Abstract LB288: Reducing the effect of cellular stress in murine oral tumors with pharmacological blockade of β-catenin/CBP activity
Authors: Khan Mohammed Muzamil , Reed Eric , Kroehling Lina , Nomoto Kenichi , Matsui Junji , Bais Manish , Varelas Xaralabos , Kukuruzinska Maria , Monti Stefano 
Published: April 2024

Oral squamous cell carcinoma (OSCC), a primary subtype of head and neck squamous cell carcinoma (HNSCC), is a complex malignancy comprising multiple anatomical sites. OSCC ranks among the deadliest cancers globally, with a 5-year survival rate of ~65%. In previous studies, we have shown that pharmacological blockade of Wnt/β-catenin/CBP activity with small molecule inhibitors effectively abolished oncogenic cell phenotypes in OSCC. However, the underlying mechanisms promoting changes in OSCC cell identities remain unknown. To address this knowledge gap, we used an immunocompetent mouse model of OSCC induced by a tobacco-derived carcinogen, 4-nitroquinoline-1-oxide (4NQO), to interrogate cell states by single-cell RNA sequencing (scRNAseq) of tongue tissues from healthy mice (n=2), 4NQO-derived mouse tongue OSCC (n=2), and from 4NQO-derived mouse tongue tissues treated with an inhibitor of β-catenin/CBP (n=4), and generated a high-quality dataset comprising ~50K cells across all conditions. We performed multiple analyses of the generated data to catalogue the cell type repertoire and its changes among conditions. We observed significant changes in cellular composition between the 4NQO-induced and inhibitor-treated groups. The proportion of epithelial cells decreased upon treatment consistent with greatly diminished tumor volumes, while endothelial and fibroblast populations increased compared to the 4NQO control group. Epithelial sub-typing using known markers revealed a decrease in basal cancer stem-like cells (Krt5+, Krt14+) concomitant with an increase in cycling cells (Top2a+, Cdc20+). In addition, we identified a decrease in a stress cell phenotype associated with the AP-1 complex (Jun+, Fos+). The latter subgroup exhibited a positive enrichment for the “stress” module (n=100 genes) derived from human HNSCC patients described by Puram et al. To further validate the relationship between the stress subtype with β-catenin/CBP activity and its relation to the AP-1 complex, we projected the Puram stress module and the down-regulated signature from the β-catenin/CBP inhibitor-treated group compared with the 4NQO-control group onto independent cohorts of human subjects with HPV-negative HNSCC. We used both the bulk TCGA RNA-seq subset (n=367 patients) and a previously published scRNAseq dataset from Choi et al., specifically profiling the epithelial compartment (3K cells, n = 16 patients). We observed highly significant positive association of the stress program and β-catenin/CBP activity in both datasets (pearson ρ = 0.74 and 0.70, respectively), indicating that inhibition of β-catenin/CBP activity reduces the stress response. Our results suggest that mitigation of tumorigenic profiles in 4NQO-induced tumors upon inhibition of β-catenin/CBP signaling in OSCC may serve as an effective treatment strategy for the benefit of human patients and shed further light into the molecular mechanisms driving treatment response.

Citation Format: Mohammed Muzamil Khan, Eric Reed, Lina Kroehling, Kenichi Nomoto, Junji Matsui, Manish Bais, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Reducing the effect of cellular stress in murine oral tumors with pharmacological blockade of β-catenin/CBP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB288.