Abstract
Pyrvinium is a quinoline-derived cyanine dye and an approved anti-helminthic drug reported to inhibit WNT signaling and have anti-proliferative effects in various cancer cell lines. To further understand the mechanism by which pyrvinium is cytotoxic, we conducted a pooled genome-wide CRISPR loss-of-function screen in the human HAP1 cell model. The top drug–gene sensitizer interactions implicated the malate–aspartate and glycerol-3-phosphate shuttles as mediators of cytotoxicity to mitochondrial complex I inhibition including pyrvinium. By contrast, perturbation of the poorly characterized gene C1orf115/RDD1 resulted in strong resistance to the cytotoxic effects of pyrvinium through dysregulation of the major drug efflux pump ABCB1/MDR1. Interestingly, C1orf115/RDD1 was found to physically associate with ABCB1/MDR1 through proximity-labeling experiments and perturbation of C1orf115 led to mis-localization of ABCB1/MDR1. Our results are consistent with a model whereby C1orf115 modulates drug efflux through regulation of the major drug exporter ABCB1/MDR1.
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Data availability
The datasets generated and analyzed during the current study are available in figures included in this article. Source data are provided with this paper.
Code availability
No advanced custom code was used for analyses in this study, and all tools and algorithms used are specified in the Methods.
Change history
24 August 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41589-022-01146-9
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Acknowledgements
We thank the Donnelly sequencing center for assistance with sequencing. We thank the flow cytometry facility at the Temerty Faculty of Medicine for providing the BD LSR-Fortessa cell analyzer to assess mitochondrial membrane potential and drug efflux. We thank the Microscopy Imaging Laboratory at the Temerty faculty of medicine for providing the Zeiss LSM 880 for imaging experiments. We thank the SPARC BioCentre at Sickkids for assistance with mitochondrial stress test experiments. We thank Agios for assistance with metabolite profiling experiments. We thank the NBCC Proteomics Facility for assistance with mass spectrometry. Funds for this study were provided by CIHR to J.M. (PJT-463531) and the Ontario Research Fund RE9 Program to J.M. and C.B. J.M was a Canada Research Chair in Functional Genomics of Cancer and the Anne and Max Tanenbaum Chair in Molecular Medicine in the Temerty Faculty of Medicine at the University of Toronto.
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S.N.M., M.C., M.A., X.Z., P.M., and D.A.P. performed experiments and analyzed data. S.N.M., M.C., D.A.P., O.Z., J.R.M.-B., and G.A.S. helped perform and analyze the metabolomics experiments. G.T. and C.B. performed yeast experiments. M.A., Z.-Y.L., C.J.W., S.N.M., J.M. and A.-C.G. helped perform and analyze BioID experiments. J.M. supervised the project. S.N.M., M.C., P.M. and J.M. made the figures and wrote the manuscript with input from all co-authors.
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J.M. is an advisor and shareholder of Century Therapeutics and Aelian Biotechnology.
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Extended data
Extended Data Fig. 1 Fitness assays reveal a role for mitochondrial respiration following pyrvinium treatment.
(a) The effect of pyrvinium or LGK974 on cell viability at the indicated concentrations after a 5-day treatment in HPAF-II cells cultured in standard cell growth medium. (b) The effect of phenformin on cell viability at the indicated concentrations after a 5-day treatment in HAP1 cells cultured in either glucose or galactose containing medium. The effect of pyrvinium on cell viability at the indicated concentrations after a 5-day treatment in (c) HCT116 or (d) A375 cells cultured in either glucose or galactose containing medium. Viability was measured using Alamar blue staining at the end point. All measurements are relative to untreated cells. All data are represented as means ± SD (n = 3 replicates).
Extended Data Fig. 2 Validation of GOT1 as a sensitizer upon pyrvinium treatment in Cas9-expressing cells.
Cas9 expressing clonal (a) HAP1, (b) HCT116, or (c) A375 cell lines infected with lentiviruses carrying expression cassettes for non-targeting LacZ or different GOT1 gRNAs, as indicated. After puromycin selection, cells were seeded for a proliferation assay and treated with pyrvinium at the indicated doses. Cells were manually counted after 5 days of drug treatment. (d, e, f) cell viability of clonal KO’s of GOT1, PGP, and parental HAP1 cells assessed under pyrvinium treatment in limiting growth conditions. Cells were seeded in (d) minimal medium (MM), (e) MM supplemented with 10 mM aspartate, or (f) MM supplemented with 1 mM alpha-ketobutyrate (AKB). Cell viability was measured using Alamar blue after 5 days of treatement. All measurements are relative to untreated cells and data are represented as means ± SD (n = 3 replicates). (g) HAP1, (h) HCT116, or (i) A375 cell lines infected with lentiviruses expressing both Cas9 and a gRNAs targeting LacZ, the targeting control locus AAVS1, or PGP. After puromycin selection, cells were seeded for a proliferation assay and treated with pyrvinium at the indicated doses. Viability was measured using Alamar blue staining at the end point. All data are represented as means ± SD (n = 3 replicates). (j) Schematic depicting the function of PGP in glycerophospholipid/free fatty (GL/FFA) cycle (left) and as a metabolite repair enzyme in glycolysis (middle), and the pentose phosphate pathway (PPP) (right). Cell viability assay showing the effect of indicated doses of pyrvinium on (k) HAP1 parental, (l) PGPΔ-C1 and (m) PGPΔ-C2 cells after a 4-day pyrvinium treatment in different media conditions supplemented with indicated concentrations of glycerol. Viability was measured using Alamar blue staining. All measurements are relative to untreated cells. All data are represented as means ± SD (n = 3 replicates). (n) Levels of F-1,6-BP measured by LC-MS metabolite profiling of HAP1, PGPΔ, and GOT1Δ cells in the presence or absence of pyrvinium. Two-way ANOVA, **p < 0.001, ***p < 0.0001. Measurements consist of 3 biological replicates. Data represented as relative to untreated HAP1 parental cells as means ± SD. G-6-P, glucose-6-phosphate; F-6-P, frcutose-6-phosphate; F-1,6-BP, fructose-1,6-bisphosphate; F-2,6-BP, fructose-2,6-bisphosphate; G-3-P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetonephosphate; 1,3-BP-glycerate, 1,3-biphosphoglycerate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Glycerol-3-P, glycerol-3-phosphate; 2-P-lactate, 2-phospho-L-lactate; FA-CoA, fatty acyl coenzyme A; GL/FFA, glycerolipid/free fatty acid; G6PD, glucose 6-phosphate dehydrogenase; PFK2, phosphofructokinase 2; 6PGL, 6-phosphoglucolactonase; 6PGDH, 6-phosphogluconate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PK, pyruvate kinase; GK, glycerokinase; Acyl-pase, acylphosphatase.
Extended Data Fig. 3 Validation of PGP as a sensitizer upon pyrvinium treatment in Cas9-expressing cells.
(a) Schematic of different metabolic pathways required for cancer cell growth. Relative abundance of indicated metabolites measured by LC/MS in glycolysis, (b) Essential amino acids, (c) Non-essential amino acids in parental HAP1 cells untreated (black) or treated (gray) with 100 nM pyrvinium for 5 hours. All measurements are relative to untreated parental HAP1 cells. All data are represented as means ± SD (n = 3 replicates). *p < 0.05; two-tailed unpaired t-test. (d) schematic showing the effect of different mitochondrial respiratory complex inhibitors. (e-j) Cell viability assays of parental, PGPΔ and GOT1Δ cells treated with indicated doses of different mitochondrial inhibitors including (e) rotenone, (f) phenformin, (g) metformin, (h) antimycin, (i) sodium azide and (j) oligomycin. Rotenone, metformin and phenformin are complex I inhibitors, antimycin inhibits complex III, sodium azide inhibits complex IV and oligomycin inhibits complex V or ATP synthase. Viability was measured using Alamar Blue staining. All measurements are relative to untreated cells. All data are represented as means ± SD (n = 3 replicates). (k, l) Cell viability assays in HCT116 parental and HCT116 NDI1 overexpressing cells (top panel) across indicated doses of either phenformin or pyrvinium. Cell viability assays in PGPΔ cells, as well as PGPΔ cells expressing NDI1 across indicated doses of phenformin or pyrvinium. All measurements presented as relative to DMSO control assessed by alamar blue (n = 4 biological replicates times 3 technical replicates).
Extended Data Fig. 4 C1orf115 mediates resistance to pyrvinium.
(a) Schematic of C1orf115 protein. (b) Top alteration frequencies of the C1orf115 gene across cancer types retrieved from cBioportal using the TCGA PanCan data. (c) and (d) show Kaplan Meier plots for clear cell renal carcinoma (ccRCC) and ovarian cancer patient outcomes predicted by C1orf115 expression. (e) HAP1-Cas9 cells were transduced with lentiviruses expressing the indicated gRNAs targeting C1orf115. After puromycin selection, cells were seeded for a viability assay and treated with pyrvinium at the indicated doses. Relative abundance of (f) lactate and (g) pyruvate in parental and C1orf115 knockout cells in the presence or absence of 100 nM pyrvinium treatment for 5-hours. (h, i) Clonal C1orf115- KO cell lines were subjected to different doses of (h) phenformin, (i) metformin, and (j) antimycin for 5 days. C1orf115-V5 overexpression did not rescue the effects of antimycin on cell viability. (k) Cell viability of C1orf115-KO cell lines assessed in either glucose or galactose media conditions with pyrvinium treatment. Cell viability was measured using Alamar Blue staining at the end point. All measurements are relative to untreated cells. All data are represented as means ± SD (n = 3 replicates). ∗p < 0.05; two-tailed unpaired t test.
Extended Data Fig. 5 Functional enrichment of BioID data.
(a) Bar plot illustrating the cellular compartments for preys captured by either BioID2 (gray) or mini-Turbo (black) C-terminal tagged C1orf115 HAP1 cells.
Extended Data Fig. 6 ABCC1 localization in relation to C1orf115 expression.
(a) ABCC1 localization (green) and DAP1 visualizing nuclei (blue) across genetic backgrounds (HAP1 parental, C1orf115/RDD1 KO clones C10, C3, C1orf115-V5 overexpression, and ABCC1 KO cells). In contrast to ABCB1 localization, no apparent changes in localization are evident for ABCC1 across C1orf115 genetic backgrounds. (b) FACs results of drug efflux of rhodamine-123, data representative of two independent biological replicates. Light gray bars depict drug load at 4 °C, green bars depict drug efflux at 37 °C. Results illustrate that C1orf115 attenuates drug efflux.
Extended Data Fig. 7 Quantitative measurements of drug efflux.
(a) Gating strategy applied to percentage efflux measurements shown in Fig. 6d. Data is representative of 3 independent biological replicates measuring DiOC(2)3 drug efflux. (b) Barplot representing percent drug efflux of DiOC(2) 3 in either 4 °C or 37 °C conditions across cell lines. (c) Gating strategy for quantifying drug efflux.
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Masud, S.N., Chandrashekhar, M., Aregger, M. et al. Chemical genomics with pyrvinium identifies C1orf115 as a regulator of drug efflux. Nat Chem Biol 18, 1370–1379 (2022). https://doi.org/10.1038/s41589-022-01109-0
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DOI: https://doi.org/10.1038/s41589-022-01109-0
