(649) Functional analysis of Trikafta-refractory Class II mutations by yeast phenomic profiling

Background: Like F508del-CFTR, most CFTR Class II mutations have been approved for treatment with Trikafta. FDA approval for Trikafta treatment is based on studies in cell models demonstrating improved processing and function, presumably due to improved folding. However, several Class II mutations have not been approved for Trikafta therapy. Thus, an important goal of PTAC is to understand the molecular basis underlying this differential therapeutic response. We are investigating this question using S. cerevisiae as a genetic model to obtain relatively comprehensive and quantitative profiles of genetic factors to discern functional clues regarding differential biogenesis of F508del and CF-causing mutations that are refractory to Trikafta treatment.

Methods: Our research group has developed yeast phenomic methods to measure gene-gene interaction effects that modify cell proliferation phenotypes. To model molecular defects underlying cystic fibrosis, we have used the closest yeast homolog to CFTR, YOR1. Yor1 protein has CFTR-like topology, with two 6TM domains, and two NBDs, and functions as a pump that extrudes oligomycin extracellularly from the cytoplasm. For phenomic analysis, haploid S. cerevisiae strains, harboring Yor1 mutations analogous to CF-causing alleles of CFTR, are mated with the haploid genomic collection of yeast gene deletion strains to obtain haploid double mutants by the synthetic genetic array method. With these derived libraries, mutation-specific profiles of Yor1 genetic modifiers of oligomycin resistance are obtained by high throughput phenotyping. For the phenotypic profiling, we use custom growth curve technology, quantitative high throughput cell array phenotyping (Q-HTCP) to quantify differential Yor1 function and thus to discover evolutionarily conserved genetic, molecular, and cellular pathways predicted to influence analogous CFTR mutations in CF cell models. CRISPR genome editing was used to construct the relevant YOR1 mutant alleles analogous to F508del (F670del-Yor1) and some not approved for Trikafta: N1303K (N1307K-Yor1), R560T (R722T-Yor1 ), R560K (R722K-Yor1), and A559T (A721T-Yor1).

Results: A background strain, constructed by chromosomal integration to permit Tetracycline-repressible (Tet-OFF) Yor1 expression, was confirmed phenotypically by titratable oligomycin sensitivity with addition of doxycycline. In the Tet-OFF-Yor1 strain, the F670del mutation was introduce by CRISPR editing, followed by introduction of a R116T missense mutation. Similarly, an N1307K allele was constructed. In the absence of doxycycline, the F670del/R1116T strain has been indistinguishable from the yor1∆0 (complete ORF knockout) control. This was unexpected based on our prior studies, where addition of only 2-10 ng/ml doxycycline was resulted in oligomycin sensitivity. This finding is being clarified by sequencing the entire gene. The N1307K allele, with addition of .1 ug/mL doxycycline, showed intermediate oligomycin sensitivity relative to the Yor1 reference and yor1∆0 controls, consistent with partial disruption of N1303K-CFTR function. The other alleles above are being made presently. Construction of the respective double-mutant libraries will proceed with completion and validation of the mutants.

Conclusions: Results from ongoing yeast phenomic profiling studies to functionally distinguish F508del from alleles refractory to Trikafta therapy will be presented.

Acknowledgements: CFF HARTMA21

NIH R01 HL136414

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