Functional adaptive responses (i.e. due to cellular machinery modulations) could contribute to targeted treatment resistance. We hypothesize that understanding tumor cell adaptive responses induced by cabozantinib (C) in renal cell cancer (RCC) cells could provide a rational base for selecting treatment combinations and optimise drug doses and schedules.
We evaluated functional proteomic changes induced in VHL-mutated 786-O RCC cell line after in vitro exposure to low-dose C using reverse phase protein array (RPPA). A linear model analysis was performed on normalized intensity data from RPPA, in order to identify proteins and phosphoproteins undergoing significant treatment-induced changes. Then, we evaluated in vitro the efficacy of the interaction between C and drugs selected on the basis of RPPA analysis by mean of dose matrix tests.
We exposed 786-O cells to HGF alone or in combination with C at 40 nM for 24 hours and then we performed RPPA analysis. Despite the low dose of C used, we observed a significant variation (i.e. with a log2 fold change for intensity of < 0.5 or > 1.5) in expression or phosphorylation of several protein targets. We observed inhibition of protein phosphorylation downstream of C targets (among which AXL, VEGFR−2, components of the PI3K/AKT/mTOR pathway and MEK1), which validates the cell model. Unexpectedly, we detected a significantly increased intensity signal for several protein targets involved in DNA repair process (RBBP8, RPA32_pS4_S8, BABAM1, BAP1, CDKN1A). We thus tested in vitro the association of C and inhibitors of the DNA repair proteins ATM, ATR and Wee1. We could observe that while the combination of C with KU60019 (an ATM inhibitor) is additive, the combinations of C with VE822 (an ATR inhibitor) or MK1775 (a Wee1 inhibitor) are synergistic in 2D and 3D cell culture.
Analysis of functional proteomic changes induced in vitro by C helped us to select targets for combination targeted therapy in RCC. Overall, our data suggest that cellular adaptive responses to drugs play a role in tumor resistance, and that elucidating them could help in designing drug combinations suitable for testing in the clinical setting.
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Institut du Cancer de Montpellier, Université de Montpellier, Montpellier, France.
D. Tosi: Other, Personal and Institutional, Research Grant: Ipsen; Other, Personal, Other, Travel support for scientific meetings: Ipsen. All other authors have declared no conflicts of interest.