Fig 2: Multiple CRISPR knockout screens identify nucleotide metabolism genes that are selectively depleted in shp16/shCdkn2a cells.(A) Schematic of our CRISPR screens. Isogenic p16/Cdkn2a wildtype cells were infected with lentiviruses expression shGFP control (shCont) or shp16 (human) or shCdkn2a (mouse). These cells were infected with the CRISPR gRNA libraries at an MOI of 0.2–0.3. After 14 days in culture, gDNA was harvested and sequenced. (B-C) Analysis of 128 genes related to nucleotide synthesis, salvage, and catabolism identified multiple genes that are negatively selected in shp16/shCdkn2a vs. shControl in human SKMEL28 (B) and mouse Yumm5.2 (C) melanoma cells.

Fig 3: p16/CDKN2A negatively correlates with mRNA and protein expression of multiple nucleotide metabolism genes.(A-B) SKMEL28 human melanoma cells were infected with lentivirus expressing a short hairpin RNA (shRNA) targeting p16 (shp16). shGFP was used as a control (shCont). (A) Expression of the 128 nucleotide metabolism genes from RNA-Seq. (B) Polysome fractionation was performed and both heavy (>2 ribosomes) and total mRNA were sequenced, and the ratio of Heavy to Total was used to assess transcripts with increased translation. (C) Genes that are transcriptionally or translationally upregulated in shp16 SKMEL28 cells. (D-E) DepMap data of cutaneous melanoma cell lines. (D) mRNA expression of 31 genes identified in the CRISPR screens. (E) Protein expression of 31 genes identified in the CRISPR screens.

Fig 4: Multiple anti-folates induce apoptosis in p16 knockdown cells.(A-B) SKMEL28 human melanoma cells were infected with lentivirus expressing a short hairpin RNA (shRNA) targeting p16 (shp16). shGFP was used as a control (shCont). (A) Cells were treated with the indicated inhibitors and cytotoxicity was assessed using IncuCyte Cytotox Green reagent. (B) Cells were treated with the indicated inhibitors, and apoptosis was assessed using Annexin V/PI staining by flow cytometry.