Haploinsufficiency underlies the neurodevelopmental consequences of SLC6A1 variants.

Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.

Determinants of DDX3X sensitivity uncovered using a helicase activity in translation reporter.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5' UTRs to measure DDX3X mediated translational activity in cells. To dissect the structural underpinnings of DDX3X dependent translation, we first used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then employed HART to investigate how their perturbation impacts DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the ribosome complex as well as its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally encoded measurement of DDX3X-dependent translation in cells.

The Combination of Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Delays KMT2A-Rearranged Leukemia Progression.

Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.

Curcumin and its cyclohexanone analogue inhibited human Equilibrative nucleoside transporter 1 (ENT1) in pancreatic cancer cells.

Our group investigated combining the phytochemical curcumin and gemcitabine in a liposome, to improve gemcitabine's activity against pancreatic tumours. While optimising the curcumin: gemcitabine ratio for co-encapsulation, we found that increasing curcumin concentrations relative to gemcitabine resulted in antagonistic interactions. As curcumin is a promiscuous transporter inhibitor; we suspected that increased resistance occurred via inhibition of Equilibrative nucleoside transporter 1 (ENT1)-mediated gemcitabine uptake. To test our hypothesis, we determined whether curcumin and a related analogue, 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone (or A13), inhibited ENT1-mediated accumulation of [3H]uridine and [3H]gemcitabine into pancreatic cancer cells. We then confirmed the inhibition of gemcitabine accumulation by investigating whether curcumin/A13 could increase gemcitabine resistance in growth inhibition assays. We found that curcumin and A13 concentration-dependently inhibited the ENT1-mediated accumulation of both uridine and gemcitabine in MIA PaCa-2 and PANC-1 cells. We also found that non-toxic concentrations of curcumin and A13 significantly increased the resistance of both cell lines to gemcitabine. Increased resistance only occurred when curcumin/A13 was co-incubated with gemcitabine, and not with sequential exposure (i.e., curcumin first, followed by gemcitabine, or vice versa). We also found that the curcumin analogue (3E,5E)-3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (or EF24) did not inhibit gemcitabine accumulation, making it more suitable in combinations than curcumin/A13. From these results, we concluded that curcumin and A13 are inhibitors of the ENT1 transporter, but only at high concentrations (2-20µM). Curcumin is unlikely to inhibit gemcitabine uptake in tumours but may interfere with the oral absorption of ENT1 substrates due to high gut concentrations readily achievable from over-the-counter tablets/capsules.

Heterocyclic cyclohexanone monocarbonyl analogs of curcumin can inhibit the activity of ATP-binding cassette transporters in cancer multidrug resistance.

Curcumin (CUR) is a phytochemical that inhibits the xenobiotic ABC efflux transporters implicated in cancer multidrug resistance (MDR), such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins 1 and 5 (MRP1 and MRP5). The use of CUR in the clinic however, is complicated by its instability and poor pharmacokinetic profile. Monocarbonyl analogs of CUR (MACs) are compounds without CUR's unstable ß-diketone moiety and were reported to have improved stability and in vivo disposition. Whether the MACs can be used as MDR reversal agents is less clear, as the absence of a ß-diketone may negatively impact transporter inhibition. In this study, we investigated 23 heterocyclic cyclohexanone MACs for inhibitory effects against P-gp, BCRP, MRP1 and MRP5. Using flow cytometry and resistance reversal assays, we found that many of these compounds inhibited the transport activity of the ABC transporters investigated, often with much greater potency than CUR. Overall the analogs were most effective at inhibiting BCRP and we identified three compounds, A12 (2,6-bis((E)-2,5-dimethoxy-benzylidene)cyclohexanone), A13 (2,6-bis((E)-4-hydroxyl-3-methoxybenzylidene)-cyclohexanone) and B11 (3,5-bis((E)-2-fluoro-4,5-dimethoxybenzylidene)-1-methylpiperidin-4-one), as the most promising BCRP inhibitors. These compounds inhibited BCRP activity in a non-cell line, non-substrate-specific manner. Their inhibition occurred by direct transporter interaction rather than modulating protein or cell surface expression. From these results, we concluded that MACs, such as the heterocyclic cyclohexanone analogs in this study, also have potential as MDR reversal agents and may be superior alternatives to the unstable parent compound, CUR.

Modulatory effects of curcumin on multi-drug resistance-associated protein 5 in pancreatic cancer cells.

PURPOSE: Chemotherapy of pancreatic cancer often fails due to the development of intrinsic and acquired resistance during drug treatment. Recent studies have suggested that MRP5 conferred resistance to first-line drugs 5-fluorouracil and gemcitabine by active efflux of drugs from the cell. Our aim was to evaluate whether curcumin could reverse this multi-drug resistance by inhibition of MRP5-mediated efflux. METHODS: MRP5 protein was detected and localized by immunocytochemistry using a monoclonal antibody in MRP5 over-expressing HEK293 (HEK293/MRP5) cells and two pancreatic cancer cell lines PANC-1 and MiaPaCa-2. The cellular accumulation of a specific MRP5 fluorescent substrate 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) into these cells was measured by flow cytometry and the cell proliferation determined by a 72-h CyQuant assay. RESULTS: The cellular accumulation of BCECF in HEK293/MRP5 cells and in PANC-1 and MiaPaCa-2 cells was significantly increased by curcumin in a concentration-dependent manner. Curcumin and a MRP5 inhibitor MK571 had no apparent effects on cellular accumulation of BCECF in parental HEK293 cells. In the proliferation assays, curcumin caused a concentration-dependant increase in the sensitivity to the cytotoxic drug 5-fluorouracil in HEK293/MRP5 cells, PANC-1 and MiaPaCa-2 pancreatic cancer cells, but not in parental HEK293 cells. CONCLUSIONS: Our results suggest that curcumin is an inhibitor of MRP5 and may be useful in the reversal of multi-drug resistance in pancreatic cancer chemotherapy.

Interactions of dietary phytochemicals with ABC transporters: possible implications for drug disposition and multidrug resistance in cancer.

Common foods, such as fruits and vegetables, contain a large variety of secondary metabolites known as phytochemicals, many of which have been associated with health benefits. However, there is a limited knowledge of the processes by which these, mainly charged, phytochemicals (and/or their metabolites) are absorbed into the body, reach their biological target, and how they are eliminated. Recent studies have indicated that some of these phytochemicals are substrates and modulators of specific members of the superfamily of ABC transporting proteins. In this review, we present the reported interactions between the different classes of phytochemicals and ABC transporters and the mechanism by which they modulate the activity of these transporters. We also discuss the implications that such interactions may have on the pharmacokinetics of xenobiotics and the possible role of phytochemicals in the reversal of multidrug resistance in cancer chemotherapy.