321 Genome-scale CRISPR KO screen identifies PCBP2 as a tumor-intrinsic regulator of cancer cell sensitivity to human CD4 + T cell killing
Year: 2026
Type: conference-abstract
Abstract: Background A major roadblock preventing durable responses to CD8 + T cell-based immunotherapies in melanoma and other solid cancers is loss or downregulation of human leukocyte antigen class I (HLA-I) on tumor cells. However, a subset of solid cancers aberrantly express HLA-II, potentially enabling HLA-II restricted tumor-specific CD4+ T cells to overcome HLA-I resistance. Unlike HLA-I, the mechanisms governing response and resistance to HLA-II antigen presentation by tumor cells have previously not been studied in a systematic fashion.Methods We performed a genome-scale CRISPR knockout (KO) screen to interrogate the molecular mechanisms of direct CD4 + T cell engagement of tumor cells. Using the HLA-II+/HLA-I+ melanoma cell line A375, we identified genes that either confer sensitivity or resistance to primary human CD4+ T cells expressing an HLA-II restricted TCR.Results Tumor killing was preserved upon KO of genes relevant for HLA-I antigen presentation, including B2M, TAP1/2, and TAPBP. This highlights that HLA-II restricted TCRs can function as an orthogonal strategy to overcome CD8+ T cell resistance. We further discovered that KO of the RNA-binding protein PCBP2 led to enhanced depletion under CD4+ T cell selection. We established the generalizability of this finding through focused CRISPR KO screens in n = 3 unique HLA-II+ cell lines using distinct guide RNA sequences and a different HLA-II restricted TCR. Mechanistically, we found that deletion of PCBP2 sensitized tumor cells to apoptosis induced by the CD4+ T cell-derived cytokines TNF and IFNγ. PCBP2 encodes multiple protein isoforms that differ in a linker region separating two RNA binding KH domains. In genetic complementation experiments, we discovered that these isoforms can be functionally grouped by inclusion (i.e. PCBP2 long) and exclusion (i.e. PCBP2 short) of exon 8. Only re-expression of PCBP2 short reverted the cytokine-sensitized phenotype of PCBP2 KO tumor cells. Integration of RNA-seq, proteomics, and isoform-specific eCLIP experiments revealed that the core spliceosome component U2AF2 is a PCBP2 short-selective target that is downregulated in PCBP2 KO cells. Re-expression of U2AF2 largely phenocopied complementation of PCBP2 short and protected PCBP2 KO tumor cells from cytokine-induced cell death.Conclusions In conclusion, we establish that CD4 + T cells can be employed to overcome acquired resistance to CD8+ T cell killing and describe a previously unknown isoform-specific role of the RNA-binding protein PCBP2 in regulating antitumor immunity. These findings establish a novel, mechanism-based immunotherapeutic approach to augment the antitumor efficacy of HLA-II restricted CD4+ T cells.
Authors: Korbinian Kropp, Jeetayu Biswas, Inaki Etxeberria, Paul Chapman, Smita S Chandran, Rebecca Nadler, Michael Gormally, John Klement, Jahan A Rahman, Michael Mu, Salima Benbarche, Fei Yi, Marco Ruzzo, Lauren B Banks, Ralph Garippa, Clemens Hinterleitner, Cecilia Lezcano, Alexander N Shoushtari, Benjamin Izar, Michael A Postow, Omar Abdel-Wahab, Christopher A Klebanoff
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225141044