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. 2023 Nov 16;30(11):1453-1467.e8.
doi: 10.1016/j.chembiol.2023.07.017. Epub 2023 Aug 21.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules

Thu P Nguyen  1 , Min Fang  2 , Jiwoong Kim  3 , Baiyun Wang  1 , Elisa Lin  1 , Vishal Khivansara  1 , Neha Barrows  1 , Giomar Rivera-Cancel  1 , Maria Goralski  1 , Christopher L Cervantes  1 , Shanhai Xie  1 , Johann M Peterson  1 , Juan Manuel Povedano  4 , Monika I Antczak  2 , Bruce A Posner  5 , Colin J B Harvey  6 , Brian T Naughton  6 , David G McFadden  7 , Joseph M Ready  5 , Jef K De Brabander  8 , Deepak Nijhawan  9
Affiliations

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules

Thu P Nguyen et al. Cell Chem Biol. .

Abstract

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Keywords: Forward Genetics; High throughput screens; Mechanism of Action; Molecular Glue; Target Identification; Targeted protein degradation.

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Conflict of interest statement

Declaration of interests C.J.B.H. and B.T.N. are employees and own equity in Hexagon Bio. D.N. is a consultant and owns equity in Hexagon Bio.

Figures

Figure 1.
Figure 1.. Engineering inducible forward genetics system for compound resistance mutation discovery.
(A) Western blot of lysates derived from Hela, HCT116, and HCT116-MLH1. (B, E) Cell viability. Outliers (clones) defined as median z-Score >15 (dashed line). (C) Western blot of lysates derived from HCT116 and iHCT116 treated with 500 μM indole-3-acetic acid (IAA). (D) Inducible forward genetics system screening schematic. Asterisk (*) denotes non-specific band.
Figure 2.
Figure 2.. Mutation in tubulin renders cells resistant to PVHD303.
(A) PVHD303. (B) Cell viability. Outliers (clones) defined as median z-Score >10 (dashed line) (C) Whole-exome sequencing (WES) analysis results for PVHD303 resistant clones. (D) Viability assay of A673WT and A673TUBB4B T238A cells treated with PVHD303 in dose-response. Points and error bars represent mean +/− SEM of two technical replicates.
Figure 3.
Figure 3.. Mutations in PARP1 render cells resistant to AZ9482.
(A) AZ9482. (B) Cell viability. Outliers (clones) defined as median z-Score >5 (dashed line). (C) WES analysis results for AZ9482 resistant clones. (D) Y896 and A898 (red) residues are mapped on PARP-1 (gray) co-crystal structure with Talazoparib (green) (PDB:4UND). (E) Western blot of lysates derived from iHCT116 and AZ9482 resistant clones. (F) Crystal violet stain of transfected HCT116 cells following 350 nM AZ9482 treatment. (G) Viability assay of HCT116 harboring non-target guide or PARP1 KO clones.
Figure 4.
Figure 4.. Mutations in ATP6V1B2 render cells resistant to destruxin B and E.
(A) Destruxin B and destruxin E. (B) Cell viability. Outliers (clones) defined as median z-Score >10 (dashed line). (C) WES analysis results for Destruxin E resistant clones. (D) Compound resistant mutations are mapped to v-ATPase crystal structure (PDB: 6WM2). Gray is ATP6V1E1, yellow is ATP6V1B2. The mutated residues are highlighted in blue. (E) Viability assay of HCT116WT and HCT116ATP6V1B2 D359N treated with destruxin B, destruxin E, or Bafilomycin A1 in dose-response. Points and error bars represent mean +/− SEM of two technical replicates. (F) Confocal images of HCT116WT and HCT116ATP6V1B2 D359N treated with bafilomycin A1 or destruxin E. Nuclei are blue. Lysotracker is red. Bar-scale represents 22 μm.
Figure 5.
Figure 5.. Forward genetics reveals multiple chemical scaffolds targeting Nicotinamide phosphoribosyltransferase (NAMPT).
(A) MM201, MM202, and MM204. (B) Cell viability. Outliers (clones) defined as median z-Score >10 (dashed line). (C) WES analysis results for MM201, MM202, and MM204 resistant clones. (D, E) Compound resistant mutations (red or orange) are mapped to NAMPT co-crystal structure with FK-866 (green) (PDB: 2GVJ). Gray and yellow are 2 chains forming the homodimer. G383GGLLQ388 helical motif is blue. (F) Summary of all clone sensitivity against MM201, 202, and 204. Values reported are fold-resistance in comparison to parental iHCT116. (G) FK-866. (H) Viability assay of HCT-116, clone 4MM201, and clone 5MM204 treated with FK-866 in dose-response. Points and error bars represent mean +/− SEM of two technical replicates (I) in vitro NAMPT activity assay in the presence of MM201, MM202, or MM204 in dose-response. Points and error bars represent mean +/− SEM of three technical replicates.
Figure 6.
Figure 6.. Loss-of-function mutations in DDB1 render cells resistant to SW394703.
(A) SW394703 and SW394704. (B) Cell viability. Outliers (clones) defined as median z-Score >4 (dashed line). (C) WES analysis results for SW394703 resistant clones. (D) Western blot of lysates derived from iHCT116 and SW394703 resistant clones. (E, F) Western blot of lysates derived from iHCT116 treated with SW394703 or SW39704 in the absence or presence of MLN4924 or Bortezomib pretreatment. (G) Immunoprecipitation and western blot of lysates derived from vector or 3xFlag-CDK12 expressing HCT116, in the presence or absence of SW394703.
Figure 7.
Figure 7.. Engineering inducible mutagenesis system in mismatch repair proficient cell line.
(A) Western blot of lysates derived from S462 and iS462 treated with 500 μM IAA. (B) Cell viability. Outliers (clones) defined as median z-Score >15 (dashed line).
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