Brain metastatic outgrowth and osimertinib resistance are potentiated by RhoA in EGFR-mutant lung cancer.

The brain is a major sanctuary site for metastatic cancer cells that evade systemic therapies. Through pre-clinical pharmacological, biological, and molecular studies, we characterize the functional link between drug resistance and central nervous system (CNS) relapse in Epidermal Growth Factor Receptor- (EGFR-) mutant non-small cell lung cancer, which can progress in the brain when treated with the CNS-penetrant EGFR inhibitor osimertinib. Despite widespread osimertinib distribution in vivo, the brain microvascular tumor microenvironment (TME) is associated with the persistence of malignant cell sub-populations, which are poised to proliferate in the brain as osimertinib-resistant lesions over time. Cellular and molecular features of this poised state are regulated through a Ras homolog family member A (RhoA) and Serum Responsive Factor (SRF) gene expression program. RhoA potentiates the outgrowth of disseminated tumor cells on osimertinib treatment, preferentially in response to extracellular laminin and in the brain. Thus, we identify pre-existing and adaptive features of metastatic and drug-resistant cancer cells, which are enhanced by RhoA/SRF signaling and the brain TME during the evolution of osimertinib-resistant disease.

Preclinical Comparison of the Blood-brain barrier Permeability of Osimertinib with Other EGFR TKIs.

PURPOSE: Osimertinib is a potent and selective EGFR tyrosine kinase inhibitor (EGFR-TKI) of both sensitizing and T790M resistance mutations. To treat metastatic brain disease, blood-brain barrier (BBB) permeability is considered desirable for increasing clinical efficacy. EXPERIMENTAL DESIGN: We examined the level of brain penetration for 16 irreversible and reversible EGFR-TKIs using multiple in vitro and in vivo BBB preclinical models. RESULTS: In vitro osimertinib was the weakest substrate for human BBB efflux transporters (efflux ratio 3.2). In vivo rat free brain to free plasma ratios (Kpuu) show osimertinib has the most BBB penetrance (0.21), compared with the other TKIs (Kpuu ≤ 0.12). PET imaging in Cynomolgus macaques demonstrated osimertinib was the only TKI among those tested to achieve significant brain penetrance (C max %ID 1.5, brain/blood Kp 2.6). Desorption electrospray ionization mass spectroscopy images of brains from mouse PC9 macrometastases models showed osimertinib readily distributes across both healthy brain and tumor tissue. Comparison of osimertinib with the poorly BBB penetrant afatinib in a mouse PC9 model of subclinical brain metastases showed only osimertinib has a significant effect on rate of brain tumor growth. CONCLUSIONS: These preclinical studies indicate that osimertinib can achieve significant exposure in the brain compared with the other EGFR-TKIs tested and supports the ongoing clinical evaluation of osimertinib for the treatment of EGFR-mutant brain metastasis. This work also demonstrates the link between low in vitro transporter efflux ratios and increased brain penetrance in vivo supporting the use of in vitro transporter assays as an early screen in drug discovery.

Adaptive Protein Translation by the Integrated Stress Response Maintains the Proliferative and Migratory Capacity of Lung Adenocarcinoma Cells.

The integrated stress response (ISR) is a conserved pathway that is activated by cells that are exposed to stress. In lung adenocarcinoma, activation of the ATF4 branch of the ISR by certain oncogenic mutations has been linked to the regulation of amino acid metabolism. In the present study, we provide evidence for ATF4 activation across multiple stages and molecular subtypes of human lung adenocarcinoma. In response to extracellular amino acid limitation, lung adenocarcinoma cells with diverse genotypes commonly induce ATF4 in an eIF2α-dependent manner, which can be blocked pharmacologically using an ISR inhibitor. Although suppressing eIF2α or ATF4 can trigger different biological consequences, adaptive cell-cycle progression and cell migration are particularly sensitive to inhibition of the ISR. These phenotypes require the ATF4 target gene asparagine synthetase (ASNS), which maintains protein translation independently of the mTOR/PI3K pathway. Moreover, NRF2 protein levels and oxidative stress can be modulated by the ISR downstream of ASNS. Finally, we demonstrate that ASNS controls the biosynthesis of select proteins, including the cell-cycle regulator cyclin B1, which are associated with poor lung adenocarcinoma patient outcome. Our findings uncover new regulatory layers of the ISR pathway and its control of proteostasis in lung cancer cells. IMPLICATIONS: We reveal novel regulatory mechanisms by which the ISR controls selective protein translation and is required for cell-cycle progression and migration of lung cancer cells.

Extracellular Matrix Receptor Expression in Subtypes of Lung Adenocarcinoma Potentiates Outgrowth of Micrometastases.

Mechanisms underlying the propensity of latent lung adenocarcinoma (LUAD) to relapse are poorly understood. In this study, we show how differential expression of a network of extracellular matrix (ECM) molecules and their interacting proteins contributes to risk of relapse in distinct LUAD subtypes. Overexpression of the hyaluronan receptor HMMR in primary LUAD was associated with an inflammatory molecular signature and poor prognosis. Attenuating HMMR in LUAD cells diminished their ability to initiate lung tumors and distant metastases. HMMR upregulation was not required for dissemination in vivo, but enhanced ECM-mediated signaling, LUAD cell survival, and micrometastasis expansion in hyaluronan-rich microenvironments in the lung and brain metastatic niches. Our findings reveal an important mechanism by which disseminated cancer cells can coopt the inflammatory ECM to persist, leading to brain metastatic outgrowths. Cancer Res; 77(8); 1905-17. ©2017 AACR.