however, its modulatory effect on multidrug resistance, which is (MDR) mediated by ATP-binding cassette (ABC) drug transporters, has not been investigated. In this study, we examine the interaction between hydroxygenkwanin, ABCB1, and ABCG2, which are two of the most well-characterized ABC transporters known to contribute to clinical MDR in cancer patients. Hydroxygenkwanin is not an efflux substrate of either ABCB1 or ABCG2. We discovered that, in a concentration-dependent manner, hydroxygenkwanin significantly reverses ABCG2-mediated resistance to multiple cytotoxic anticancer drugs in ABCG2-overexpressing multidrug-resistant cancer cells. Although it inhibited the drug transport function of ABCG2, it had no significant effect on the protein expression of this transporter in cancer cells. Experimental data showing that hydroxygenkwanin stimulates the ATPase activity of ABCG2, and in silico docking analysis of hydroxygenkwanin binding to the inward-open conformation of human ABCG2, further indicate that hydroxygenkwanin sensitizes ABCG2-overexpressing cancer cells by binding to the substrate-binding pocket of ABCG2 and attenuating the transport function of ABCG2. This study demonstrates the potential use of hydroxygenkwanin as an effective inhibitor of ABCG2 in drug combination therapy trials for patients with tumors expressing higher levels of ABCG2.

ATP-binding cassette (ABC) drug transporters in the developing blood-brain barrier: role in fetal brain protection.

The blood-brain barrier (BBB) provides essential neuroprotection from environmental toxins and xenobiotics, through high expression of drug efflux transporters in endothelial cells of the cerebral capillaries. However, xenobiotic exposure, stress, and inflammatory stimuli have the potential to disrupt BBB permeability in fetal and post-natal life. Understanding the role and ability of the BBB in protecting the developing brain, particularly with respect to drug/toxin transport, is key to promoting long-term brain health. Drug transporters, particularly P-gp and BCRP are expressed in early gestation at the developing BBB and have a crucial role in developmental homeostasis and fetal brain protection. We have highlighted several factors that modulate drug transporters at the developing BBB, including synthetic glucocorticoid (sGC), cytokines, maternal infection, and growth factors. Some factors have the potential to increase expression and function of drug transporters and increase brain protection (e.g., sGC, transforming growth factor [TGF]-β). However, others inhibit drug transporters expression and function at the BBB, increasing brain exposure to xenobiotics (e.g., tumor necrosis factor [TNF], interleukin [IL]-6), negatively impacting brain development. This has implications for pregnant women and neonates, who represent a vulnerable population and may be exposed to drugs and environmental toxins, many of which are P-gp and BCRP substrates. Thus, alterations in regulated transport across the developing BBB may induce long-term changes in brain health and compromise pregnancy outcome. Furthermore, a large portion of neonatal adverse drug reactions are attributed to agents that target or access the nervous system, such as stimulants (e.g., caffeine), anesthetics (e.g., midazolam), analgesics (e.g., morphine) and antiretrovirals (e.g., Zidovudine); thus, understanding brain protection is key for the development of strategies to protect the fetal and neonatal brain.

Interactions of organophosphorus pesticides with ATP-binding cassette (ABC) drug transporters

Although pharmaceutical companies have to study drug-transporter interaction, environmental contaminant interactions with these transporters are not well characterised. In this study, we demonstrated using in vitro transfected cell line that some organophosphorus pesticides are able to interact with drug efflux transporters like P-glycoprotein, BCRP and MRPs. According to our results, dibrom was found to inhibit only Hoechst binding site of P-gp with an IC50 closed to 77 µM, phosmet inhibited BCRP efflux with an IC50 of 42 µM and only profenofos was able to inhibit BCRP, MRPs and P-gp at two binding sites. As profenofos appeared to be a potent ABC transporter inhibitor, we studied its potential substrate property towards P-gp. Using a docking approach, we developed an in silico tool to study pesticide properties to be a probe or inhibitor of P-gp transporter. From both in silico and in vitro results, profenofos was not considered as a P-gp substrate. Combining both in vitro and docking methods appears to be an attractive approach to select pesticides that would not pass into the blood systemic circulation.

Sophoraflavanone G Resensitizes ABCG2-Overexpressing Multidrug-Resistant Non-Small-Cell Lung Cancer Cells to Chemotherapeutic Drugs.

Elevated expression of the ATP-binding cassette (ABC) drug transporter ABCG2 in cancer cells contributes to the development of the multidrug resistance phenotype in patients with advanced non-small-cell lung cancer (NSCLC). Due to the lack of U.S. Food and Drug Administration (FDA)-approved synthetic inhibitors of ABCG2, significant efforts have been invested in discovering bioactive compounds of plant origin that are capable of reversing ABCG2-mediated multidrug resistance in cancer cells. Sophoraflavanone G (SFG), a phytoncide isolated from the plant species Sophora flavescens, is known to possess a wide spectrum of pharmacological activities, including antibacterial, anti-inflammatory, antimalarial, and antiproliferative effects. In the present study, the chemosensitizing effect of SFG in ABCG2-overexpressing NSCLC cells was investigated. Experimental results demonstrate that at subtoxic concentrations SFG significantly reversed ABCG2-mediated multidrug resistance in a concentration-dependent manner. Additional biochemical data and in silico docking analysis of SFG to the inward-open conformation of human ABCG2 indicate that SFG inhibited the drug transport function of ABCG2 by interacting with residues within the transmembrane substrate-binding pocket of ABCG2. Collectively, these findings provide evidence that SFG has the potential to be further tested as an effective inhibitor of ABCG2 to improve the efficacy of therapeutic drugs in patients with advanced NSCLC.

Use of photoimmunoconjugates to characterize ABCB1 in cancer cells.

Accurate detection of ATP-binding cassette drug transporter ABCB1 expression is imperative for precise identification of drug-resistant tumors. Existing detection methods fail to provide the necessary molecular details regarding the functional state of the transporter. Photo-immunoconjugates are a unique class of antibody–dye conjugates for molecular diagnosis and therapeutic treatment. However, conjugating hydrophobic photosensitizers to hydrophilic antibodies is quite challenging. Here, we devise a photoimmunoconjugate that combines a clinically approved benzoporphyrin derivative (BPD) photosensitizer and the conformational-sensitive UIC2 monoclonal antibody to target functionally active human ABCB1 (i.e., ABCB1 in the inward-open conformation). We show that PEGylation of UIC2 enhances the BPD conjugation efficiency and reduces the amount of non-covalently conjugated BPD molecules by 17%. Size exclusion chromatography effectively separates the different molecular weight species found in the UIC2–BPD sample. The binding of UIC2–BPD to ABCB1 was demonstrated in lipidic nanodiscs and ABCB1-overexpressing triple negative breast cancer (TNBC) cells. UIC2–BPD was found to retain the conformation sensitivity of UIC2, as the addition of ABCB1 modulators increases the antibody reactivity in vitro. Thus, the inherent fluorescence capability of BPD can be used to label ABCB1-overexpressing TNBC cells using UIC2–BPD. Our findings provide insight into conjugation of hydrophobic photosensitizers to conformation-sensitive antibodies to target proteins expressed on the surface of cancer cells.

Branebrutinib (BMS-986195), a Bruton's Tyrosine Kinase Inhibitor, Resensitizes P-Glycoprotein-Overexpressing Multidrug-Resistant Cancer Cells to Chemotherapeutic Agents.

The overexpression of P-glycoprotein (P-gp/ABCB1), an ATP-binding cassette (ABC) drug transporter, often contributes to the development of multidrug resistance (MDR) in cancer cells. P-gp mediates the ATP hydrolysis-dependent efflux of a wide range of chemotherapeutic agents out of cancer cells, thereby reducing the intracellular drug accumulation and decreasing the chemosensitivity of these multidrug-resistant cancer cells. Studies with tyrosine kinase inhibitors (TKIs) in P-gp-overexpressing cells have shown that certain TKIs could reverse MDR mediated by P-gp, while some TKIs are transported by P-gp. In the present work, we explored the prospect of repositioning branebrutinib (BMS-986195), a highly selective inhibitor of Bruton’s tyrosine kinase (BTK), to resensitize P-gp-overexpressing multidrug-resistant cancer cells to chemotherapeutic agents. Our results demonstrated that branebrutinib is capable of reversing P-gp-mediated MDR at sub-toxic concentrations, most likely by directly inhibiting the drug transport function of P-gp. Our findings were supported by the result of branebrutinib stimulating the ATPase activity of P-gp in a concentration-dependent manner and the in silico study of branebrutinib binding to the substrate-binding pocket of P-gp. In addition, we found that branebrutinib is equally cytotoxic to drug-sensitive parental cell lines and the respective P-gp-overexpressing multidrug-resistant variants, suggesting that it is unlikely that the overexpression of P-gp in cancer cells plays a significant role in reduced susceptibility or resistance to branebrutinib. In summary, we discovered an additional pharmacological action of branebrutinib against the activity of P-gp, which should be investigated further in future drug combination studies.

Interleukin-6 reverses Adriamycin resistance in nasal NK/T-cell lymphoma via downregulation of ABCC4 and inactivation of the JAK2/STAT3/NF-κB/P65 pathway

Chemotherapy is generally effective for extranodal natural killer (NK)/T-cell lymphoma (ENKTCL), nasal type. Nevertheless, multidrug resistance (MDR) remains a key challenge in treating nasal NK/T-cell lymphoma. Interleukin-6 (IL-6) is reportedly an important regulator of MDR in many cancers, implicating a role of IL-6 in the chemotherapy response. However, the effects and mechanism of IL-6 in nasal NK/T-cell lymphoma remain unclear. Herein, we demonstrated that the IL-6 serum level was decreased in nasal NK/T-cell lymphoma patients compared to chronic rhinitis patients. Lower serum levels of IL-6 were closely correlated with Ki67 expression and patient survival. ATP-binding cassette (ABC) drug transporter ABCC4 in patients was abnormally upregulated. IL-6 significantly inhibited resistance to Adriamycin (ADM) in ADM-resistant SNK-6 cells (SNK-6/ADM). Moreover, IL-6 resulted in cell cycle arrest and led to apoptosis in SNK-6/ADM cells. Furthermore, IL-6 decreased ABCC4, p-JAK2, p-STAT3, and phospho-NF-κB p65 expression in SNK-6/ADM cells. IL-6 in combination with ADM inhibited tumor growth and increased the survival of SNK-6/ADM xenograft mice. In conclusion, our findings suggest that IL-6 can inhibit the upregulation of ABCC4 and inactivate the JAK2/STAT3/NF-κB/P65 pathway to sensitize NK/T-cell lymphoma to ADM, indicating that combination therapy with IL-6 and other chemotherapeutic drugs may be effective in reversing acquired resistance in nasal NK/T-cell lymphoma.

The third-generation EGFR inhibitor almonertinib (HS-10296) resensitizes ABCB1-overexpressing multidrug-resistant cancer cells to chemotherapeutic drugs

The overexpression of the human ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein, P-gp) or ABCG2 (breast cancer resistance protein, BCRP) in cancer cells often contributes significantly to the development of multidrug resistance (MDR) in cancer patients. Previous reports have demonstrated that some epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) could modulate the activity of ABCB1 and/or ABCG2 in human cancer cells, whereas some EGFR TKIs are transport substrates of these transporters. Almonertinib (HS-10296) is a promising, orally available third-generation EGFR TKI for the treatment of EGFR T790M mutation-positive non-small cell lung cancer (NSCLC) in patients who have progressed on or after other EGFR TKI therapies. Additional clinical trials are currently in progress to study almonertinib as monotherapy and in combination with other agents in patients with NSCLC. In the present work, we found that neither ABCB1 nor ABCG2 confers significant resistance to almonertinib. More importantly, we discovered that almonertinib was able to reverse MDR mediated by ABCB1, but not ABCG2, in multidrug-resistant cancer cells at submicromolar concentrations by inhibiting the drug transport activity of ABCB1 without affecting its expression level. These findings are further supported by in silico docking of almonertinib in the drug-binding pocket of ABCB1. In summary, our study revealed an additional activity of almonertinib to re-sensitize ABCB1-overexpressing multidrug-resistant cancer cells to conventional chemotherapeutic drugs, which may be beneficial for cancer patients and warrant further investigation.

Antigiardial Activity of Acetylsalicylic Acid Is Associated with Overexpression of HSP70 and Membrane Transporters.

Giardia lamblia is a flagellated protozoan responsible for giardiasis, a worldwide diarrheal disease. The adverse effects of the pharmacological treatments and the appearance of drug resistance have increased the rate of therapeutic failures. In the search for alternative therapeutics, drug repositioning has become a popular strategy. Acetylsalicylic acid (ASA) exhibits diverse biological activities through multiple mechanisms. However, the full spectrum of its activities is incompletely understood. In this study we show that ASA displayed direct antigiardial activity and affected the adhesion and growth of trophozoites in a time-dose-dependent manner. Electron microscopy images revealed remarkable morphological alterations in the membrane, ventral disk, and caudal region. Using mass spectrometry and real-time quantitative reverse transcription (qRT-PCR), we identified that ASA induced the overexpression of heat shock protein 70 (HSP70). ASA also showed a significant increase of five ATP-binding cassette (ABC) transporters (giABC, giABCP, giMDRP, giMRPL and giMDRAP1). Additionally, we found low toxicity on Caco-2 cells. Taken together, these results suggest an important role of HSPs and ABC drug transporters in contributing to stress tolerance and protecting cells from ASA-induced stress.

The Role of Specific ATP-Binding Cassette Transporters in the Acquired Resistance to Pyrrolobenzodiazepine Dimer-Containing Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) containing pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematologic and solid tumors. These include ADCT-301 (camidanlumab tesirine) and ADCT-402 (loncastuximab tesirine) in pivotal phase II trials that contain the payload tesirine, which releases the PBD dimer warhead SG3199. An important consideration in future clinical development is acquired resistance. The aim was to generate and characterize PBD acquired resistant cell lines in both hematologic and solid tumor settings. Human Karpas-299 (ALCL) and NCI-N87 (gastric cancer) cells were incubated with increasing IC50 doses of ADC (targeting CD25 and HER2, respectively) or SG3199 in a pulsed manner until stable acquired resistance was established. The level of resistance achieved was approximately 3,000-fold for ADCT-301 and 3-fold for SG3199 in Karpas-299, and 8-fold for ADCT-502 and 4-fold for SG3199 in NCI-N87. Cross-resistance between ADC and SG3199, and with an alternative PBD-containing ADC or PBD dimer was observed. The acquired resistant lines produced fewer DNA interstrand cross-links, indicating an upstream mechanism of resistance. Loss of antibody binding or internalization was not observed. A human drug transporter PCR Array revealed several genes upregulated in all the resistant cell lines, including ABCG2 and ABCC2, but not ABCB1(MDR1). These findings were confirmed by RT-PCR and Western blot, and inhibitors and siRNA knockdown of ABCG2 and ABCC2 recovered drug sensitivity. These data show that acquired resistance to PBD-ADCs and SG3199 can involve specific ATP-binding cassette drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations.

Overexpression of ABCB1 and ABCG2 contributes to reduced efficacy of the PI3K/mTOR inhibitor samotolisib (LY3023414) in cancer cell lines

LY3023414 (samotolisib) is a promising new dual inhibitor of phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR). Currently, multiple clinical trials are underway to evaluate the efficacy of LY3023414 in patients with various types of cancer. However, the potential mechanisms underlying acquired resistance to LY3023414 in human cancer cells still remain elusive. In this study, we investigated whether the overexpression of ATP-binding cassette (ABC) drug transporters such as ABCB1 and ABCG2, one of the most common mechanisms for developing multidrug resistance, may potentially reduce the efficacy of LY3023414 in human cancer cells. We demonstrated that the intracellular accumulation of LY3023414 in cancer cells was significantly reduced by the drug efflux function of ABCB1 and ABCG2. Consequently, the cytotoxicity and efficacy of LY3023414 for inhibiting the activation of the PI3K pathway and induction of G0/G1 cell-cycle arrest were substantially reduced in cancer cells overexpressing ABCB1 or ABCG2, which could be restored using tariquidar or Ko143, respectively. Furthermore, stimulatory effect of LY3023414 on the ATPase activity of ABCB1 and ABCG2, as well as in silico molecular docking analysis of LY3023414 binding to the substrate-binding pockets of these transporters provided additional insight into the manner in which LY3023414 interacts with both transporters. In conclusion, we report that LY3023414 is a substrate for ABCB1 and ABCG2 transporters implicating their role in the development of resistance to LY3023414, which can have substantial clinical implications and should be further investigated.

Unique metabolite preferences of the drug transporters OAT1 and OAT3 analyzed by machine learning

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from Oat-knockout mice (Oat1 and Oat3KO) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a "logical" statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent "brute force" approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways in vivo As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which "multispecific" kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 versus OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug-metabolite interactions.

Proton Pump Inhibitors Associated with Higher Incidence of Mucositis in Patients Receiving Methotrexate for Graft Vs Host Disease Prophylaxis

<h3>Introduction</h3> Graft-versus-host disease (GVHD) is a major complicating factor in determining morbidity and mortality following allogeneic stem cell transplant. To prevent GVHD, our institution administers calcineurin inhibitors and low doses of methotrexate (MTX) (5-15 mg/m²) on days +1, +3, +6, +11 for GVHD prophylaxis. Proton pump inhibitors (PPIs) are used for the treatment of gastroesophageal reflux disease, pyrosis, and/or peptic ulcer disease. PPIs have been shown to inhibit the ATP-binding cassette drug transporter Breast Cancer Resistance Protein's (BCRP/ABCG2) ability to excrete methotrexate into the urine in a concentration-dependent manner. Delayed excretion of methotrexate can lead to adverse events such as renal impairment, increased severity of mucositis, and increased infection rates. <h3>Objectives</h3> The purpose of this retrospective, single center study is to assess the clinical outcomes of patients receiving MTX and PPIs compared to MTX and no PPI for GVHD prophylaxis. <h3>Methods</h3> A single-site retrospective analysis of patients 18 years of age or older who underwent allogeneic stem cell transplantation from 2013 to 2018. Only patients that received methotrexate for GVHD prophylaxis were included. Cases were identified from clinical databases and were divided into two cohorts, those on PPIs, and those not on PPIs (no-PPI). The primary endpoint was grade of mucositis in each cohort. Secondary endpoints were total parenteral nutrition (TPN) use, need for patient controlled analgesia (PCA), time to engraftment of absolute neutrophil count (ANC) and hospital length of stay (LOS). <h3>Results</h3> 284 cases were identified, 119 (42%) patients were on a PPI at the time of MTX administration. The median age of patients on PPI was younger than those not on PPI (60 vs 54 p <0.0001). There were no differences in sex, BSA, conditioning regimen intent, dose of MTX, degree of match, or stem cell source. Patients on PPI had a higher incidence of grade 1-4 mucositis compared to no-PPI (Table 1) and a higher percentage of grade 3-4 mucositis (Figure 1). Patients on PPIs had a higher incidence of TPN use and need for PCA. Use of PPI did not increase the time to ANC engraftment or LOS. <h3>Conclusions</h3> The concurrent use of PPIs and MTX was associated with a higher incidence and severity of mucositis with increased need for PCA, and TPN. This study was limited by retrospective review. Grading of mucositis was based on clinician assessment and documentation. This study raises the question if PPIs should be discontinued or patients transitioned to a histamine blocker prior to MTX administration to improve severity of mucositis and reduce costs associated with TPN/PCA use.