CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A

Abstract

Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.

Keywords: CRISPR/Cas9; chemical biology; eIF4A; rocaglates; translational control.

Figure 1. Generation and Characterization of a Rocaglate-Resistant eIF4A1 Allele

(A) Schematic illustrating conserved motifs of the DEAD-box helicase family. Sequences of the conserved motifs are denoted with motifs involved in RNA binding highlighted in bold red. The structure of eIF4A indicates it to be dumbbell in shape with two domains (I and II) linked via a flexible linker sequence. The inset shows a ribbon diagram of eIF4A1 (PDB 2ZU6) aligned to eIF4A3 (not shown; PDB 2HYI). The residues targeted for mutagenesis are highlighted and the single-stranded RNA substrate (positioned relative to the eIF4A3 crystal structure) is shown in orange. (B) Coomassie stain of purified recombinant eIF4A1 proteins. (C) Assessment of ATP hydrolysis by recombinant proteins via thin layer chromatography. (D) Kinetics of ATP hydrolysis by eIF4A1 and eIF4A1(F163L). ATPase assays were performed with 1 µg protein and varying ATP concentrations. Graph represents the Michaelis-Menten fit from two independent experiments. (E) RNA binding activity of eIF4A1 and eIF4A1(F163L) using [³²P]-labeled RNA generated from pSP/CAT (see Supplemental Experimental Procedures). Assays were performed in the presence of 0.5% DMSO or 1 µM silvestrol and the retained eIF4A:RNA complexes are set relative to DMSO controls. n = 3 biological replicates performed in triplicate ±SEM; *p < 0.001. (F) Quantitation of eIF4A1 and eIF4A1(F163L) helicase activity performed with 0.5 µg recombinant eIF4A1 and an 11-nt radiolabeled RNA duplex in the presence of DMSO or 50 µM silvestrol. n = 3 biological replicates ±SEM; *p < 0.05. (G) DSF analysis of eIF4A1 or eIF4A1(F163L) in the presence of DMSO or (−)-SDS-1-021.

Figure 2. Ectopic Expression of eIF4A1(F163L) Confers Resistance to Rocaglates in Mammalian Cells

(A) Schematic diagram of RCV designed to simultaneously express an shRNA-resistant His₆-eIF4A1 allele while suppressing endogenous eIF4A1. (B) Representative western blot of NIH/3T3 cells transduced with RCVs. The dashed line separates the two sets of western blots. (C) Viability assay of RCV-transduced NIH/3T3 cells. Cells were exposed to the indicated concentrations of silvestrol and relative viability was assessed 6 days later by sulforhodamine B (SRB). n = 2 biological replicates performed in duplicates ±SEM. (D) Competition assay of transduced NIH/3T3 cells. Transduced cells (GFP⁺) were mixed with parental cells (GFP⁻) and cultured in the presence of 20 nM silvestrol. The percentage of GFP⁺ cells was determined on the indicated days. n = 2 biological replicates performed in triplicate ±SEM. (E) Cells expressing eIF4A1(F163L) are resistant to translation inhibition by silvestrol. Transduced cells were incubated with the indicated concentrations of silvestrol for 1 hr and labeled with [³⁵S]-methionine/cysteine during the last 15 min. n = 4 biological replicates ±SEM. (F) Polysome profiles of transduced NIH/3T3 cells following exposure to 200 nM silvestrol for 30 min. P/M represents the polysome/monosome ratio. n = 3 biological replicates ±SEM. See also Figure S1.

Figure 3. Cas9-Mediated Editing of Eif4a1

(A) Strategy for introducing the Eif4a1(F163L) mutant allele. The sequence of two sgRNAs targeting exon 5 and the partial sequence of the ssODN donor are shown. The PAMs are shaded, and the nucleotide changes in the ssODN donor that abolishes their presence are indicated in red. The targeted TTT (F) codon is indicated by a dashed orange box, and engineered CTC (L) change in the ssODN donor is indicated in green. (B) Sequence analysis of the PCR products from eIF4A1^em1JP and eIF4A1^em2JP cells indicating loss of the wild-type Eif4a1 allele and composition of mutant alleles. (C) Relative translation rates in Rosa26^em1JP, eIF4A1^em1JP, eIF4A1^em2JP cells transduced with the indicated retroviruses. (D) Western blot assessing His₆-eIF4A1 and total eIF4A1 in the cell lines used in (C). (E) CETSA of Rosa26^em1JP and eIF4A1^em1JP cells. Cells were incubated with 1 µM (−)-SDS-1-021 or DMSO for 1 hr at 37°C and heated at the indicated temperatures for 3 min. Soluble lysates were prepared and used for western blotting. n = 4 biological replicates ±SEM. See also Figure S2.

Figure 4. Cas9-Mediated Editing of the Eif4a1 Locus Confirms the Drug-Target Hypothesis

(A) Colony formation assay of Myr-Akt-transformed Rosa26^em1JP or eIF4A1^em1JP cells in the presence of silvestrol. (B) Response of Myr-Akt-transformed Rosa26^em1JP or eIF4A1^em1JP xenografts in vivo to silvestrol. On the indicated days, mice were treated with silvestrol (0.2 mg/kg) following tumor appearance. n = 6–7 mice/cohort ±SEM. (C) Bar graph of the percentage of apoptotic nuclei from tumor sections. Three hours before harvesting of tumors, mice were treated with vehicle or 0.2 mg/kg silvestrol. n = 2 biological replicates (with ~7,000 nuclei analyzed per tumor) ±SD; *p < 0.05; ns, not significant. (D) Polysome profiles of Myr-Akt-transformed Rosa26^em1JP or eIF4A1^em1JP cells exposed to 10 nM silvestrol for 1 hr. (E) Distribution of mRNAs in polysome fractions shown in (D). See also Figures S3 and S4.