Abstract P1-05-02: CRISPR/Cas9-guided editing of spliceosome factors enhances major histocompatibility complex proteins in triple-negative breast cancer
Authors: Bustos MA , Orozco JIJ , Salomon MP , Hoon DSB , Marzese DM 
Published: February 2018

Triple-negative breast cancer (TNBC) exhibits an extraordinary plasticity allowing adaptation to unfamiliar microenvironments and survival despite hindrances imposed by aggressive therapeutic approaches and the immune system. Alternative mRNA splicing (AS), catalyzed by spliceosome factors (SFs), is a major source of transcriptional diversity and phenotypic plasticity for normal and cancer cells. We ascertained that patients with TNBC present up-regulation of specific subsets of SFs mainly involving the epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) and the polypyrimidine tract binding proteins (PTBP1/2). Methods and Results: Through the integration of in-house and publicly-available gene expression profiles (n=890), we evaluated the correlation between the levels of 290 SFs in patients with TNBC. This analysis identified nine subsets of TNBC based on SFs expression profiles with diverse clinical and pathological characteristics. These findings were further validated using data generated by the TCGA-BRCA (n=1,105) and METABRIC projects (n=2,509). Interestingly, up-regulation of PTBP1 was significantly associated with a shorter relapse-free survival interval for patients with TNBC (n=305; HR=1.58 (1.07 − 2.33); p-value=0.02). To systematically identify PTBP1-regulated AS events, we generated and clonally selected CRISPR/Cas9-guided PTBP1 knock-out (KO) TNBC cell lines. Analysis of our RNA-sequencing data at the gene level revealed a significant enrichment of inflammatory response and antigen presentation pathways. Evaluation of potential upstream transcriptional regulators for the enriched molecular pathways and predicted PTBP1 targets identified SMARCA4, a member of the SWI/SNF chromatin remodeling complex, to be significantly activated after PTBP1KO (q-value&lt;0.01). Integration of ENCODE ChIP-sequencing and JASPAR transcription factor binding profile databases revealed clusters of SMARCA4 binding sites upstream of several members of the major histocompatibility (MHC) class I and MHC class II genes. Mechanistically, we identified that PTBP1 induces SMARCA4 exon 30 retention leading to the full length transcript variant 1, which has lower affinity for HLA gene promoter regions. Significant up-regulation of HLA-A, HLA-B, HLA-DPA1, and HLA-DRA genes in CRIPSR-guided PTBP1KO TNBC cells was further demonstrated by either western blot or indirect immunofluorescence. Finally, a negative correlation between PTBP1 and HLA genes expression was also identified in multiple breast cancer gene expression datasets. Conclusions: This study suggests that alterations in the PTBP1-associated splicing programming lead to a reduction of the antigen presentation capability of TNBC cells. Due to the limited therapeutic alternatives for patients with TNBC, beyond chemotherapy, further understanding and modulation of this novel alteration may expand the applications of immunotherapy for patients with TNBC.

Citation Format: Bustos MA, Orozco JIJ, Salomon MP, Hoon DSB, Marzese DM. CRISPR/Cas9-guided editing of spliceosome factors enhances major histocompatibility complex proteins in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-02.