Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma.

BACKGROUND: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.

Efficient prioritization of CRISPR screen hits by accounting for targeting efficiency of guide RNA.

BACKGROUND: CRISPR-based screens are revolutionizing drug discovery as tools to identify genes whose ablation induces a phenotype of interest. For instance, CRISPR-Cas9 screening has been successfully used to identify novel therapeutic targets in cancer where disruption of genes leads to decreased viability of malignant cells. However, low-activity guide RNAs may give rise to variable changes in phenotype, preventing easy identification of hits and leading to false negative results. Therefore, correcting the effects of bias due to differences in guide RNA efficiency in CRISPR screening data can improve the efficiency of prioritizing hits for further validation. Here, we developed an approach to identify hits from negative CRISPR screens by correcting the fold changes (FC) in gRNA frequency by the actual, observed frequency of indel mutations generated by gRNA. RESULTS: Each gRNA was coupled with the "reporter sequence" that can be targeted by the same gRNA so that the frequency of mutations in the reporter sequence can be used as a proxy for the endogenous target gene. The measured gRNA activity was used to correct the FC. We identified indel generation efficiency as the dominant factor contributing significant bias to screening results, and our method significantly removed such bias and was better at identifying essential genes when compared to conventional fold change analysis. We successfully applied our gRNA activity data to previously published gRNA screening data, and identified novel genes whose ablation could synergize with vemurafenib in the A375 melanoma cell line. Our method identified nicotinamide N-methyltransferase, lactate dehydrogenase B, and polypyrimidine tract-binding protein 1 as synergistic targets whose ablation sensitized A375 cells to vemurafenib. CONCLUSIONS: We identified the variations in target cleavage efficiency, even in optimized sgRNA libraries, that pose a strong bias in phenotype and developed an analysis method that corrects phenotype score by the measured differences in the targeting efficiency among sgRNAs. Collectively, we expect that our new analysis method will more accurately identify genes that confer the phenotype of interest.