KE-39

Identifying Upstream and Downstream Genes in KRAS-Amp Cancer Cell Lines

Gastroesophageal adenocarcinomas (GEAs) harbor recurrent amplification of KRAS, leading to marked overexpression of WT KRAS protein. SHP2 phosphatase, which acts to promote KRAS and downstream MAPK pathway activation, is a target in these tumors when combined with MEK inhibition. A custom, focused, secondary screening, CRISPR/Cas9 sgRNA library, was designed. In addition to those targeting genes of interest (Fig. 1A), 3 KRAS-amp cancer cell lines were screened, including 2 cancer cell lines (KE-39 and YCC-1) and 1 patient-derived cell line (CAT12) (Fig.1B-D).

The KE-39 cell line was first evaluated. BRAF, CRAF (RAF1), and SHOC2 were among the top depleted genes (denoting sensitization to SHP2i), and NF1 and PTEN were among the top enriched hits (denoting resistance). The composite group of KRAS-amp models was then evaluated from the secondary screen. Again, NF1 was the top enriched gene when silenced. On average, the top 10 depleted genes were SHOC2, RIC1, GAREM1, CRKL, RAPGEF1, RUNX1, BRAF, TCF7L2, CHD2, and VPS16 in the KRAS-amp cells. These included genes related to the MAPK pathway but also others, including RIC1, GAREM1, CRKL, RUNX1, TCF7L2, CHD2, and VPS16.

Fig. 1 Both genes upstream and downstream of KRAS are potential targets to enhance SHP2i efficacy. (Li T, et al., 2023)

Correlations Between Ploidy Defects and DDR Pathway Activity in Gastric Cancer Cell Lines

Five gastric cancer cell lines (AGS, HGC27, HUGC3, GSU, and KE39) that showed varying levels of aneuploidy (Fig. 2A) and one non-neoplastic diploid cell line (RPE1) were evaluated. Levels of pH2AX and pKAP1 were determined in these cell lines to assess intrinsic DNA DSBs/replication stress. In response to DNA damage and replication stress, KAP1 is phosphorylated at serine 473. As expected, RPE1 cells showed minimal staining for pH2AX and pKAP1. Similarly, low levels of pH2AX and pKAP1 were observed in AGS, a TP53 WT gastric cancer cell line displaying the least aneuploidy defects. In contrast, strong and moderate pH2AX and pKAP1 staining in high (KE39 and GSU) and intermediate (HGC27 and NUGC3) aneuploidy cell lines, respectively (Fig. 2B). Importantly, since dsDNA breaks correlate with DDR response, these markers tracked with one another. As such, pH2AX and pKAP1 levels positively correlated with the degree of various ploidy defect measurements and p53 mutation status (Fig. 2C). GSU and KE39 not only demonstrated greater baseline levels of DNA DSB and replication stress compared to RPE1 and AGS measured by pH2AX and pKAP1 but also showed a more dramatic, dose-dependent induction when treated with prexasertib, a well-known Chk1/2 inhibitor (Fig. 2D-2F).

Fig. 2 Aneuploidy correlates with DDR pathway activity in gastric cancer. (Scarpa ES, et al., 2020)