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Published on: August 11, 2017
Cellular responses after (neratinib plus pemetrexed) exposure in NSCLC cells
Laurence Booth1, Andrew Poklepovic2, John F Hancock3
1Department of Biochemistry and Molecular Biology, Virginia Commonwealth University.
Abstract:
We previously demonstrated that neratinib interacted with pemetrexed to kill non-small cell lung cancer (NSCLC) cells. From developing other drug combinations, we observed that several days following exposure, cells activated survival mechanisms to counteract drug toxicity. The present studies attempted to define mechanisms that evolve to reduce the efficacy of neratinib and pemetrexed. Neratinib and pemetrexed synergized to kill NSCLC cells expressing wild-type RAS proteins, mutant KRAS (G12S; Q61H; G12A and G12C) or mutant NRAS (Q61K) or mutant ERBB1 (L858R; L858R T790M and exon 19 deletion). Neratinib and pemetrexed interacted in a greater than additive fashion to kill after 24 h, and after a further 24 h culture in the absence of drugs. Mutant KRAS G12V was more cytoprotective than either activated MEK1 or activated AKT. Knockdown of mutant KRAS reduced drug combination killing at the 48 h timepoint. Despite culture for 24 h in the absence of the drugs, the expression and activities of ERBB1, ERBB2 and ERBB4 remained significantly lower as did the activities of mammalian target of rapamycin (mTOR) C1 and mTORC2. The drug combination reduced KRAS and NRAS levels for 24 h, however, in the absence of the drugs, RAS levels had normalized by 48 h. Expression of Beclin1 and ATG5 remained elevated and of MCL1 and BCL-XL lower. No evolutionary activations of survival signaling by ERBB3, c-KIT, c-MET or PDGFRβ or in intracellular signaling pathways were observed. These findings argue against the development of 'early' resistance mechanisms after neratinib and pemetrexed exposure. Future studies will be required to understand how NSCLC cells become resistant to neratinib and pemetrexed.
Insights
Neratinib and pemetrexed effectively kill non-small cell lung cancer (NSCLC) cells, including those with specific RAS and ERBB1 mutations. Studies suggest early resistance mechanisms do not develop, but further research is needed to understand long-term resistance to these drugs.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Neratinib and pemetrexed previously showed efficacy in killing non-small cell lung cancer (NSCLC) cells.
- Cells can activate survival mechanisms to counteract drug toxicity after exposure.
- Understanding resistance mechanisms is crucial for optimizing cancer therapy.
Purpose of the Study:
- To define the mechanisms that reduce the efficacy of neratinib and pemetrexed in NSCLC cells.
- To investigate the role of RAS mutations in drug response and resistance.
- To identify potential early resistance pathways to the drug combination.
Main Methods:
- Synergistic killing assays with neratinib and pemetrexed in NSCLC cells with various mutations.
- Assessment of cell survival mechanisms, including RAS, MEK, AKT, ERBB receptors, and mTOR pathways.
- Analysis of protein expression and activity at different time points post-drug exposure.
- Gene knockdown experiments to evaluate the role of specific mutations, such as KRAS.
Main Results:
- Neratinib and pemetrexed synergized to kill NSCLC cells with wild-type RAS, mutant KRAS (G12S, Q61H, G12A, G12C), mutant NRAS (Q61K), and mutant ERBB1 (L858R, L858R T790M, exon 19 deletion).
- Mutant KRAS G12V exhibited greater cytoprotective effects than activated MEK1 or AKT.
- Knockdown of mutant KRAS reduced the combination's killing efficacy at 48 hours.
- While RAS levels normalized by 48 hours in the absence of drugs, ERBB1, ERBB2, ERBB4, mTORC1, and mTORC2 activities remained low.
- Expression of Beclin1 and ATG5 remained elevated, while MCL1 and BCL-XL were lower.
- No evidence of 'early' resistance via activation of ERBB3, c-KIT, c-MET, PDGFRβ, or other intracellular signaling pathways was observed.
Conclusions:
- The drug combination of neratinib and pemetrexed demonstrates synergistic efficacy against a range of NSCLC mutations.
- Mutant KRAS appears to play a significant role in cytoprotection against this drug combination.
- Findings suggest that early resistance mechanisms do not readily develop following neratinib and pemetrexed exposure.
- Further investigation is warranted to elucidate the mechanisms underlying eventual resistance to neratinib and pemetrexed in NSCLC.
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