Loss of Cyclin C or CDK8 provides ATR inhibitor resistance by suppressing transcription-associated replication stress.

The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.

Map of synthetic rescue interactions for the Fanconi anemia DNA repair pathway identifies USP48.

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.

High specificity in CheR methyltransferase function: CheR2 of Pseudomonas putida is essential for chemotaxis, whereas CheR1 is involved in biofilm formation.

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14-2.2 µM) than the substrate S-adenosylmethionine (KD of 22-43 µM), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

Genes encoding Cher-TPR fusion proteins are predominantly found in gene clusters encoding chemosensory pathways with alternative cellular functions.

Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be monomeric, which rules out a role of the TPR domain in self-association.

CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells.

Functional aminophospholipid translocases are composed of at least two proteins: an alpha subunit from the P4 subfamily of P-type ATPases and a beta subunit from the CDC50-Lem3p family. Over-expression and knockdown of the human beta subunit CDC50A in KB cells enhanced and decreased, respectively, the uptake of both fluorescent aminophospholipid analogues and the anticancer alkyl-phospholipid perifosine. Confocal microscopy showed that CDC50A-V5 was localized at the endoplasmic reticulum and the Golgi complex of both KB (perifosine-sensitive) and KB PER-R (perifosine-resistant, alkyl-phospholipid uptake deficient) cells, but was only widely distributed in the early and late endosomes in KB cells. Biotinylation of cell surface proteins allowed CDC50A-V5 to be detected in the plasma membrane of KB cells but not in KB PER-R cells, thereby suggesting a defect in CDC50A trafficking that could explain the inability of KB PER-R to uptake perifosine. Over-expression of CDC50A in HeLa and HEK293T cells did not increase uptake, since the protein was retained at the endoplasmic reticulum and Golgi. However, when CDC50A was co-expressed with the P4-ATPase Atp8b1, the two proteins co-localized at the plasma membrane and the uptake of aminophospholipids and perifosine increased strikingly in both cell lines. These findings suggest that CDC50A plays a key role in perifosine uptake in human cells, presumably by forming a functional plasma membrane translocator in combination with a P4-ATPase.

Novel dihydro-beta-agarofuran sesquiterpenes as potent modulators of human P-glycoprotein dependent multidrug resistance.

P-Glycoprotein (Pgp) overexpression is one factor contributing to multidrug resistance (MDR) in cancer cells and represents one drawback in the treatment of cancer. In an attempt to find more specific and less toxic anticancer MDR-reversal agents, we report herein the isolation, structure elucidation and biological activity of nine new (, and ) and seven known (, and ) dihydro-beta-agarofuran sesquiterpenes from the leaves of Celastrus vulcanicola. Their stereostructures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies and biogenetic means. All the compounds were assayed on human MDR1-transfected NIH-3T3 cells, in order to determine their ability to reverse the MDR phenotype due to Pgp overexpression. Six compounds from these series (, , , , and ) showed an effectiveness that was similar to (or higher than) the classical Pgp reversal agent verapamil for the reversal of resistance to daunomycin and vinblastine. The structure-activity relationships are discussed.

Bis-pyranobenzoquinones as a new family of reversal agents of the multidrug resistance phenotype mediated by P-glycoprotein in mammalian cells and the protozoan parasite Leishmania.

We have synthesized a set of bis-pyranobenzoquinones through a direct and highly efficient approach based on a double intramolecular domino Knoevenagel hetero Diels-Alder reaction. These bis-pyranobenzoquinone derivatives are compounds whose skeletons have similarities to those of some anticancerous and leishmanicidal drugs. Considering that these drugs are substrates for some members of the ATP-binding cassette (ABC) family of proteins that confers a multidrug resistance (MDR) phenotype, we have carried out the biological evaluation of 20 bis-pyranobenzoquinones as modulators of the MDR phenotype in mammalian cell lines overexpressing P-glycoprotein, MRP1, or BCRP. Moreover, we also tested some of these compounds as potential MDR modulators in a Leishmania tropica line overexpressing a P-glycoprotein-like transporter. Compounds 9 and 10 are, in this work, the most promising reversal agents of MDR in human cancer cell lines, while compounds 4 and 20 showed potent reversal activity of MDR phenotype in the protozoan parasite Leishmania.

The anti-tumor alkylphospholipid perifosine is internalized by an ATP-dependent translocase activity across the plasma membrane of human KB carcinoma cells.

Perifosine is a promising anticancer alkylphospholipid (ALP) that induces apoptosis in tumor cells. Here we report evidences against a role of endocytosis in perifosine uptake by human KB carcinoma cells. We have generated a KB cell line resistant to perifosine (KB PER(R) clone10), which shows cross-resistance to the ALPs miltefosine and edelfosine, a marked impairment in the uptake of (14)C-perifosine at both 37 degrees C and 4 degrees C, and no signs for active efflux of the drug. KB PER(R) clone10 cells show a similar rate of raft-dependent endocytosis with respect to the parental cells, and silencing of both clathrin and dynamin in the latter causes only minor changes in the rate of perifosine uptake. Perifosine uptake is a temperature- and ATP-dependent, N-ethylmaleimide- and orthovanadate-sensitive process in parental cells. Accumulation of (14)C-perifosine and the fluorescent phospholipid analogue 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylethanolamine (NBD-PE) is inhibited by perifosine in a concentration-dependent manner in parental cells. Moreover, NBD-PE accumulation is slower in PER(R) clone10 cells and correlated with phosphatidylserine exposure in their plasma membrane surface. Together, all these data suggest a role of plasma membrane translocation by a putative phospholipid translocase, rather than endocytosis, as the true mechanism for ALPs uptake in KB carcinoma cells.

Biological evaluation, structure-activity relationships, and three-dimensional quantitative structure-activity relationship studies of dihydro-beta-agarofuran sesquiterpenes as modulators of P-glycoprotein-dependent multidrug resistance.

Multidrug resistance (MDR) is one of the main challenges in the chemotherapy of cancer, malaria, and other important diseases. Here, we report the inhibitory activity of a series of 76 dihydro-beta-agarofuran sesquiterpenes, tested on NIH-3T3 cells expressing the human P-glycoprotein (Pgp) multidrug transporter, to establish quantitative comparisons of their respective abilities to block the drug transport activity. The screening was performed on the basis of the ability of sesquiterpenes to modulate the intracellular accumulation of the classical Pgp substrate daunorubicin. To understand the structural basis for inhibitory activity and guide the design of more potent Pgp inhibitors, we have performed a three-dimensional quantitative structure-activity relationship model using the comparative molecular similarity indices analysis (CoMSIA). The most salient features of these requirements are in the region of the substituents at the C-2, C-3, and C-8 positions, which seem to be critical for determining the overall effectiveness of sesquiterpenes as Pgp inhibitors.

Combination of suboptimal doses of inhibitors targeting different domains of LtrMDR1 efficiently overcomes resistance of Leishmania spp. to Miltefosine by inhibiting drug efflux.

Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-beta-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein.

Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.

Flavonoid structure-activity studies identify 6-prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein ABCG2.

Overexpression of breast cancer resistance protein ABCG2 confers multidrug resistance in cancer cells. The GF120918-sensitive drug efflux activity of human wild-type (R482) ABCG2-transfected cells was used for rational screening of inhibitory flavonoids and establishment of structure-activity relationships. Flavones were found more efficient than flavonols, isoflavones, and flavanones. Differentially substituted flavone derivatives indicated positive OH effects at position 5, in contrast to positions 3 and 7. A methoxy at position 7 was slightly positive in tectochrysin, whereas a strong positive effect was produced by prenylation at position 6. The potency of 6-prenylchrysin was comparable with that of GF120918 (IC50 = 0.3 micromol/L). Both 6-prenylchrysin and tectochrysin seemed specific for ABCG2 because no interaction was detected with either P-glycoprotein or MRP1. The ABCG2 resistance profile in vitro is altered by mutation at amino acid 482. The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Whereas GF120918 strongly inhibited the ATPase activity of wild-type ABCG2, neither 6-prenylchrysin nor tectochrysin altered the activity. In contrast, all three inhibitors stimulated the ATPase activity of mutant ABCG2. 6-Prenylchrysin at 0.5 micromol/L efficiently sensitized the growth of wild-type ABCG2-transfected cells to mitoxantrone, whereas higher concentrations were required for the mutant ones. In contrast, 1 micromol/L tectochrysin was sufficient to fully sensitize mutant ABCG2-transfected cells, whereas higher concentrations were required for the wild-type ones. Both flavones exhibited a lower intrinsic cytotoxicity than GF120918 and were apparently not transported by ABCG2. 6-Prenylchrysin and tectochrysin therefore constitute new and promising inhibitors for the reversal of ABCG2-mediated drug transport.

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.