Cellular responses after (neratinib plus pemetrexed) exposure in NSCLC cells

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.

Autophagy as a therapeutic mechanism to kill drug-resistant cancer cells

“…More surprising was that we subsequently discovered that neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, KRAS and NRAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells [16,[36][37][38][39]. This suggested the drugs each have individual off-target effectors.…”