Fig. 2: Immune RNA-based synthetic lethality as a mechanism to selectively eliminate target cells, demonstrated with CASP3 overexpression.
a, Left: illustration of the coculture used to test top hits in a different cancer type and with a different TCR and antigen. HPV16+ CaSki cells express the HPV16 oncoprotein E7 and present it on major histocompatibility complex (MHC)-I (HLA-A2 allele), allowing E7 TCR-specific cytotoxicity. Validations of this model are depicted in Extended Data Fig. 3. Right: percentage of surviving CaSki cells (y axis) in coculture with E7 TCR T cells, shown for CaSki cells transduced to express different ORFs (mean ± s.d., n = 6 technical replicates per group). The P value is from two-way ANOVA and Dunnett’s multiple-comparison test. b, Confluency of different A375 (left) and CaSki ORF (right) lines (that is, transduced with different ORFs for constitutive expression of different hits) measured via Incucyte. Cells were monitored for 16 h, with images taken every 1.5 h (mean ± s.d., n = 4–6 technical replicates per group). c, Viability of A375 ORF lines measured via PrestoBlue after 24-h treatment with varying doses (x axis) of IFNγ (left) or TNF (right) (mean ± s.d., n = 2–3 technical replicates per group). d,e, Primary CD8 T cells transduced with lentivirus for ORF-based overexpression of CASP3. CASP3 expression was measured via quantitative PCR (qPCR) and normalized to GAPDH (d, left); cytotoxicity was measured in 24-h coculture with A375 cells at 0.5:1 and 1:1 E-to-T ratio (d, right). Data are presented as mean values (n = 3 technical replicates per group). **P < 0.01, two-tailed Student’s t-test. e, T cell counts over 6 d in monoculture (mean ± s.d., n = 2–3, technical replicates per group). NS, two-way ANOVA. f, Delivery of CASP3 RNA via cationic lipid-based or polymer-based nanoparticle in A375 cancer cell coculture with NY-ESO-1 T cells. CASP3 expression in the A375 cells was measured via qPCR and normalized to GAPDH (left) and cancer cell survival with RNA delivery in coculture versus monoculture measured via PrestoBlue (right). Data are presented as mean values (n = 3 technical replicates per group). ****P < 0.0001, unpaired, two-tailed Student’s t-test. g, Western blot of pro-caspase-3 and cleaved caspase-3 protein levels measured in control and CASP3OE A375 cells that were untreated in monoculture, treated with etoposide (24 h at 2.5 μM) in monoculture or untreated in cocultured with NY-ESO-1 TCR CD8 T cells (1:1 E-to-T ratio, 24 h; Methods). The experiment was conducted once to demonstrate this protein-level property of caspase-3 in accordance with the screen data and other experiments conducted here. h, Proposed model of CASP3 RNA-based synthetic lethality with TCR-specific cytotoxicity. Illustrations in a and h created in BioRender; Jerby Lab https://biorender.com/qdelxmb (2026).
