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Weight loss diet Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy

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AbstractCancer chemotherapy targeting frequent loss of heterozygosity events is an attractive concept, since tumor cells may lack enzymatic activities present in normal constitutional cells. To find exploitable targets, we map prevalent genetic polymorphisms to protein structures and identify 45 nsSNVs (non-synonymous small nucleotide variations) near the catalytic sites of 17 enzymes frequently lost in cancer. For proof of concept, we select the gastrointestinal drug metabolic enzyme NAT2 at 8p22, which is frequently lost in colorectal cancers and has a common variant with 10-fold reduced activity. Small molecule screening results in a cytotoxic kinase inhibitor that impairs growth of cells with slow NAT2 and decreases the growth of tumors with slow NAT2 by half as compared to those with wild-type NAT2. Most of the patient-derived CRC cells expressing slow NAT2 also show sensitivity to 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA) treatment. These findings indicate that the therapeutic index of anti-cancer drugs can be altered by bystander mutations affecting drug metabolic genes.

IntroductionRecent targeted anti-cancer therapies exploit acquired genetic differences between cancer and normal cells, such as activation by mutation of a specific oncogene, inactivation by mutation of a tumor suppressor gene, or perturbation of pathways involved in the maintenance of genome integrity to achieve preferential killing of cancer cells1. Drugs targeting protein tyrosine kinases are mainstays of clinical cancer care2,3,4, and strategies addressing loss-of-function of tumor suppressor genes such as TP535,6 and RB7 are in development. Collateral lethality, exploiting vulnerabilities left in cancer cells after a passenger gene in the vicinity of a tumor suppressor is lost, has emerged as a new therapeutic avenue. The frequent 1p36 deletion in glioblastoma, entailing loss of ENO1, sensitizes tumor cells to ENO2 inhibition8. Similarly, complete losses of POLR2A, MTAP, and ME39,10,11,12, and partial losses of PSMC213, SF3B114, and MAGOH15 have been identified as potential therapeutic targets in human cancers. Targeting of the loss of heterozygosity (LOH) events occurring as cancer genomes inactivate tumor suppressor genes has been achieved by allele-specific inhibition, where variants of essential genes such as the 70-kDa subunit of replication protein A (RPA70) near TP53 are silenced using antisense oligonucleotides16. However, allele-specific LOH therapy targeting proteins tractable to inhibition by systemically administered agents has previously not been demonstrated. We here demonstrate that a recurring loss of heterozygosity event affecting a drug metabolic activity (NAT2) can increase the sensitivity to a low molecular weight cytotoxic compound.ResultsIdentification of N-acetyltransferase 2 (NAT2) as a target for allele-specific inhibitionTo identify target proteins, we mapped variants observed in the 1092 individuals in the 1000 Genomes project to functional domains and crystal structures to identify those that could alter protein structure in catalytic or substrate binding sites (Fig. 1a). The variants were first mapped to transcripts, localizing 482,280 small nucleotide variants (SNVs) to protein coding regions. Of these, 78% were likely to be present in only one population17. To enrich for prevalent targets of higher utility in LOH-directed therapies, 23,532 non-synonymous SNVs in functional protein domains with allele frequency ≥0.5% were selected. Of these, 1367 SNVs (~5.8%) mapping to 566 crystal structures had both the SNV and active or substrate binding sites defined in the structure. After visual inspection, 56 SNVs in 45 intracellular proteins resulted in amino-acid substitutions near catalytic residues or substrate binding pockets, including 36 SNVs in 26 genes with >5% heterozygosity in all 1000 Genomes populations (Supplementary Data 1). Finally, retaining only the genes with >15% LOH frequency in common human cancers18 and with a gene expression profile matching the tissues of interest, yielded 17 potential target enzymes for LOH-directed cancer therapies (Supplementary Table 1). From the putative target genes, we selected polymorphisms in NAT2, AKR7A2, and SULT1A1 for validation as these genes were known to be involved in drug metabolism and are non-essential for cell survival. Other genes with common variants were considered but not prioritized because of likely limited chemical space of substrates (HSD17B), small reduction in catalytic activity by the variant (GSTP1), unknown importance (HAAO), known substrate redundancy with related enzymes (GSTP1, SULTs), or too few LOH events in CRC (ABP1 and AKR7A). To confirm the expected prevalence of candidate SNVs and frequency of LOH events, we genotyped the selected SNVs in 74 patients with chromosomally unstable colorectal cancers (CRCs) and could detect all in heterozygous states (Supplementary Table 2). Next, tumors from heterozygous individuals were assessed for somatic LOH events. The highest frequency of allelic loss was observed for NAT2, where ~7% heterozygous for the rs1799930 polymorphism retained only one allele in their tumors because of LOH (Fig. 1b; Supplementary Table 2). We observed a similar likelihood for CRCs to lose either allele (Fig. 1b), in an LOH event detected in stage II as well as stage III and IV CRCs (Supplementary Fig. 1), in line with NAT2 being a bystander gene on 8p which is lost early in CRC development19. After genotyping, based on the frequency of the LOH events NAT2 was prioritized as a proof of concept gene over AKR7A2 and SULT1A1. The NAT2 gene encodes one of the two human N-acetyltransferases involved in xenobiotic metabolism of arylamines and hydrazine compounds. NAT2 is highly polymorphic with 108 allelic variants identified in human populations [http://nat.mbg.duth.gr/]. The SNV rs1799930 defines the NAT2*6 group of variant alleles (R197Q) with ≥10-fold reduced activity compared with wild-type NAT2, considered to encode a rapid acetylator phenotype20,21 (Fig. 1c). Whereas NAT1 is expressed in essentially all tissues, NAT2 is confined to the liver and gastrointestinal tract (GI)22,23. From the putative target proteins identified, we selected NAT2 because of its (a) known role in drug metabolism, (b) non-redundant and well defined substrate specificity, (c) GI restricted expression, (d) location on a chromosome arm frequently lost during transition from colorectal adenoma to carcinoma24, (e) 10-fold activity difference between the gene products encoded by the wild-type and a common variant allele, and (f) clinically relevant substrates as ~10 commonly used drugs are subject to NAT2 metabolism, among them the cytotoxic drug amonafide25, where rapid acetylators have ~4.5-fold higher plasma ratio of N-acetyl-amonafide to amonafide than slow acetylators leading to increased systemic toxicity26.Fig. 1: N-acetyltransferase 2 (NAT2) is a target for allele-specific inhibition.a Identification of enzymes with prevalent alternative alleles resulting in amino-acid substitution near functional sites. A series of filtering steps was applied to identify the subset of SNVs causing amino-acid substitutions near active sites in publicly available 3D protein structures. To enrich potential target proteins for LOH-based tumor targeting approaches, the candidate genes were further selected based on expression profile and the frequency of LOH in common cancer types. b Loss of heterozygosity at 8p22 can render CRCs deficient in NAT2 function. To confirm the prevalence of rs1799930 and the frequency of LOH at NAT2, genomic DNA from 74 CIN CRCs and patient-matched normal tissues was genotyped. Left, genotype distribution of normal tissues. Right, LOH events observed in the tumors of heterozygous individuals. c Structure of human NAT2 co-crystallized with the cofactor coenzyme A (light blue) (PDB: 2PFR). The side chains of the active site amino-acid residues involved in substrate transformation (Asp122, Cys68, and His107) and cofactor binding (Gly104, Thr103, Thr214, Tyr208, and Ser287) (dark blue) and the side chain of Arg197 (rs1799930, red) are shown. The figure was designed in PyMOL (v. 2.3.2). d Exploiting loss of a rapid NAT2 allele in tumor cells for anti-cancer therapy. Eligible patients are heterozygous for the slow (A, blue) and rapid (G, red) NAT2 alleles. During cancer progression, cancer cells may undergo loss of heterozygosity (LOH) and lose the rapid NAT2 allele (red). Treatment with a cytotoxic compound (triangle) that can only be processed by the rapid NAT2 enzymatic variant lost in cancer cells will result in selective tumor death.Discovery of compounds selectively toxic to slow NAT2 cellsWe reasoned that anti-cancer drugs rendered less toxic by NAT2 metabolism are also likely to exist and that CRC cells having lost a rapid NAT2 allele through LOH could be sensitized to treatment with a cytotoxic NAT2 substrate relative to other constitutional cells retaining the rapid allele (Fig. 1d). Therefore, cell systems for small molecule library screening were engineered in human CRC RKO and DLD-1 cells by transfection with NAT2 expression vectors encoding slow NAT2*6A (rs1799930) or rapid NAT2*13A (wt) alleles (Figs. 2a, b). Both cell lines are homozygous for the slow NAT2*6A allele and have low endogenous NAT2 expression. The acetylation velocities of the NAT2 substrates amonafide and procainamide were ≥8-fold higher in rapid NAT2 clones when compared with slow NAT2 in RKO (p 239 (amonafide), 326>281 (N-acetyl-amonafide), 236>163 (Procainamide), and 278>205 (N-acetyl-procainamide).Cell-based screen for cytotoxic drugs metabolized by NAT2To enrich for potential NAT2 substrates, the database of the compound collection (189,018 compounds) of CBCS (Chemical Biology Consortium Sweden) was analyzed for primary arylamines yielding 1150 compounds of which 156 were available for screening. Of the 156 compounds, 120 had shown cytotoxicity in previous cell-based screens at CBCS. In addition, 20 primary heteroarylamines that had previously shown cytotoxicity were included. In total, 176 compounds were selected based on chemical structure and their potential to impair cell growth. Each of the compounds was evaluated at three concentrations (0.5, 2, and 10 μM) using a rapid acetylator RKO clone and a vector control clone. Cells were seeded at 8000 cells per well in 96-well plates and incubated with the different compounds for 72 h. Next, cell viability was scored in an MTT assay according to the manufacturer’s protocol by addition of resazurin (Sigma-Aldrich) to each well and incubation of the plates at 37 °C. Fluorescence was read at 590 nm 2 h following the addition of resazurin using a Victor2 1420 multilabel counter (Wallac).Quantification of NAT2 catalytic activity toward APARKO and DLD-1 cells were plated at a density of 18,000 cells per well in a 96-well plate and treated with 0.1–50 µM of APA (AKos GmbH). The NAT2 catalytic activity was quenched after 30 and 60 min (0.1–1 µM APA) or after 15 and 30 min (2–50 µM APA) by addition of 99.8% methanol. APA and N-acetyl APA (NAPA, AKos GmbH) were detected by LC-MS/MS on a XEVO TQ (Waters) coupled to an Acquity UPLC (Waters) using a HSS T3 column (1.7 µm, 2 × 50 mm; Waters). The mobile phases consisted of 0.05% heptafluorobutyric acid and propionic acid (A), and 0.05% heptafluorobutyric acid and propionic acid in acetonitrile (B). The following m/z transitions were monitored: 353>323 (APA) and 395>143 (NAPA).In vitro ADME profiling of APA and NAPAMetabolic stability was assessed by incubating APA and NAPA at a final concentration of 1 μM with 0.5 mg⋅ml−1 pooled CD-1 mouse liver microsomes (XenoTech, cat. no. M1000) in 100 mM potassium phosphate buffer pH 7.4. The reaction was initiated by addition of NADPH at a final concentration of 1 mM and stopped at 0, 5, 10, 20, 40, and 60 min by transferring a 50 µl sample to 100 μl ice-cold acetonitrile. Before LC-MS/MS analysis, samples were centrifuged at 3000 × g for 20 min at 4 °C. In vitro half-life was estimated as previously described35. Bufuralol, diclofenac and midazolam were used as controls. Solubility in phosphate buffer was determined by adding 5 μl of 10 mM DMSO stock solution of the compounds to 500 μl of 100 mM potassium phosphate buffer pH 7.4. After overnight incubation of the samples at 37 °C, the samples were centrifuged at 3000 × g for 30 min at 37 °C and analyzed by LC-MS/MS. Ketoconazole and nicardipine were used as controls. Plasma protein binding was measured by incubating the compounds at a final concentration of 10 μM with pooled CD-1 mouse plasma (Innovative Research) in a Rapid Equilibrium Dialysis device (Thermo Fisher Scientific) against isotonic 67 mM phosphate buffer pH 7.4. After 4 h incubation at 37 °C, samples were 10-fold diluted in acetonitrile. Before LC-MS/MS analysis, samples were centrifuged at 3000 × g for 20 min at 4 °C. Fraction unbound in plasma was calculated as the ratio of compound concentration in the buffer sample and the plasma sample. Diclofenac and propranolol were used as controls.Binding affinities of APA and NAPA toward protein kinasesThe binding affinities of APA and NAPA were evaluated in the scanMAXsm Kinase Assay Panel (DiscoverX) at a final concentration of 10 µM. The compounds were tested for steric or allosteric binding to kinases in solution. The kinase was captured from the solution through an immobilized kinase-specific ligand and the kinase recovery was quantified. The amount of recovered kinase in the presence or absence of APA or NAPA was measured by quantitative PCR compared with the kinase capture in absence of the compound. Any compound reducing the binding affinity of a kinase to the respective immobilized ligand was scored as a potential hit, and the binding affinity of the compound toward a kinase is interpreted as the difference between the kinase recovery in the absence and presence of the compound. The inhibitory binding constants (Kd) of APA and NAPA to AURKA were evaluated in a KdELECT Kinase Assay (DiscoverX). The Kd was derived from independent duplicate of 11-point dose-response curves ranging from 0.85 nM to 50 µM for each compound. Data acquisition followed the same methodology as for the scanMAXsm Kinase Assay Panel.siRNA knockdown of DYRK1A, AURKA, and CDK7
The RKO and DLD-1 CRC cells were plated at 10,000 or 5000 cells, respectively, per well in a 96-well plate. After 16 h, the cells were exposed to serum- and antibiotics-free McCoy’s 5A medium and transfected with SMARTpool ON-TARGETplus control siRNA or siRNAs targeting DYRK1A, AURKA, or CDK7 using the DharmaFECT 2 transfection reagent (Dharmacon). After 5 h the cells were supplemented with FBS to 10% final concentration and incubated for 72 h. The cell viability was evaluated in an MTT assay as described above.siRNA knockdown of NAT2 in HCT116 cellsHCT116 (ATCC) CRC cells were plated at 4500 cells per well in a 96-well plate, let attach for 16 h and transfected with SMARTpool ON-TARGETplus control siRNA or siRNAs targeting NAT2 using the DharmaFECT 2 transfection reagent (Dharmacon) in serum- and antibiotics-free McCoy’s 5A medium. After 5 h, the cells were supplemented with FBS to 10% final concentration and incubated for 24 h. Finally, cells were treated with APA or 5-FU in different concentrations and cell viability was evaluated in an MTT assay after 68 h.RT-qPCR analysisDirect cDNA extractions were performed from cell lysates using the TaqMan® Gene Expression Cells-to-CT™ Kit (Thermo Fisher Scientific). The expression of NAT2, DYRK1A, AURKA, or CDK7 transcripts was detected by qPCR using TaqMan probes against NAT2 (Hs04194721_s1), DYRK1A (Hs00176369_m1), AURKA (Hs01582072_m1), or CDK7 (Hs00361486_m1) with ACTB (Hs01060665_g1) as an internal reference gene. The quantification was performed according to the comparative Ct (ΔΔCT) standard protocol in StepOnePlus instrument (Applied Biosciences).Validation of APA as a NAT2-specific substrateThe acetylation rate of APA (AKos GmbH), 4-aminoasylic acid (PAS) (Sigma-Aldrich) and procainamide (Sigma-Aldrich) was determined by incubation with recombinant human N-acetyltransferases 1 and 2 (Corning-Supersomes™). Briefly, triethanolamine pH 7.5 (50 mM), acetyl-CoA (0.1 mM), acetyl-d,l-carnitine (4.6 mM), EDTA (1 mM), DTT (1 mM), and carnitine acetyltransferase (0.6 U/mL) were combined with 10 µM of the substrate. After incubation at 37 °C for 5 min, the enzymatic reaction was initiated by addition of 0.005 mg/ml of the respective recombinant NAT enzyme. NAT activity was quenched by addition of 200 µl of acetonitrile to 100 µl of the incubation mix at indicated timepoints followed by centrifugation at 10,000 × g for 3 min and analysis of the supernatant. NAT2 catalytic activity toward APA and NAPA was quantified by LC-MS/MS as described above.Liposomal preparation of APAA mixture of HEPC:Chol:DSPE-PEG2000 at a molar ratio of 50:45:5 was solubilized in chloroform, dried overnight to a thin film under rotary evaporation, hydrated with 1 M sodium citrate (pH 3) and submerged into a 65 °C sonication bath to allow formation of large multilamellar vesicles. The lipid suspension was extruded 10 times through a double stack of 0.1 μm Nuclepore filters (Whatman) using a Lipex Thermobarrel Extruder (Northern Lipids). The resulting colloidal suspension of single unilamellar vesicles (SUVs) was then dialyzed against 300 mM sucrose at 4 °C. The mean size of the SUVs was determined by quasi-elastic light scattering using a Malvern Zetasizer 3000 (Malvern). APA was actively loaded into liposomes using a citrate pH gradient. To encapsulate APA, the compound was dissolved in DMSO and mixed with liposomes at a drug:lipid molar ratio of 1:3 following incubation at 65 °C for 30 min. Drug-loaded liposomes were stored at 4 °C and diluted to 8.8 mg mL−1 of APA in PBS before use. Encapsulation efficiency was 95.5% as determined by disruption of liposomes with 0.1% Triton X-100 and fluorometric measurement (excitation at 380 nm, emission at 450 nm) using a fluorescence plate reader (Tecan Infinite 200). Concentrations were derived by reference to an APA standard curve. Free APA was prepared by solubilization with HCl followed by dilution with PBS to achieve a final concentration of 8.8 mg mL−1.APA treatment of mouse xenograft modelsThe BALB/c nude mice are rapid Nat acetylators (Nat1*6, Nat2*8, and Nat3*1), and the scarcity of genetic polymorphisms in the Nat mouse genes36 supports the biological relevance of this animal model in recreating APA targeting in a NAT2-proficient organism. Mouse Nat2 and human NAT1 are orthologous and believed to share similar substrate specificity and tissue expression profiles37. All animal experiments were overseen and approved by the Institutional Animal Care and Use Committee of Temasek Life Sciences Laboratory at the National University of Singapore (#TLL-14-022). Statistical power analysis was performed to determine the minimum number of animals per experimental arm required to demonstrate a statistically significant difference in treatment outcomes. Dose escalation studies revealed 88 mg kg−1 as maximum tolerated dose of free APA. Six to eight week female NCr nude mice (In Vivos) were each injected subcutaneously with 4 × 106 RKO NAT2 slow cells in the left flank and 4 × 106 RKO NAT2 rapid cells on the right flank. The xenografts were grown for 10 days before randomization and non-blinded treatment. A minimum of eight animals were used for each experimental arm, consisting of liposomal APA (88 mg kg−1), free APA (88 mg kg−1) and control treatment (empty liposomes suspended in PBS). Tumor volumes were calculated as length × width2 × 0.5. All drug and control treatments were administered at the indicated timepoints as tail vein injections in volumes of 0.2 mL. weight loss remained

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