Introduction: Triple-negative and inflammatory breast cancer (TNBC and IBC) are the most aggressive breast cancer subtypes. Novel actionable targets and complementary therapies are critically needed. AXL, a receptor tyrosine kinase, drives pleiotropic phenotypes of TNBC and IBC aggressiveness. Targeting AXL reduces IBC tumor growth in vivo and inhibits polarization of M2 macrophages. To identify novel tumor microenvironment (TME) targets to enhance the efficacy of AXL-targeted therapy, we conducted a synthetic lethal kinome siRNA screening. We identified TBK1 as a candidate to synergize with AXL growth inhibition. TBK1 is a serine/threonine protein kinase that regulates innate, adaptive responses and antitumor immune responses. We hypothesized that inhibition of TBK1 enhances the antitumor effect of AXL-targeted therapy in aggressive breast cancers. Methods: We used knockdown, knockout (KO) and inhibitors to suppress AXL and TBK1 pathways and tested the synergistic effect of targeting AXL and TBK1 on the growth of human SUM149 and BCX010 IBC cells, and HS578T TNBC cells in vitro. To determine the synergistic effect of targeting both pathways in vivo, we inoculated control or TBK1 KO murine TNBC 4T1.2 cells into BALB/c mice. We assessed the activity of AXL inhibitor TP-0903 in reducing tumor growth. Using multicolor flow cytometry, we studied the effects of targeting AXL and TBK1 on the TME. We tested the activity of TP-0903 combined with a TBK1 inhibitor in another TNBC mouse model, E0771. We used RNA-sequencing and real-time PCR/Western blot to determine the molecular mechanisms of how TBK1 inhibition synergizes AXL-targeted therapy in these cancers. Results: Compared with AXL or TBK1 suppression alone, genomic or pharmacologic suppression of AXL and TBK1 significantly reduced the growth of SUM149, BCX010, and HST578T cells in vitro. In both 4T1.2 and E0771 syngeneic mouse models, TP-0903 was more active in reducing tumor growth in TBK1-inhibited tumors than in control tumors. Tumors with TBK1 inhibition and treated with TP-0903 had a significantly higher population of cytotoxic T cells than control tumors. Depletion of CD8+ T cells blocked the synergistic effect of targeting AXL and TBK1 pathways on reducing tumor growth, suggesting that cytotoxic T cells contributed to the anti-tumor synergy of targeting AXL and TBK1. Mechanistically, TBK1 induced M2 macrophage migration via IRF3-regulated CCL5 secretion in SUM149 and HS578T cells, and AXL KO attenuated the polarization and migration of M2 macrophages by inhibiting the CCR5/CCL5 axis. Conclusions: Targeting TBK1 enhances the efficacy of AXL-targeted therapy in aggressive breast cancer by suppressing the paracrine effect of CCR5/CCL5 axis. This combination represents a novel and effective therapy modulating the TME of aggressive breast cancer, which warrants further investigation in the clinical setting. Citation Format: Lan Phi, Takashi Semba, Ngu V. Trinh, Fang Zou, Jason M. Foulks, Steven L. Warner, Savitri Krishnamurthy, James P. Long, James M. Reuben, Debu Tripathy, Naoto T. Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3635.

Background: Novel mechanism-oriented targeted therapies are needed for aggressive breast cancers such as triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC). We previously showed the oncogenic receptor tyrosine kinase AXL crucially contributes to the aggressiveness of TNBC and TN-IBC, which suggests that AXL is a potential therapeutic target. However, AXL-targeted therapy is insufficient to control the progress of aggressive breast cancer in preclinical models. Therefore, to further enhance the efficacy of AXL-targeted therapy in aggressive breast cancer, we conducted a synthetic lethality screen of pooled kinome siRNA and identified TANK-binding kinase 1 (TBK1) as a potential target. TBK1 is a necessary serine/threonine protein kinase that regulates innate and adaptive immune responses, T cell migration, and anti-tumor immune responses. In the present work, we validated that AXL and TBK1 inhibition have a synergistic anti-tumor effect by modulating the tumor microenvironment (TME). Methods: To determine the synergistic effect of AXL and TBK1 inhibition on cell proliferation and colony formation in vitro, we first used siRNA-mediated knockdown (KD) or CRISPR/Cas9 knockout (KO) to independently silence AXL and TBK1 expression. We also used pharmacological inhibitors to inactivate AXL and TBK1 signaling and conducted similar studies. To determine the effect of targeting TBK1 and AXL pathways in vivo, we injected control or TBK1 KO murine 4T1.2 TNBC cells into BALB/c mice and assessed the efficacy of the AXL inhibitor TP-0903 in reducing the tumor growth. We used multicolor flow cytometry and multiplexed immunostaining to examine the population of TME components in control and TBK1 KO 4T1.2 tumors treated with vehicle or TP-0903. We also used an in vitro migration assay to test the effect of targeting AXL and TBK1 on the recruitment of CD8+ T cells. We performed a mouse chemokine array to identify intratumoral chemokines involved in regulating infiltrating immune cells and used RT-PCR to validate these findings. Results: Compared with AXL or TBK1 suppression alone, the genomic or pharmacological suppression of both AXL and TBK1 resulted in a significantly greater reduction of TNBC and TN-IBC cell growth and colony-forming ability (P < 0.01). In our syngeneic 4T1.2 TNBC mouse tumor model, TP-0903 was more effective at controlling tumor growth from TBK1-KO cells than from control cells (P < 0.0005). TBK1-KO 4T1.2 tumors treated with TP-0903 had a significantly higher population of functional cytotoxic T cells (GranzymeB+CD3+CD8+) than control KO tumors treated with vehicle or TP-0903. Furthermore, our migration assay showed that TBK1-KO/AXL-KD SUM149 cell-conditioned media attracted more CD8+ T cells than control cell-conditioned media, suggesting the inhibition of AXL/TBK1 signaling enhances the recruitment of CD8+ T cells through a paracrine effect. Our chemokine array revealed increased CXCL16 levels in TP-0903-treated TBK1-KO tumors. Conclusion: Targeting TBK1 could enhance the sensitivity of aggressive breast cancer cells to AXL-targeted therapy in vitro and in vivo. Our preliminary TME data suggest that combined AXL and TBK1 inhibition enhances the anti-tumor immune environment by upregulating secretion of CXCL16 by tumor cells, resulting in the increased recruitment of cytotoxic CD8+ T cells. In future studies, we will further elucidate the role that CXCL16 upregulation plays in this novel synergy. Significance of this findings: This study will enable us to develop hypothesis-driven novel combination therapies for patients with aggressive breast cancer that can be rapidly tested as a clinical trial, leading to an improvement of treatment and patients’ outcome. Citation Format: Lan Phi, Takashi Semba, Jason Foulks, Steven Warner, David Bearss, Savitri Krishnamurthy, James Long, James Reuben, Debu Tripathy, Naoto Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy in aggressive breast cancer preclinical models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-10.