Journal: eBioMedicine

Article Title: Loss of heterozygosity of CYP2D6 enhances the sensitivity of hepatocellular carcinomas to talazoparib

doi: 10.1016/j.ebiom.2024.105368

Figure Lengend Snippet: A graphical summary illustrates the strategy used to identify CYP2D6 as a target for collateral lethality, driven by prevalent loss of heterozygosity (LOH) in cancer genomes .

Article Snippet: Proteins were transferred to nitrocellulose membranes and incubated at room temperature in 5% non-fat dry milk in 1 × PBST for 1 h. Membranes were then incubated overnight at 4 °C with anti-FlAG primary antibody (peptide sequence DYKDDDDK, #F3165, Mouse, Sigma) to detect the overexpressed CYP2D6∗1 (56 kDa) as well as CYP2D6∗4 (∼20 kDa) proteins, anti-CYP2D6 to detect endogenous CYP2D6 (#ab137426), anti-POR (#sc-25270, Mouse, Santa Cruz Biotechnology), anti-CYB5A (#NBP2-49284, Rabbit, NovusBio) and β-Actin (#sc-47778, Mouse, Santa Cruz Biotechnology).

Techniques:

Journal: eBioMedicine

Article Title: Loss of heterozygosity of CYP2D6 enhances the sensitivity of hepatocellular carcinomas to talazoparib

doi: 10.1016/j.ebiom.2024.105368

Figure Lengend Snippet: Establishment of cell models expressing functional CYP2D6∗1 or loss-of-function CYP2D6∗4 alleles . a . Upper panel: as part of our drug discovery toolset, we generated models expressing either the wild-type CYP2D6 or the loss-of-function CYP2D6 allele associated with rs3892097 ( CYP2D6∗4 ). CYP2D6∗1 allele encodes the wild-type sequence, producing a transcript that is translated into a 497 amino acid (aa) protein (green). In contrast, the CYP2D6∗4 variant creates a cryptic acceptor site at the start of exon 4 (yellow), creating a frameshift when transcription and splicing occurs. The resulting aberrant transcript is missing 1 bp in exon 4 and is translated into a truncated peptide of 181 amino acids (grey). Lower panel: genotyping of the generated clones confirmed that they encode either the wild-type CYP2D6∗1 or the CYP2D6∗4 allele resulting from the 1846 G > A splice variant. The coding part of exon 3 (blue) and exon 4 (orange) is shown. b. Detection of CYP2D6 catalytic activity in HEK293T cells overexpressing wild-type or LoF CYP2D6 alleles, and transiently overexpressing cofactors POR and CYB5A. The formation of dextrorphan was measured by LC-MS/MS at indicated time points after incubation with 10 μM of the CYP2D6 specific substrate dextromethorphan. One representative experiment with three technical replicates is shown (mean ± SD, POR MOI:0/CYB5A MOI:0, 90′ incubation p = 0.024; POR MOI:0.5/CYB5A MOI:0.5, 90′ incubation p = 0.015; POR MOI:0.5/CYB5A MOI:2.0, 90′ incubation p = 0.002; POR MOI:0.5/CYB5A MOI:5.0, 90′ incubation p = 0.002; unpaired t-test, Two-stage step-up (Benjamini, Krieger, and Yekutieli)). c. Schematic of POR and CYB5A in relation to the CYP family CYP2D6 and CYP3A4. POR donates electrons to CYPs, which in turn donates them to molecular oxygen as the terminal electron acceptor thereby mediating catalysis. In some cases, CYB5A facilitates interaction of POR with CYPs, and may act as an alternative donor for the second, but not the first, electron in the P450 cycle; thus, the action of POR is essential for all CYPs. d. Confirmation of HEK293T CYP2D6∗ 1+POR + B5A and CYP2D6∗ 4+POR + B5A cell models. Expression of FLAG-tagged wild-type CYP2D6∗1 and LoF CYP2D6∗4, POR and CYB5A was confirmed by immunoblotting with actin as loading control. e. Dose response to the CYP2D6 specific substrate rucaparib in HEK293T CYP2D6∗ 1+POR + B5A and CYP2D6∗ 4+POR + B5A cells. One representative experiment with three technical replicates is shown (mean ± SD, rucaparib 65 μM, p = 0.024; rucaparib 130 μM, p = 0.010; unpaired t-test, Two-stage step-up (Benjamini, Krieger, and Yekutieli)). f. Workflow of the cell-based drug screen. A total of 525 compounds from the approved oncology drug set and known kinase inhibitors set were assessed in a cell-based screen to find compounds that exhibited selective toxicity to either HEK293T cells with CYP2D6∗ 1+POR + B5A (thereafter HEK293T CYP2D6∗1 in the figure) or CYP2D6∗ 4+POR + B5A (thereafter HEK293T CYP2D6∗4 in the figure). g. Dose response to 12 identified hit compounds in the HEK293T cell model. Name of 12 compounds: AZD-3463, CYC-16, etoposide, everolimus, GDC-0349, lenvatinib, MK-8776, PHA-680632, talazoparib, tyrphostin 9, VX-702 and WZ-3146. The half-maximal inhibitory concentration (IC 50 ) was calculated using Dose–Response inhibition (inhibition vs normalized response).

Article Snippet: Proteins were transferred to nitrocellulose membranes and incubated at room temperature in 5% non-fat dry milk in 1 × PBST for 1 h. Membranes were then incubated overnight at 4 °C with anti-FlAG primary antibody (peptide sequence DYKDDDDK, #F3165, Mouse, Sigma) to detect the overexpressed CYP2D6∗1 (56 kDa) as well as CYP2D6∗4 (∼20 kDa) proteins, anti-CYP2D6 to detect endogenous CYP2D6 (#ab137426), anti-POR (#sc-25270, Mouse, Santa Cruz Biotechnology), anti-CYB5A (#NBP2-49284, Rabbit, NovusBio) and β-Actin (#sc-47778, Mouse, Santa Cruz Biotechnology).

Techniques: Expressing, Functional Assay, Drug discovery, Generated, Sequencing, Variant Assay, Clone Assay, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Incubation, Western Blot, Control, Concentration Assay, Inhibition

Journal: eBioMedicine

Article Title: Loss of heterozygosity of CYP2D6 enhances the sensitivity of hepatocellular carcinomas to talazoparib

doi: 10.1016/j.ebiom.2024.105368

Figure Lengend Snippet: Talazoparib and MK-8776 exhibit CYP2D6 dependent cytotoxicity in hepatocellular carcinoma cells . a. Relative expression of CYP2D6 in parental HepG2 cells and two CRISPR/Cas9 CYP2D6 KO clones was determined by qPCR. ∗p < 0.05 by two-tailed t-test. RQ: relative quantification. b. Expression levels of endogenous CYP2D6 protein was determined in HepG2 parental and CYP2D6 KO clones by immunoblotting using actin as loading control. Recombinant CYP2D6 is pure CYP2D6 protein thus no actin was detected. c. Catalytic activity of CYP2D6 in HepG2 parental and CYP2D6 KO cells. The formation of dextrorphan was measured by LC-MS/MS at indicated time points after incubation with 10 μM of the CYP2D6 specific substrate dextromethorphan. One representative experiment with three technical replicates is shown (mean ± SD, HepG2 parental 30′ vs 0′ incubation, p = 0.036; HepG2 parental 60′ vs 0′ incubation, p = 0.0004, unpaired t-test, Two-stage step-up (Benjamini, Krieger, and Yekutieli)). d. The total amount of the CYP2D6-specific substrate dextromethorphan in the cell pellet was determined by LC-MS/MS after 90min incubation with 10 μM dextromethorphan. One representative experiment with three technical replicates is shown (mean ± SD, ∗p < 0.05 by two-tailed t-test). e. Dose–response to rucaparib in HepG2 parental and CYP2D6 KO clones. One representative experiment with three technical replicates is shown (mean ± SD, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, unpaired t-test, Two-stage step-up (Benjamini, Krieger, and Yekutieli)). f. The top 10 down-regulated KEGG signalling pathways in HepG2 CYP2D6 KO cells compared to HepG2 parental cells (full list Supplementary Table S6 ). g. The enriched genes in the KEGG pathway of drug metabolsim-CYP450. h. The top 10 up-regulated KEGG signalling pathways in HepG2 CYP2D6 KO cells compared to HepG2 parental cells (full list Supplementary Table S7 ). i. The enriched genes in the MAPK signalling pathway. j. Dose–response of talazoparib and MK-8776 in HepG2 parental and CYP2D6 KO clones. Cell viability of KO clone 1 vs parental, and KO clone 2 vs parental, unpaired t-test, Two-stage step-up (Benjamini, Krieger, and Yekutieli), only show p-values <0.05).

Article Snippet: Proteins were transferred to nitrocellulose membranes and incubated at room temperature in 5% non-fat dry milk in 1 × PBST for 1 h. Membranes were then incubated overnight at 4 °C with anti-FlAG primary antibody (peptide sequence DYKDDDDK, #F3165, Mouse, Sigma) to detect the overexpressed CYP2D6∗1 (56 kDa) as well as CYP2D6∗4 (∼20 kDa) proteins, anti-CYP2D6 to detect endogenous CYP2D6 (#ab137426), anti-POR (#sc-25270, Mouse, Santa Cruz Biotechnology), anti-CYB5A (#NBP2-49284, Rabbit, NovusBio) and β-Actin (#sc-47778, Mouse, Santa Cruz Biotechnology).

Techniques: Expressing, CRISPR, Clone Assay, Two Tailed Test, Quantitative Proteomics, Western Blot, Control, Recombinant, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: eBioMedicine

Article Title: Loss of heterozygosity of CYP2D6 enhances the sensitivity of hepatocellular carcinomas to talazoparib

doi: 10.1016/j.ebiom.2024.105368

Figure Lengend Snippet: Talazoparib exhibits cytotoxicity against cells with deficient CYP2D6 activity in 3D culture models and hepatocellular carcinoma organoids . a. Dose response to talazoparib and MK-8776 in spheroids formed by HEK293T CYP2D6∗ 1+POR + B5A and CYP2D6∗ 4+POR + B5A cells, or in spheroids formed by HepG2 parental and CYP2D6 KO cells. One representative experiment with three technical replicates is shown (mean ± SD, ∗p < 0.05, ∗∗p < 0.01, unpaired t-test, Two-stage step-up (Benjamini, Krieger, and Yekutieli)). b. Representative images for HEK293T CYP2D6∗ 1+POR + B5A and CYP2D6∗ 4+POR + B5A cells were taken by Incucyte after 72 h incubation with indicated drugs. Diameters were measured and spheroid volumes were calculated using the formula V = (4/3) ∗π ∗Rˆ3, where π is approximately 3.14, and R represents the spheroid radius. One representative experiment with three technical replicates is shown (mean ± SD, two-tailed t-test, only show p-values <0.05). Bar scale = 300 μm. c. Dose response of human HCCOs to talazoparib. HCCO-C975, HCCO-D045, HCCO-D046, HCCO-D324, HCCO-D386, HCCO-D415, and HCCO-D455 were determined as normal metabolizers; HCCO-C798, HCCO-C948, HCCO-C949, and HCCO-D359 were determined as intermediate metabolizers. ∗p < 0.05, using unpaired t test with Welch's correction. d . Targeting CYP2D6 allelic loss in tumors. CYP2D6 is located on chromosome 22 (p-arm, blue; centromere, grey; q-arm, yellow), which is acrocentric (shown by the two black lines). Eligible patients are heterozygous for the wild-type CYP2D6 allele ( CYP2D6∗1 , pink) and the LoF allele ( CYP2D6∗4 , dark grey). Throughout cancer progression, tumor cells undergo LOH and may lose the wild-type CYP2D6 allele. Treatment with a cytotoxic CYP2D6 substrate (pink) will result in selective killing of tumor cells, as they cannot process the drug and die. Normal cells which still express CYP2D6 are able to detoxify the drug and survive.

Article Snippet: Proteins were transferred to nitrocellulose membranes and incubated at room temperature in 5% non-fat dry milk in 1 × PBST for 1 h. Membranes were then incubated overnight at 4 °C with anti-FlAG primary antibody (peptide sequence DYKDDDDK, #F3165, Mouse, Sigma) to detect the overexpressed CYP2D6∗1 (56 kDa) as well as CYP2D6∗4 (∼20 kDa) proteins, anti-CYP2D6 to detect endogenous CYP2D6 (#ab137426), anti-POR (#sc-25270, Mouse, Santa Cruz Biotechnology), anti-CYB5A (#NBP2-49284, Rabbit, NovusBio) and β-Actin (#sc-47778, Mouse, Santa Cruz Biotechnology).

Techniques: Activity Assay, Incubation, Two Tailed Test

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet: Number of CFUs of bacteria per fly infected with Ecc15 (A) and EcN (B). Each invasive organism was screened in axenic flies and gnotobiotic flies colonized with Lp, At, and LpAt. Bacterial load was determined at 3 h, 24 h, and 48 h post-feeding infection. Each point represents bacterial load from an individual fly; bars and error bars represent the median and 95% confidence intervals; limit of detection is 2 × 10 2 CFUs/fly. n = 15 flies per treatment per time point over 3 biological replicates. Statistical significance was determined between flies containing different microbiomes by the Mann-Whitney method. p values are represented as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques: Bacteria, Infection, MANN-WHITNEY

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet: (A) Scheme describing the multi-organism interaction assay procedure. (B) Multi-organism interaction assays display growth effects of gut microbes Lp and At on invasive organisms Pe, Ecc15, and EcN. Microbiome members were grown in perpendicular streaks for 3 days, and invasive organisms were added to the adjacent quadrants and allowed to grow for an additional day. Zones of inhibition are indicated by dashed lines. (C) Co-culture analysis of invasive organisms with microbiome members. Pe, Ecc15, and EcN were grown in mono-culture or in media previously inoculated with Lp, At, or LpAt. Bars and error bars represent the mean concentration of invasive organisms in CFUs/mL ± SEM. N = 3 biological replicates per condition.

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques: Inhibition, Co-Culture Assay, Concentration Assay

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet: (A) Multi-organism interaction assay showing the effects of Lp and At on EcN growth on media containing the pH indicator bromophenol blue. After 3 days of growth, a yellow acidic zone appears around Lp, but it tapers off near the At interaction point. When EcN is added, the zone of inhibition overlaps with the acidic region. (B) pH measurement of microbiome mono-cultures and co-cultures reveals a sharp acidification of culture media by Lp. (C) Density of invasive organisms in media adjusted to pH 4.0 with lactic acid with or without 24 h of prior growth with At. Each bar represents mean CFUs/mL ± SEM of 3 biological replicates. (D) Microbial concentration of invasive organisms in media buffered to pH 6.0 with phosphate buffer with or without 24 h of prior growth with Lp NAB1 . Each bar represents mean CFUs/mL ± SEM for 3 biological replicates. (E) Ratio of Ecc15 microbial load in flies infected on buffered vs. standard fly diets, n = 15 flies per treatment per time point over three biological replicates. Statistical significance was determined for flies of each microbiome status between standard and buffered diets using the Mann-Whitney method. p values are represented as follows: NS, p > 0.05, * p ≤ 0.05, and ** p ≤ 0.01. (F) The pH of the copper cell region of the intestine was approximated by feeding axenic and gnotobiotic flies food soaked with 2% bromophenol blue. Guts were dissected and imaged immediately. A yellow/green color in the copper cell region indicates an acidified environment (pH < 4.6).

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques: Inhibition, Concentration Assay, Infection, MANN-WHITNEY

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet: (A) Multi-organism interaction assay showing the effects of Lp WCFS1 Lp TF103 ( ΔldhL ldhD::cat ) and At on EcN growth. Neither an acidic zone nor a zone of inhibition appears around Lp TF103 . (B) pH measurement of Lp WCFS1 and Lp TF103 cultures. (C) Analysis of the effect of Lp LDH activity on EcN growth levels during co-culture. EcN was grown in mono-culture or in medium previously inoculated with Lp WCFS1 or Lp TF103 . Bars and error bars represent the mean concentration of invasive organisms in CFUs/mL ± SEM. N = 3 biological replicates per condition. (D) Number of CFUs of Ecc15 per fly. Ecc15 was screened in axenic flies and gnotobiotic flies colonized with Lp WCFS1 or Lp TF103 . Bacterial load was determined at 3 h, 24 h, and 48 h post-feeding infection. Each point represents bacterial load from an individual fly; bars and error bars represent the median and 95% confidence intervals; limit of detection is 2 × 10 2 CFU/fly. n = 15 flies per treatment per time point over 3 biological replicates. Statistical significance was determined between flies containing different microbiomes by the Mann-Whitney method. p values are represented as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques: Inhibition, Activity Assay, Co-Culture Assay, Concentration Assay, Infection, MANN-WHITNEY

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet: (A) pH analysis of fly food after 3 days of growth of Lp, At, or 1:1 LpAt, with culture medium added as a control. Bars represent the mean pH of three biological replicates ± SEM. (B) pH analysis of fly food 3 days after the addition of 40 male flies of different microbiome statuses (axenic, Lp, At, LpAt). Bars represent the mean pH ± SEM of three biological replicates. (C) Bacterial load of Pe, Ecc15, and EcN present on fly food initially inoculated with microbiome members Lp, At, or 1:1 LpAt, with culture medium as a control. Each bar represents the mean CFUs/g fly food ± SEM for three biological replicates. (D) Pictorial representation of microbe-microbe interactions between microbiome members and gram-negative invasive bacteria on fly food and during consumption by the host.

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques: Control, Bacteria

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet:

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques:

Journal: Cell reports

Article Title: Microbiome-derived acidity protects against microbial invasion in Drosophila

doi: 10.1016/j.celrep.2024.114087

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Pectobacterium carotovorum pOM1-GFP (Ecc15) (ATCC 25270, pOM1-GFP) , Basset et al., 2003 , N/A.

Techniques: Virus, Recombinant, Lactate Assay, Software

Journal: Scientific reports

Article Title: Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform.

doi: 10.1038/s41598-024-51781-6

Figure Lengend Snippet: Figure 2. Characterization of translationally active CHO lysates derived from genetically modified CHO cells after cell-free synthesis of CYP3A4. CYP3A4 (57 kDa) and CPR (82 kDa) were identified in the translation mixture through SDS-PAGE (10%) and subsequent western blotting with anti CPR antibodies (A) and anti CYP3A4 antibodies (B) followed by a secondary HRP linked antibody. Cell-free synthesis of CYP3A4 was compared to no-template-controls (NTC) in each sample. An autoradiograph (C) allows the detection of 14C-Leucine labeled cell-free synthesized proteins in the cell-free reaction. The activity of CYP3A4 per µg synthesized protein in the different lysates was determined by an IPA-Luciferin assay after CYP3A4 cell-free synthesis. Synthesized protein was quantified through 14C-labeling. Background activity from the NTC was subtracted. Measurements were performed as triplicates (n = 3). Blots and autoradiographs visible in each individual sub-image were created simultaneously and treated equally.

Article Snippet: The primary antibody used for the detection of CPR was “CYPOR (F-10): sc-25270” (Santa Cruz Biotechnology, Dallas, Texas, USA), the primary antibody used for the detection of CYP3A4 was “CYP3A4 (HL3): sc-53850” (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Derivative Assay, Genetically Modified, SDS Page, Western Blot, Autoradiography, Labeling, Synthesized, Activity Assay