TLSB-09 TARGETED P16 KNOCKOUT CONFERS AN IMMUNOSUPPRESSIVE MICROENVIRONMENT IN PRECLINICAL MODELS OF TRIPLE NEGATIVE BREAST CANCER BRAIN METASTASIS
Abstract Loss of the 9p21 chromosomal locus, which contains CDKN2A, has been associated with non-response to immune checkpoint inhibition (ICI) across multiple human tumor histologies, yet the underlying mechanisms remain elusive. Given the high frequency of CDK pathway alterations in human brain metastases (BM), especially those arising from triple negative breast cancer (TNBC), we sought to explore the relationship between loss of p16, a CDKN2A isoform, and ICI resistance associated with 9p21 loss. We used CRISPR-Cas9 to generate an isogenic p16-knockout derivative (EMT6 sgRNA3) from ICI-sensitive murine TNBC cell line EMT6, as well as a non-targeting control (EMT6 sgNT). With these cell lines, we established an in vivo model in BALB/c mice to assess intracranial response to anti-PD-1 treatment. Preliminary results revealed a trend toward diminished intracranial ICI efficacy in mice bearing EMT6 sgRNA3 tumors compared to EMT6 sgNT controls (n = 10/group, p=0.08). To validate this finding, we generated ex vivo organotypic spheroids (OTS) from intracranial tumors at survival endpoint and assessed anti-PD-1-induced cell death by lactate dehydrogenase release. Compared to IgG control (10 μg/mL), anti-PD-1 (10 μg/mL) treatment produced a significant increase in cytotoxicity in EMT6 sgNT OTS but not EMT6 sgRNA3 OTS (p=0.001 vs. p=0.76, respectively) after 72 hours. To interrogate the mechanistic basis of these findings, we performed bulk RNAseq on intracranial tumors (n = 4/group), which revealed a significant decrease in expression of chemokines (Cxcl1, Cxcl10, Cxcl11) and immune-related genes (Cd274, Vcam1, Il2ra) in EMT6 sgRNA3 tumors, suggesting that p16 loss is associated with an immunosuppressive microenvironment characterized by decreased innate immune signaling and immune cell recruitment. These findings suggest a role for p16 loss as a candidate functional driver of ICI resistance associated with 9p21 loss in TNBC-BM and warrant further investigation given the need to nominate biomarkers for ICI response to inform patient care.