Progress in characterizing ABC multidrug transporters in zebrafish.

Zebrafish have proved to be invaluable for modeling complex physiological processes shared by all vertebrate animals. Resistance of cancers and other diseases to drug treatment can occur owing to expression of the ATP-dependent multidrug transporters ABCB1, ABCG2, and ABCC1, either because of expression of these transporters by the target cells to reduce intracellular concentrations of cytotoxic drugs at barrier sites such as the blood-brain barrier (BBB) to limit penetration of drugs into privileged compartments, or by affecting the absorption, distribution, and excretion of drugs administered orally, through the skin, or directly into the bloodstream. We describe the drug specificity, cellular localization, and function of zebrafish orthologs of multidrug resistance ABC transporters with the goal of developing zebrafish models to explore the physiological and pathophysiological functions of these transporters. Finally, we provide context demonstrating the utility of zebrafish in studying cancer drug resistance. Our ultimate goal is to improve treatment of cancer and other diseases which are affected by ABC multidrug resistance transporters.

A role for ceramide glycosylation in resistance to oxaliplatin in colorectal cancer.

There is growing evidence to support a role for the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in resistance to a variety of chemotherapeutic agents. Whether GCS contributes to oxaliplatin resistance in colorectal cancer (CRC) has not yet been determined. We have addressed this potentially important clinical issue by examining GCS function in two panels of oxaliplatin-resistant, isogenic CRC cell lines. Compared to parental cell lines, oxaliplatin-resistant cells have increased expression of GCS protein associated with increased levels of the pro-survival ceramide metabolite, glucosylceramide (GlcCer). Inhibition of GCS expression by RNAi-mediated gene knockdown resulted in a reduction in cellular GlcCer levels, with restored sensitivity to oxaliplatin. Furthermore, oxaliplatin-resistant CRC cells displayed lower ceramide levels both basally and after treatment with oxaliplatin, compared to parental cells. GlcCer, formed by GCS-mediated ceramide glycosylation, is the precursor to a complex array of glycosphingolipids. Differences in cellular levels and species of gangliosides, a family of glycosphingolipids, were also seen between parental and oxaliplatin-resistant CRC cells. Increased Akt activation was also observed in oxaliplatin-resistant CRC cell lines, together with increased expression of the anti-apoptotic protein survivin. Finally, this study shows that GCS protein levels are greatly increased in human CRC specimens, compared to matched, normal colonic mucosa, and that high levels of UGCG gene expression are significantly associated with decreased disease-free survival in colorectal cancer patients. These findings uncover an important cellular role for GCS in oxaliplatin chemosensitivity and may provide a novel cellular target for augmenting chemotherapeutic drug effectiveness in CRC.

Targeting mitochondrial hexokinases increases efficacy of histone deacetylase inhibitors in solid tumor models.

Hexokinase 1 and 2 have been shown to inhibit Bak- and Bax-mediated apoptosis, leading us to combine the histone deacetylase inhibitor romidepsin with clotrimazole or bifonazole, two compounds that reportedly decrease mitochondrial localization of hexokinases. Cancer cell lines derived from breast, kidney, lung, colon or ovarian cancers were treated with a short-term exposure to 25 ng/ml romidepsin combined with either clotrimazole or bifonazole. The combination of romidepsin with 25 µM clotrimazole or bifonazole resulted in increased annexin staining compared to cells treated with any of the drugs alone. Cell death was caspase-mediated, as the pan-caspase inhibitor Q-VD-OPh was found to inhibit apoptosis induced by the combination. A549 lung cancer cells or HCT-116 cells deficient in Bak and Bax were also resistant to apoptosis with the combination implicating the intrinsic apoptotic pathway. We found that a 24 h treatment with clotrimazole or bifonazole decreased total hexokinase 2 expression, resulting in a 76% or 60% decrease, respectively, of mitochondrial expression of hexokinase 2. Mitochondrial hexokinase 1 levels increased 2-fold or less. Our work suggests that the combination of a short-term romidepsin treatment with bifonazole or clotrimazole leads to increased apoptosis, most likely due to decreased mitochondrial expression of hexokinase 2.

Leptin Signaling Affects Survival and Chemoresistance of Estrogen Receptor Negative Breast Cancer.

Estrogen-receptor-negative breast cancer (BCER-) is mainly treated with chemotherapeutics. Leptin signaling can influence BCER- progression, but its effects on patient survival and chemoresistance are not well understood. We hypothesize that leptin signaling decreases the survival of BCER- patients by, in part, inducing the expression of chemoresistance-related genes. The correlation of expression of leptin receptor (OBR), leptin-targeted genes (CDK8, NANOG, and RBP-Jk), and breast cancer (BC) patient survival was determined from The Cancer Genome Atlas (TCGA) mRNA data. Leptin-induced expression of proliferation and chemoresistance-related molecules was investigated in triple-negative BC (TNBC) cells that respond differently to chemotherapeutics. Leptin-induced gene expression in TNBC was analyzed by RNA-Seq. The specificity of leptin effects was assessed using OBR inhibitors (shRNA and peptides). The results show that OBR and leptin-targeted gene expression are associated with lower survival of BCER- patients. Importantly, the co-expression of these genes was also associated with chemotherapy failure. Leptin signaling increased the expression of tumorigenesis and chemoresistance-related genes (ABCB1, WNT4, ADHFE1, TBC1D3, LL22NC03, RDH5, and ITGB3) and impaired chemotherapeutic effects in TNBC cells. OBR inhibition re-sensitized TNBC to chemotherapeutics. In conclusion, the co-expression of OBR and leptin-targeted genes may be used as a predictor of survival and drug resistance of BCER- patients. Targeting OBR signaling could improve chemotherapeutic efficacy.

A High-Throughput Screen of a Library of Therapeutics Identifies Cytotoxic Substrates of P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.

Spatial control of oxygen delivery to three-dimensional cultures alters cancer cell growth and gene expression.

Commonly used monolayer cancer cell cultures fail to provide a physiologically relevant environment in terms of oxygen delivery. Here, we describe a three-dimensional (3D) bioreactor system where cancer cells are grown in Matrigel in modified six-well plates. Oxygen is delivered to the cultures through a polydimethylsiloxane (PDMS) membrane at the bottom of the wells, with microfabricated PDMS pillars to control oxygen delivery. The plates receive 3% oxygen from below and 0% oxygen at the top surface of the media, providing a gradient of 3-0% oxygen. We compared growth and transcriptional profiles for cancer cells grown in Matrigel in the bioreactor, 3D cultures grown in 21% oxygen, and cells grown in a standard hypoxia chamber at 3% oxygen. Additionally, we compared gene expression of conventional two-dimensional monolayer culture and 3D Matrigel culture in 21% oxygen. We conclude that controlled oxygen delivery may provide a more physiologically relevant 3D system.

Coexpression of ABCB1 and ABCG2 in a Cell Line Model Reveals Both Independent and Additive Transporter Function.

Although overexpression of multiple ATP-binding cassette transporters has been reported in clinical samples, few studies have examined how coexpression of multiple transporters affected resistance to chemotherapeutic drugs. We therefore examined how coexpression of ABCB1 (P-glycoprotein) and ABCG2 contributes to drug resistance in a cell line model. HEK293 cells were transfected with vector-encoding full-length ABCB1, ABCG2, or a bicistronic vector containing both genes, each under the control of a separate promoter. Cells transfected with both transporters (B1/G2 cells) demonstrated high levels of both transporters, and uptake of both the ABCB1-specific substrate rhodamine 123 and the ABCG2-specific substrate pheophorbide a was reduced when examined by flow cytometry. B1/G2 cells were also cross-resistant to the ABCB1 substrate doxorubicin, the ABCG2 substrate topotecan, as well as mitoxantrone and the cell cycle checkpoint kinase 1 inhibitor prexasertib, both of which were found to be substrates of both ABCB1 and ABCG2. When B1/G2 cells were incubated with both rhodamine 123 and pheophorbide a, transport of both compounds was observed, suggesting that ABCB1 and ABCG2, when coexpressed, can function independently to transport substrates. ABCB1 and ABCG2 also functioned additively to transport the common fluorescent substrates mitoxantrone and BODIPY-prazosin, as it was necessary to inhibit both transporters to prevent efflux from B1/G2 cells. ABCG2 expression was also found to decrease the efficacy of the ABCB1 inhibitor tariquidar in B1/G2 cells. Thus, ABCB1 and ABCG2 can independently and additively confer resistance to substrates, underscoring the need to inhibit multiple transporters when they are coexpressed.

MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor.

To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

Abcg2a is the functional homolog of human ABCG2 expressed at the zebrafish blood-brain barrier.

Background: A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. Methods: To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish. Results: We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. Conclusions: These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for the studying the role of ABCG2 at the BBB.

Abcg2a is the functional homolog of human ABCG2 expressed at the zebrafish blood-brain barrier.

BACKGROUND: A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2) on the lumenal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies human P-gp. Comparatively little is known about the four zebrafish homologs of the human ABCG2 gene: abcg2a, abcg2b, abcg2c, and abcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. METHODS: To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. To assess the expression of transporter homologs, we used a combination of RNAscope in situ hybridization probes and immunohistochemistry to stain paraffin-embedded sections of adult and larval zebrafish. RESULTS: We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. We identified abcg2a as the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. CONCLUSIONS: These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.

The thiol methyltransferase activity of TMT1A (METTL7A) is conserved across species.

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7%, and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170, and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model.

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib's effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1-10 µM and 25 mg/kg) and in combination with doxil (10-100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell-cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a - 53% versus - 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib's ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Carrier-Free, Amorphous Verteporfin Nanodrug for Enhanced Photodynamic Cancer Therapy and Brain Drug Delivery.

Glioblastoma (GBM) is hard to treat due to cellular invasion into functioning brain tissues, limited drug delivery, and evolved treatment resistance. Recurrence is nearly universal even after surgery, chemotherapy, and radiation. Photodynamic therapy (PDT) involves photosensitizer administration followed by light activation to generate reactive oxygen species at tumor sites, thereby killing cells or inducing biological changes. PDT can ablate unresectable GBM and sensitize tumors to chemotherapy. Verteporfin (VP) is a promising photosensitizer that relies on liposomal carriers for clinical use. While lipids increase VP's solubility, they also reduce intracellular photosensitizer accumulation. Here, a pure-drug nanoformulation of VP, termed "NanoVP", eliminating the need for lipids, excipients, or stabilizers is reported. NanoVP has a tunable size (65-150 nm) and 1500-fold higher photosensitizer loading capacity than liposomal VP. NanoVP shows a 2-fold increase in photosensitizer uptake and superior PDT efficacy in GBM cells compared to liposomal VP. In mouse models, NanoVP-PDT improved tumor control and extended animal survival, outperforming liposomal VP and 5-aminolevulinic acid (5-ALA). Moreover, low-dose NanoVP-PDT can safely open the blood-brain barrier, increasing drug accumulation in rat brains by 5.5-fold compared to 5-ALA. NanoVP is a new photosensitizer formulation that has the potential to facilitate PDT for the treatment of GBM.

Exploiting the therapeutic vulnerability of IDH-mutant gliomas with zotiraciclib.

Isocitrate dehydrogenase (IDH)-mutant gliomas have distinctive metabolic and biological traits that may render them susceptible to targeted treatments. Here, by conducting a high-throughput drug screen, we pinpointed a specific susceptibility of IDH-mutant gliomas to zotiraciclib (ZTR). ZTR exhibited selective growth inhibition across multiple IDH-mutant glioma in vitro and in vivo models. Mechanistically, ZTR at low doses suppressed CDK9 and RNA Pol II phosphorylation in IDH-mutant cells, disrupting mitochondrial function and NAD+ production, causing oxidative stress. Integrated biochemical profiling of ZTR kinase targets and transcriptomics unveiled that ZTR-induced bioenergetic failure was linked to the suppression of PIM kinase activity. We posit that the combination of mitochondrial dysfunction and an inability to adapt to oxidative stress resulted in significant cell death upon ZTR treatment, ultimately increasing the therapeutic vulnerability of IDH-mutant gliomas. These findings prompted a clinical trial evaluating ZTR in IDH-mutant gliomas towards precision medicine ( NCT05588141 ). Highlights: Zotiraciclib (ZTR), a CDK9 inhibitor, hinders IDH-mutant glioma growth in vitro and in vivo . ZTR halts cell cycle, disrupts respiration, and induces oxidative stress in IDH-mutant cells.ZTR unexpectedly inhibits PIM kinases, impacting mitochondria and causing bioenergetic failure.These findings led to the clinical trial NCT05588141, evaluating ZTR for IDH-mutant gliomas.

The Methyltransferases METTL7A and METTL7B Confer Resistance to Thiol-Based Histone Deacetylase Inhibitors.

Histone deacetylase inhibitors (HDACi) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, treatment of solid tumors with this class of drugs has not been successful. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the likelihood of P-gp-mediated resistance. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidepsin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA-sequencing analysis revealed upregulation of the mRNA coding for the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. We demonstrate that expression of METTL7A or METTL7B confers resistance to thiol-based HDACis and that both enzymes are capable of methylating thiol-containing HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.

Overlapping substrate and inhibitor specificity of human and murine ABCG2.

ABCG2 (also known as breast cancer resistance protein) is an ATP-binding cassette (ABC) transporter localized to the plasma membrane where it mediates the efflux of xenobiotics, including potential therapeutics. Studies investigating Abcg2 function at the blood-brain barrier in mouse models are often compared with human ABCG2 function. It is critical to understand the nature of species differences between mouse and human ABCG2, since extrapolations are made from murine data to humans. Two independent drug-selected cell line pairs expressing human or mouse ABCG2 were compared for efflux of fluorescent substrates using flow cytometry. To this end, we developed and characterized a new mouse Abcg2-expressing subline that demonstrated efflux of known fluorescent ABCG2 substrates and increased resistance to mitoxantrone, which is reduced in the presence of the ABCG2 inhibitor Ko143. Our results indicate that the substrate specificity of human and mouse ABCG2 is very similar. We identified a new human and mouse ABCG2 substrate, a porphyrin analog, purpurin-18 (Pp-18), which is not a substrate for P-glycoprotein or multidrug resistance protein 1. The ability of inhibitors to block efflux activity of ABCG2 was assessed using Pp-18. Inhibitors also demonstrated similar effects on human and mouse ABCG2. Chrysin, benzoflavone, and cyclosporin A inhibited Pp-18 efflux in both human and mouse ABCG2. The similarity of the substrate and inhibitor specificity of human and mouse ABCG2 supports interpretation of mouse models in understanding the clinical, pharmacological, and physiologic roles of ABCG2.

New mechanisms of multidrug resistance: an introduction to the Cancer Drug Resistance special collection.

Cancer Drug Resistance publishes contributions to understanding the biology and consequences of mechanisms that interfere with successful treatment of cancer. Since virtually all patients who die of metastatic cancer have multidrug-resistant tumors, improved treatment will require an understanding of the mechanisms of resistance to design therapies that circumvent these mechanisms, exploit these mechanisms, or inactivate these multidrug resistance mechanisms. One example of a resistance mechanism is the expression of ATP-binding cassette efflux pumps, but unfortunately, inhibition of these transporters has not proved to be the solution to overcome multidrug resistance in cancer. Other mechanisms that confer multidrug resistance, and the confluence of multiple different mechanisms (multifactorial multidrug resistance) have been identified, and it is the goal of this Special Collection to expand this catalog of potential multidrug resistance mechanisms, to explore novel ways to overcome resistance, and to present thoughtful reviews on the problem of multidrug resistance in cancer.

Novel Therapeutic Strategies Exploiting the Unique Properties of Neuroendocrine Neoplasms.

BACKGROUND: Over the last few decades of treatment, the outcomes for at least some subsets of neuroendocrine neoplasms (NENs) have improved. However, the identification of new vulnerabilities for this heterogeneous group of cancers remains a priority. METHODS: Using two libraries of compounds selected for potential repurposing, we identified the inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) and histone deacetylases (HDAC) as the agents with the highest activity. We validated the hits in an expanded set of neuroendocrine cell lines and examined the mechanisms of action. RESULTS: In Kelly, NH-6, and NCI-H82, which are two neuroblastoma and one small cell lung cancer cell lines, respectively, metabolic studies suggested that cell death following NAMPT inhibition is the result of a reduction in basal oxidative phosphorylation and energy production. NAMPT is the rate-limiting enzyme in the production of NAD+, and in the three cell lines, NAMPT inhibition led to a marked reduction in the ATP and NAD+ levels and the catalytic activity of the citric acid cycle. Moreover, comparative analysis of the mRNA expression in drug-sensitive and -insensitive cell lines found less dependency of the latter on oxidative phosphorylation for their energy requirement. Further, the analysis of HDAC and NAMPT inhibitors administered in combination found marked activity using low sub-lethal concentrations of both agents, suggesting a synergistic effect. CONCLUSION: These data suggest NAMPT inhibitors alone or in combination with HDAC inhibitors could be particularly effective in the treatment of neuroendocrine neoplasms.

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, and expressed at the zebrafish BBB. We evaluated the luminescence of ten NanoLuc substrates, then screened the eight brightest to determine which are most efficiently effluxed by the ABC transporters. We identified one substrate efficiently pumped out by ABCB1, two by Abcb4, six by ABCG2, and four by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter.

The Multidrug Resistance Transporter P-glycoprotein Confers Resistance to Ferroptosis Inducers.

Ferroptosis is a form of cell death caused by direct or indirect inhibition of glutathione peroxidase 4 that leads to lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding resistance mechanisms, particularly their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. Given the role that ABC transporters play in absorption, distribution, and excretion of many drugs, characterizing these interactions could provide information regarding oral bioavailability and brain penetration and may predict drug-drug interactions. Using ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2, we found that P-gp overexpression was able to confer resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. Results were confirmed with OVCAR8-derived NCI/ADR-RES cells that overexpress P-gp, where the P-gp inhibitor valspodar completely inhibited resistance to the FINs. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. At a concentration of 10 µM, the FINs ML-162, GPX inhibitor 26a, and PACMA31 were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells and GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.