Fibroblast growth factor receptor facilitates recurrence of minimal residual disease following trastuzumab emtansine therapy.

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-ß1 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.

Pharmacologic Inhibition of FGFR Modulates the Metastatic Immune Microenvironment and Promotes Response to Immune Checkpoint Blockade.

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.

MgtE is a dual-function protein in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections, including chronic biofilm infections in the lungs of individuals with cystic fibrosis. We previously found that the inner-membrane protein MgtE can function both as a magnesium transporter and a virulence modulator, although the exact mechanism governing these activities is unclear. To address this issue, we carried out an experimental characterization of P. aeruginosa MgtE and generated a computer-rendered model. Our in silico analysis demonstrated the structural similarity of P. aeruginosa MgtE to that of the crystal structure of MgtE in Thermus thermophilus. Experimentally, we verified that MgtE is not essential for growth and found that it may not be involved directly in biofilm formation, even under low-magnesium conditions. We demonstrated both magnesium transport and cytotoxicity-regulating functions, and showed that magnesium-binding sites in the connecting helix region of MgtE are vital in coupling these two functions. Furthermore, limiting magnesium environments stimulated mgtE transcriptional responses. Our results suggested that MgtE might play an important role in linking magnesium availability to P. aeruginosa pathogenesis.