TKI-addicted ROS1-rearranged cells are destined to survival or death by the intensity of ROS1 kinase activity

Abstract

ROS1 rearrangement is observed in 1–2% of non-small cell lung cancers (NSCLC). The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has induced marked tumour shrinkage in ROS1-rearranged cancers. However, emergence of acquired resistance to TKI is inevitable within a few years. Previous findings indicate that cabozantinib overcomes secondary mutation–mediated crizotinib-resistance in ROS1-fusion-positive cells. Here we attempted to establish cabozantinib-resistant cells by N-ethyl-N-nitrosourea mutagenesis screening using CD74-ROS1–expressing Ba/F3 cells. Two resistant cell lines with CD74-ROS1 F2004V or F2075C mutations, which are homologous to ALK F1174 or F1245 mutations, survived in the presence of a low dose of ROS1-TKI. Removal of ROS1-TKI from these TKI-addicted cells induced excessive activation of ROS1 tyrosine kinase followed by apoptosis. We succeeded in recapturing the TKI-addicted phenotype using doxycycline-inducible CD74-ROS1 mutant over-expression in Ba/F3 cells, suggesting that excessive ROS1 oncogenic signaling itself induced apoptosis instead of cell growth. Phosphoproteomic analysis and high-throughput inhibitor screening revealed that excessive ROS1 signaling in the TKI-addicted cells phosphorylated or activated apoptosis-related molecules such as FAF1 or p38. Collectively, our findings partly clarify molecular mechanisms of excessive ROS1 oncogenic signaling that mediates paradoxical induction of apoptosis.

Dramatic Response to Lorlatinib in a Patient With CD74-ROS1-Positive Lung Adenocarcinoma With Acquired F2004V Mutation

“… 21 Instead, Ba/F3 cells expressing ROS1 F2004V survive in the presence of low-dose ROS1 inhibitor. 20 However, the patient in this report did not respond to withdrawal of brigatinib, and symptoms, namely headaches, developed from brain metastases. Disease burden in the brain was not amenable to stereotactic ablative radiation, and to avoid whole-brain radiation therapy, a decision was made to enroll the patient in a clinical trial ( ClinicalTrials.gov identifier NCT03178071) of lorlatinib, another TKI with ROS1 activity.…”

“…This mutation, along with F2075C, emerged as a possible mechanism of resistance to cabozantinib, a multikinase inhibitor with ROS1 activity in vitro. 20 Although the patient in that study did not receive cabozantinib, it is likely that F2004V is a mechanism of secondary resistance to ROS1 inhibition with entrectinib. In that work, Ogura et al 20 established Ba/F3 cells expressing CD74-ROS1 and resistance to cabozantinib by exposure to N -ethyl- N -nitrosourea, a known mutagen.…”

“… 20 Although the patient in that study did not receive cabozantinib, it is likely that F2004V is a mechanism of secondary resistance to ROS1 inhibition with entrectinib. In that work, Ogura et al 20 established Ba/F3 cells expressing CD74-ROS1 and resistance to cabozantinib by exposure to N -ethyl- N -nitrosourea, a known mutagen. Interestingly, the resistant cell lines were TKI addicted, because they required the presence of low-dose cabozantinib to survive.…”

“…In three patients, there were copy-number gains in oncogenes, which might also represent mechanisms of resistance. Figure 3 summarizes the in vitro 10 , 16 , 20 , 28 - 33 and clinical 10 , 27 , 30 , 34 data of ROS1 secondary mutations reported in the literature.…”

“…Although, to our knowledge, the ROS1 F2004V has not been described before in patients, it is analogous to the ALK F1174L mutation described in patients with neuroblastoma 19 and confers paradoxic apoptosis to Ba/F3 cells, a result of excessive oncogenic signaling through ROS1 and p38MAPK. 20 Also, a secondary F1174V mutation has been described in ALK-positive lung cancer with resistance to crizotinib. 21 Instead, Ba/F3 cells expressing ROS1 F2004V survive in the presence of low-dose ROS1 inhibitor.…”