[Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration

// Laurence Booth 1 , Jane L. Roberts 1 , Rumeesa Rais 1 , John Kirkwood 4 , Francesca Avogadri-Connors 3 , Richard E. Cutler Jr. 3 , Alshad S. Lalani 3 , Andrew Poklepovic 2 and Paul Dent 1 1 Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA 2 Department of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA 3 Puma Biotechnology Inc., Los Angeles, CA 90024, USA 4 University of Pittsburgh Cancer Institute Melanoma and Skin Cancer Program, Hillman Cancer Research Pavilion Laboratory L1.32c, Pittsburgh, PA 15232, USA Correspondence to: Paul Dent, email: paul.dent@vcuhealth.org Keywords: autophagy; receptor tyrosine kinase; neratinib; valproate Received: November 22, 2017      Accepted: December 12, 2017      Published: December 26, 2017 ABSTRACT The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo , prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.