The effectiveness of immunotherapy as a treatment for metastatic breast cancer is limited due to low numbers of infiltrating lymphocytes in metastatic lesions. Herein, we demonstrated that adjuvant therapy using FIIN4, a covalent inhibitor of fibroblast growth factor receptor (FGFR), dramatically delayed the growth of pulmonary metastases in syngeneic models of metastatic breast cancer. In addition, we demonstrated in a syngeneic model of systemic tumor dormancy that targeting of FGFR enhanced the immunogenicity of the pulmonary tumor microenvironment through increased infiltration of CD8+ lymphocytes and reduced presence of myeloid suppressor cells. Similar impacts on immune cell infiltration were observed upon genetic depletion of FGFR1 in tumor cells, which suggested a direct influence of FGFR signaling on lymphocyte trafficking. Suppression of CD8+ lymphocyte infiltration was consistent with FGFR-mediated inhibition of the T-cell chemoattractant CXCL16. Initial attempts to concomitantly administer FIIN4 with immune checkpoint blockade failed due to inhibition of immune-mediated tumor cell killing via blockade of T-cell receptor signaling by FIIN4. However, this was overcome by using a sequential dosing protocol that consisted of FIIN4 treatment followed by anti–PD-L1. These data illustrate the complexities of combining kinase inhibitors with immunotherapy and provide support for further assessment of FGFR targeting as an approach to enhance antitumor immunity and improve immunotherapy response rates in patients with metastatic breast cancer.

Inhibition of epidermal growth factor receptor (EGFR) signaling by small molecule kinase inhibitors and monoclonal antibodies has proven effective in the treatment of multiple cancers. In contrast, metastatic breast cancers (BC) derived from EGFR-expressing mammary tumors are inherently resistant to EGFR-targeted therapies. Mechanisms that contribute to this inherent resistance remain poorly defined. Here, we show that in contrast to primary tumors, ligand-mediated activation of EGFR in metastatic BC is dominated by STAT1 signaling. This change in downstream signaling leads to apoptosis and growth inhibition in response to epidermal growth factor (EGF) in metastatic BC cells. Mechanistically, these changes in downstream signaling result from an increase in the internalized pool of EGFR in metastatic cells, increasing physical access to the nuclear pool of STAT1. Along these lines, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1 phosphorylation and apoptosis. Additionally, inhibition of endosomal signaling using an EGFR inhibitor linked to a nuclear localization signal specifically prevents EGF-induced STAT1 phosphorylation and cell death, without affecting EGFR:ERK1/2 signaling. Pharmacologic blockade of ERK1/2 signaling through the use of the allosteric MEK1/2 inhibitor, trametinib, dramatically biases downstream EGFR signaling toward a STAT1-dominated event, resulting in enhanced EGF-induced apoptosis in metastatic BC cells. Importantly, combined administration of trametinib and EGF also facilitated an apoptotic switch in EGFR-transformed primary tumor cells, but not normal mammary epithelial cells. These studies reveal a fundamental distinction for EGFR function in metastatic BC. Furthermore, the data demonstrate that pharmacological biasing of EGFR signaling toward STAT1 activation is capable of revealing the apoptotic function of this critical pathway.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that efficiently delivers a highly potent microtubule inhibitor to HER2 overexpressing cells. Herein, we utilize HER2 transformed human mammary epithelial cells (HME2) to demonstrate in vitro and in vivo response and recurrence upon T-DM1 treatment. Continuous in vitro dosing of HME2 cells with T-DM1 failed to produce a spontaneously resistant cell line. However, induction of epithelial–mesenchymal transition (EMT) via pretreatment with TGF-β1 was capable of promoting emergence of T-DM1-resistant (TDM1R) cells. Flow cytometric analyses indicated that induction of EMT decreased trastuzumab binding, prior to overt loss of HER2 expression in TDM1R cells. Kinome analyses of TDM1R cells indicated increased phosphorylation of ErbB1, ErbB4, and FGFR1. TDM1R cells failed to respond to the ErbB kinase inhibitors lapatinib and afatinib, but they acquired sensitivity to FIIN4, a covalent FGFR kinase inhibitor. In vivo, minimal residual disease (MRD) remained detectable via bioluminescent imaging following T-DM1-induced tumor regression. Upon cessation of the ADC, relapse occurred and secondary tumors were resistant to additional rounds of T-DM1. These recurrent tumors could be inhibited by FIIN4. Moreover, ectopic overexpression of FGFR1 was sufficient to enhance tumor growth, diminish trastuzumab binding, and promote recurrence following T-DM1-induced MRD. Finally, patient-derived xenografts from a HER2+ breast cancer patient who had progressed on trastuzumab failed to respond to T-DM1, but tumor growth was significantly inhibited by FIIN4. Overall, our studies strongly support therapeutic combination of TDM1 with FGFR-targeted agents in HER2+ breast cancer.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that efficiently delivers a potent microtubule inhibitor into HER2 overexpressing tumor cells. However, resistance to T-DM1 is emerging as a significant clinical problem. Continuous in vitro treatment of HER2-transformed mammary epithelial cells with T-DM1 did not elicit spontaneously resistant cells. However, induction of epithelial-mesenchymal transition (EMT) via pretreatment with TGF-β1 facilitated acquisition of T-DM1 resistance. Flow cytometric analyses indicated that induction of EMT decreased trastuzumab binding, prior to overt loss of HER2 expression. Kinome analyses of T-DM1 resistant cells indicated increased phosphorylation of ErbB1, ErbB4, and fibroblast blast growth factor receptor 1 (FGFR1). T-DM1 resistant cells failed to respond to the ErbB kinase inhibitors lapatinib and afatinib, but they acquired sensitivity to FIIN4, a covalent FGFR kinase inhibitor. In vivo, T-DM1 treatment led to robust regression of HER2-transformed tumors, but minimal residual disease (MRD) was still detectable via bioluminescent imaging. Upon cessation of the ADC relapse occurred and secondary tumors were resistant to additional rounds of T-DM1, but this recurrent tumor growth could be inhibited by FIIN4. Expression of FGFR1 was upregulated in T-DM1 resistant tumors, and ectopic overexpression of FGFR1 was sufficient to enhance tumor growth, diminish trastuzumab binding, and promote recurrence following T-DM1-induced MRD. Finally, patient-derived xenografts from a HER2 + breast cancer patient who had progressed on trastuzumab failed to respond to T-DM1, but tumor growth was significantly inhibited by FIIN4. Overall, our studies strongly support therapeutic combination of TDM1 and FGFR targeted agents in HER2 + breast cancer.Suppl. Fig. S1. HER2 expression is diminished upon acquisition of resistantance to TDM1. A, Ex-vivo subculture of HME2 tumors that recurred after T-DM1-induced minimal residual disease as shown in Figure 1. These cells failed to thrive in culture. B, HME2 parental cells and their in vitro-derived T-DM1 resistant (TDM1R) counterparts were fixed, permeabilized and stained for HER2. These cells were counter stained with DAPI to visualize the nucleus. C, HME2 parental cells (Par), those treated and recovered from TGF-b1 (Post-TGF-b), those treated and recovered from TGF-b1 and subsequently selected for by continuous treatment with T-DM1 (TDM1R Invitro), and primary culture from P3 HME2 tumors selected for resistance to T-DM1, as described in Figure 7a of the main text (TDM1R In-vivo), were assayed by immunoblot for expression of HER2 and b-tubulin (b-Tub) served as a loading control. Fig. S2. Enhanced FGFR expression upon acquisition of resistance to TDM1. A, Expression levels of FGFR1-4 in the NCI-N87 cells selected for resistant to TDM1. Data are extracted from a single RNA sequencing experiment and are normalized to the untreated control cells. B, Expression levels of FGFR1-4 in four different TDM1 resistant BT474 clones (C1-3 and C6). D...

No abstract