HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody in vivo.

Laurence Booth,Alshad S Lalani,Richard E Cutler,Francesca Avogadri-Connors,Jane L Roberts,Paul Dent,Andrew Poklepovic

doi:10.18632/oncotarget.21660

Laurence Booth, Alshad S Lalani + Show 5 more

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https://doi.org/10.18632/oncotarget.21660

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Abstract

Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo, neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.

Highlights

Over-expression of the epidermal growth factor receptor (EGFR, ERBB1) has for many years been recognized as a biomarker for tumor cell growth, invasion and resistance to chemotherapy [1, and references therein]
Molecular knock down of HDAC3, HDAC6 or HDAC10 in the afatinib resistant clones significantly reduced the expression of the receptor tyrosine kinases ERBB1, ERBB3, ERBB4 and c-MET (Figure 1D)
The key discoveries made in this manuscript are that neratinib at clinically relevant concentrations kills afatinib resistant NSCLC cells and such lethality can be enhanced by histone deacetylase (HDAC) inhibitors

Summary

Introduction

Over-expression of the epidermal growth factor receptor (EGFR, ERBB1) has for many years been recognized as a biomarker for tumor cell growth, invasion and resistance to chemotherapy [1, and references therein]. In contrast to the laboratory, tumors that over-expressed ERBB1 did not in general exhibit an exquisite sensitivity to the ERBB1 inhibitory drugs [7]. In part this was because neither gefitinib nor erlotinib inhibited ERBB2, and the use of these drugs could facilitate compensatory cell-survival-signaling via the formation of ERBB2:ERBB3 complexes, with downstream www.impactjournals.com/oncotarget activation of the cytoprotective PI3K pathway [8, 9]. Other mechanisms for gefitinib / erlotinib failure at tumor control included the activation of other cyto-protective growth factor receptors, e.g. c-MET [10]

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