Pazopanib and Trametinib as a Synergistic Strategy against Osteosarcoma: Preclinical Activity and Molecular Insights

Abstract

Receptor tyrosine kinases (RTKs) inhibitors’ activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.

EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

“…The total protein extracts were obtained from six different PDX-derived fresh-frozen tumor samples. After mechanical disaggregation in the presence of Lysis Buffer 6 (R&D Systems, Biotechne, Minneapolis, MN USA), western blotting was performed as previously described [ 19 ]. Briefly, the protein concentrations were determined using a bicinchoninic acid assay (BCA Protein Assay, Thermo Fisher Scientific, Whaltam, MA, USA), and the absorbance levels were measured using a BioPhotometer (Eppendorf, Hamburg, Germany).…”

“…In our previous work, we demonstrated that combined pazopanib + trametinib treatment could significantly reduce EphA2 expression in osteosarcoma cell lines and that this was possibly a relevant mechanism of response to the combination treatment [ 19 ].…”

Eph-Ephrin Signaling Mediates Cross-Talk Within the Bone Microenvironment

“…More specific to the musculoskeletal field, a preclinical study treating osteosarcoma utilized drugs that inhibit receptor tyrosine kinase signaling, Pazopanib and Trametinib. The authors identified that this treatment down-regulated EphA2 and IL-7R, and silencing EphA2 resulted in significant reduction of cell proliferation and migration (Chiabotto et al, 2020 ).…”

Chimeric Antigen Receptor T-Cell Therapy: The Light of Day for Osteosarcoma

“…EphA2 overexpression has been widely reported in various cancer [101,102] and plays an important role in OS [103]. There are also many drugs targeting EphA2 for treating OS, and they have achieved some efficacy [104][105][106]. Related studies also have suggested the EphA2 receptor may be an attractive candidate receptor for the targeted delivery of therapeutics to OS [107].…”