The Lethality of [Pazopanib + HDAC Inhibitors] Is Enhanced by Neratinib

Abstract

Sarcomas are a diverse set of malignancies. For soft tissue sarcomas, the kinase and chaperone inhibitor pazopanib is a standard of care therapeutic. Previously, we demonstrated that HDAC inhibitors enhanced pazopanib lethality against sarcoma and other tumor cell types in vitro and in vivo . The present studies defined mechanisms of drug-combination resistance. Exposure of sarcoma and PDX ovarian carcinoma cells to [pazopanib + entinostat] caused a prolonged activation of ERBB1 and transient/prolonged activations of ERBB2, c-KIT, and c-MET, in a cell-specific fashion. The activities of mTORC1, mTORC2, GRP78, HSP90, and HSP70 were reduced, expression of Beclin1 and ATG5 enhanced, and the ATM-AMPK-ULK1-ATG13-Beclin1/ATG5 pathway activated. Inhibition of ERBB1/2/4 using neratinib or of c-MET using crizotinib significantly enhanced [pazopanib + entinostat] lethality. For neratinib with [pazopanib + entinostat], this effect correlated with reduced phosphorylation and expression of ERBB1, ERBB2, c-KIT, and c-MET and reduced expression, regardless of mutational status, of N-RAS and K-RAS. [Pazopanib + entinostat + neratinib] reduced the phosphorylation of the Hippo pathway proteins MST1/3/4 and MOB1 whereas this treatment increased the phosphorylation of LATS1, YAP, and TAZ. The activation of ATM, ULK-1, and eIF2α was further enhanced by [pazopanib + entinostat + neratinib] as was the expression of ATG5 and Beclin1. Compared to other manipulations, knock down of eIF2α or over-expression of BCL-XL significantly reduced killing by the three-drug interaction. In vivo , pazopanib and entinostat, and also neratinib and entinostat, both combined to significantly suppress the growth of sarcoma tumors.

GZ17-6.02 and Doxorubicin Interact to Kill Sarcoma Cells via Autophagy and Death Receptor Signaling

“…All cell lines were cultured at 37°C (5% (v/v CO 2 ) in vitro using RPMI supplemented with 5% (v/v) fetal calf serum and 10% (v/v) non-essential amino acids. Cells were transfected with siRNA molecules or plasmids as described in prior manuscripts ( 7 – 10 ). Cells were transfected with plasmids (0.1 μg) using lipofectamine 2000.…”

“…In the past 5 years the multi-kinase and chaperone inhibitor pazopanib was FDA approved for treatment of the majority of STS subtypes ( 6 ). Pazopanib, alone or in combination with other agents in our hands utilizes endoplasmic reticulum stress signaling and autophagosome formation as key components of its mechanisms of action ( 7 – 10 ). In preliminary studies for this manuscript initially we examined whether pazopanib and 602 could interact to kill sarcoma cells.…”

Molecular mechanisms underpinning sarcomas and implications for current and future therapy

“…189,190 For example, HDAC inhibitors enhanced the anti-cancer effect of pazopanib against sarcoma cells, and this effect was even more pronounced in combination with the TK inhibitor, neratinib. 191,192 Furthermore, results from a phase I clinical trial found panobinostat to increase the efficacy of the topoisomerase II inhibitor epirubicin (PubChem CID: 41867) and this led to clinical benefit and has the potential to reverse anthracycline resistance. 193 Interestingly, tazemetostat (PubChem CID: 66558664), a lysine methyltransferase inhibitor of the histone modification enzyme enhancer of zeste homolog 2 (EZH2), was approved by the FDA in 2020 as the first epigenetic therapy for solid tumors and is used to treat advanced or metastatic epithelioid sarcoma.…”

Emerging role of the Hippo pathway in autophagy

“…For instance, neratinib (ERBB1/2/4 inhibitor) enhanced [pazopanib (the kinase inhibitor) + entinostat (histone deacetylase inhibitor)] lethality against sarcoma and other tumor cell types in vitro and in vivo. Specifically, the triplet combination increases the phosphorylation of YAP/TAZ and promotes the conversion of LC3 and expression of Beclin1 and ATG13, which together enhance autophagosome formation 114,115 . The mammalian STK 26/ MST4 stimulates ATG4B activity and increases autophagic flux by phosphorylating ATG4B 116 .…”