Abstract B051: Deletion of the RNA-binding protein PCBP2 sensitizes tumor cells to CD4+ T cell killing
Authors: Kropp Korbinian N , Etxeberria Inaki , Chandran Smita S , Yi Fei , Nadler Rebecca , Gormally Michael V , Rahman Jahan , Benbarche Salima , Biswas Jeetayu , Ruzzo Marco V , Garippa Ralph , Abdel-Wahab Omar , Klebanoff Christopher A 
Published: February 2025

Abstract
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. We hypothesized this immune escape mechanism might be overcome by tumor-reactive cytotoxic CD4+ T cells which can directly engage tumor cells that express HLA-II. Unlike HLA-I, the molecular mechanisms governing response and resistance to HLA-II antigen presentation by tumor cells have previously not been studied in a systematic fashion. 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. CD4+ T cell killing was preserved following KO of genes that confer intrinsic resistance to CD8+ T cells, including B2M, TAP1/2, and TAPBP. This confirms that HLA-II restricted TCRs can serve 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 (i.e. sensitization) 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 CD4+ T cell-derived cytokines, including TNF and IFNγ. PCBP2 encodes multiple protein isoforms that differ in the linker region separating two of PCBP2’s three 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 coding exon 8. Only re-expression of short PCBP2 isoforms reverted the cytokine-sensitized phenotype of PCBP2 KO tumor cells. Reflecting this observation, specific deletion of PCBP2 long isoforms using CRISPR/Cas9 KO of PCBP2 exon 8 did not affect cytokine sensitivity. We further found that disabling the RNA binding capacity of the PCBP2 short isoforms’ third KH domain, but not others, blocked protection from cytokine-induced cell death. In vivo, we discovered that adoptive transfer of CD4+ T cells targeting an HLA-II restricted tumor antigen resulted in superior control of PCBP2 KO tumors compared to control tumors. 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.
Citation Format: Korbinian N Kropp, Inaki Etxeberria, Smita S Chandran, Fei Yi, Rebecca Nadler, Michael V Gormally, Jahan Rahman, Salima Benbarche, Jeetayu Biswas, Marco V Ruzzo, Ralph Garippa, Omar Abdel-Wahab, Christopher A Klebanoff. Deletion of the RNA-binding protein PCBP2 sensitizes tumor cells to CD4+ T cell killing [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B051.