Genome-wide CRISPR-Cas9 screening identified GPCR regulators RIC8A and PDCL as therapeutic targets in MLN with PDGFRB fusions Free

Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK) are rare hematologic malignancies characterized by constitutive activation of tyrosine kinase (TK) signaling. Among them, platelet-derived growth factor receptor β (PDGFRB) fusion-positive MLN (MLN-PDGFRB) typically responds well to tyrosine kinase inhibitors (TKIs) in the chronic phase (CP); however, treatment options for the blast phase (BP) remain limited, and overcoming TKI resistance in BP requires identification of other key genetic drivers. To identify novel therapeutic vulnerabilities in BP MLN-PDGFRB, we performed genome-wide CRISPR-Cas9 knockout screening using a murine Ba/F3 cell model expressing EBF1-PDGFRB.

Ba/F3 cells stably expressing EBF1-PDGFRB and Cas9 were generated and confirmed to proliferate in an IL-3 independent manner. Genome-wide CRISPR-Cas9 screening was performed using the GeCKO library, with cells split into IL-3(+) and IL-3(−) conditions for 14 days. sgRNAs depleted under IL-3(−) but not IL-3(+) conditions were prioritized as candidate dependencies specific to EBF1-PDGFRB-driven signaling. These candidate genes were further validated via targeted knockout and functional assays. Pathway enrichment analysis was also performed using MAGeCKFlute and MAGeCK pipelines.

Among the top-ranked hits under IL-3(−) condition, RIC8 guanine nucleotide exchange factor A (Ric8a) and phosducin-like (Pdcl), both of which were positive regulators of G-protein-coupled receptor (GPCR) signaling, were identified as essential genes for the survival of Ba/F3-EBF1-PDGFRB cells. Targeted knockout of either Ric8a or Pdcl significantly impaired cell proliferation only in IL-3(−) conditions. Moreover, knockout of either gene individually led to a synergistic enhancement of TKI-mediated growth inhibition. Notably, pathway enrichment analysis suggested partial reliance on the mechanistic target of rapamycin (mTOR) pathway; however, pharmacologic inhibitors targeting mTOR failed to show selective efficacy. These results suggest that multiple signaling pathways, including GPCR and mTOR, may cooperatively sustain PDGFRB-mediated oncogenesis in a complementary manner.

Importantly, neither RIC8A nor PDCL have been previously implicated in MLN-PDGFRB pathobiology, and their identification was unexpected given the upstream location of PDGFRB within RTK signaling. Functional crosstalk between RTKs and GPCRs may underlie this dependency. These results are consistent with previous reports in EGFR-mutated lung cancer, where RIC8A knockout enhances TKI sensitivity through modulation of the Hippo-YAP pathway. While Ric8a or Pdcl knockout in Ba/F3 cells expressing other RTK fusion oncogenes such as FGFR1 or PDGFRA did not result in comparable effects on cell proliferation or TKI sensitivity, similar growth-inhibitory effects were observed in Ba/F3 cells expressing another PDGFRB fusion, AGGF1-PDGFRB. These findings suggest that the observed dependency may be shared across multiple PDGFRB fusion variants, but not across other types of RTK-driven MLN-TK.

This study demonstrates the feasibility and utility of genome-wide CRISPR-Cas9 screening in Ba/F3-MLN model. We identified RIC8A and PDCL as novel therapeutic targets in MLN-PDGFRB, potentially mediating crosstalk between RTK and GPCR signaling. Further in vivo validation and extension to other PDGFRB fusion partners are warranted to assess their translational relevance.