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Neratinib augments the lethality of [regorafenib + sildenafil]
Laurence Booth1, Jane L Roberts1, Rumeesa Rais1
1Departments of Biochemistry and Molecular Biology, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia.
Abstract:
Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein-tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)-AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.
Insights
Neratinib enhances regorafenib and sildenafil combination therapy, increasing cancer cell death. This three-drug regimen shows promise for advanced solid tumors, warranting a Phase I clinical trial.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- Regorafenib is approved for colorectal cancer and hepatocellular carcinoma.
- Previous trials established the safety of combining regorafenib with sildenafil in advanced solid tumors.
- Neratinib is an approved drug that downregulates ERBB1/2/4 and RAS signaling pathways.
Purpose of the Study:
- To determine if neratinib can enhance the anti-cancer effects of the regorafenib plus sildenafil combination.
- To investigate the molecular mechanisms underlying the enhanced lethality induced by the three-drug combination.
- To evaluate the in vivo efficacy of the three-drug combination in preclinical cancer models.
Main Methods:
- Colon cancer cells were treated with regorafenib, sildenafil, and neratinib individually and in combination.
- Expression levels of mutant K-RAS, histone deacetylase (HDAC) proteins, and immunotherapy biomarkers were analyzed.
- Cellular localization of K-RAS V12 was observed using fluorescent tagging.
- Activation of signaling pathways including ATM-AMPK-ULK-1 and mTOR/AKT/p70S6K was assessed.
- Gene knockdown experiments were performed for key proteins involved in autophagy and cell death.
- Antitumor efficacy was evaluated in mouse cancer models.
Main Results:
- Neratinib significantly enhanced the lethality of regorafenib plus sildenafil in colon cancer cells in a supra-additive manner.
- The three-drug combination reduced mutant K-RAS and HDAC protein expression, leading to K-RAS V12 localization in intracellular vesicles.
- Enhanced activation of the ATM-AMPK-ULK-1 pathway and prolonged inactivation of mTOR, AKT, and p70S6K were observed.
- Approximately 70% of the enhanced lethality was dependent on ATM-AMPK signaling, and autophagy-related proteins (Beclin1, ATG5) and death receptors (FADD, CD95) were crucial.
- The combination downregulated checkpoint immunotherapy biomarkers (PD-L1, ODC, IDO-1) and upregulated MHCA, requiring autophagosome formation.
- Neratinib significantly improved the antitumor efficacy of regorafenib plus sildenafil in mouse models.
Conclusions:
- Neratinib potentiates the anti-cancer effects of regorafenib and sildenafil through modulation of K-RAS, HDACs, and autophagy.
- The combination therapy impacts key signaling pathways and immunotherapy-related biomarkers.
- The findings support a potential new three-drug Phase I clinical trial combining regorafenib, sildenafil, and neratinib for advanced solid tumors.
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