Abstract P1-06-17: Inhibition of oncogenic PTK6 kinase enhances immune response against triple negative breast cancer via EMT reversal

Abstract Background/Rationale: PTK6/Brk, a non-receptor tyrosine kinase, is highly expressed in approximately 40% of triple negative breast cancers (TNBC). Higher levels of PTK6 expression are associated with worse patient outcomes. PTK6 is a driver of oncogenic growth, survival, invasion, epithelial-mesenchymal transition (EMT), chemotherapy resistance and metastasis. Recent studies have reported an association between PTK6 expression and an immunosuppressive microenvironment in patient breast cancers. Therefore, in addition to tumor cell-intrinsic oncogenic functions, PTK6 may play a critical role in shaping the tumor immune microenvironment which could modulate sensitivity to chemo/immunotherapy of TNBC. Inhibition of PTK6 could therefore suppress TNBC tumor growth and metastasis via dual (cell autonomous and microenvironmental) mechanisms. Methods. Genetic and pharmacological approaches were used to downregulate/inhibit PTK6 in immunocompetent and immunodeficient mouse models of TNBC (MMTV-myc, 4T-1). The effects of PTK6 shRNA expression, as well as treatment with a validated PTK6 kinase inhibitor (P21d), on TNBC tumor growth were monitored. For studies using immunocompetent models, immune populations (tumor-infiltrating and systemic) were analyzed by flow cytometry and immunofluorescence. Results: MMTV-myc or 4T-1 tumor-bearing, immunocompetent mice were treated with P21d, a validated small molecule inhibitor of PTK6 kinase activity. P21d treatment significantly inhibited tumor growth and increased tumoral infiltration by activated, cytotoxic immune cells (CD8+ T cells, NK cells). These effects were phenocopied by PTK6 shRNA or SNAIL shRNA expression, both of which also promote EMT reversal. Interestingly, the effects of P21d and PTK6 shRNA treatment on tumor growth were discordant when these same tumors were grown in immunodeficient mice; PTK6 shRNA, but not P21d treatment, inhibited TNBC growth. These results indicate that P21d’s inhibitory effect on tumor growth is reliant on immune modulation and that this immune regulation is PTK6 kinase-activity dependent. These results also suggest that there are kinase activity-independent mechanisms that are important for tumor growth control by PTK6. The importance of immune modulation in P21d-dependent triple negative tumor inhibition was reinforced by the fact that co-treatment of tumors with CD8 T cell or NK cell-depleting antibodies nearly completely abrogated P21d's effects on tumor growth. We identified CXCL10 as a mediator of P21d treatment-induced tumoral immune cell infiltration and tumor growth inhibition. Summary/Conclusion: Our studies highlight novel tumor immune microenvironmental functions of PTK6 oncogene in TNBC. PTK6 inhibition leads to recruitment and activation of cytotoxic T and NK cells that are critical for TNBC growth inhbition. Ongoing studies will determine whether these functions can be leveraged to enhance efficacy of immunotherapies currently used in the care of patients with TNBC. Citation Format: Hanna Irie, Ibuki Harada, Criseyda Martinez, Koichi Ito, Eunjee Lee, Jun Zhu. Inhibition of oncogenic PTK6 kinase enhances immune response against triple negative breast cancer via EMT reversal [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-06-17.