Genetic evaluation of a combination therapy for the treatment of KRAS mutant lung adenocarcinoma

Abstract

Genetic ablation of CDK4 is known to prevent tumor development of Kras-driven lung adenocarcinoma (LUAD). Yet, elimination of the protein does not mimic pharmacological inhibition. We have utilized a genetically engineered mouse model (GEMM) that closely recapitulates this pharmacological treatment to evaluate the consequences of inactivating CDK4. In the present thesis, we have used two kinase dead (KD) isoforms, K35M and D140A, to eliminate the catalytic activity of CDK4. We demonstrated that the kinase function of CDK4 is not essential for mouse embryonic development. These mutant mice recapitulate most of the features of the complete elimination of CDK4 but later in time and with less penetrance. Mice expressing these CDK4 kinase dead isoforms are smaller than their wildtype counterparts and develop diabetes due to defects in the β-pancreatic cells development. Furthermore, CDK4 inactivation impairs lung tumor initiation, although to a lesser extent than CDK4 ablation, suggesting the existence of kinase independent functions for the maintenance of cell cycle progression. Nevertheless, inactivation of CDK4 is not sufficient to eradicate aggressive KrasG12V;Trp53-null adenocarcinomas. Hence, combinatorial targets to improve therapeutic responses have been validated. Recently, it has been reported that elimination of RAF1 decreases lung tumor progression but unfortunately and similarly to CDK4 inactivation, it is insufficient to completely eliminate the disease in a Trp53 deficient background. Here, we demonstrated that concomitant inhibition of CDK4 and RAF1 leads to regression of 100% of the Kras-driven adenocarcinomas even in the most aggressive context where the tumor suppressor Trp53 is depleted. Of note, 24% were complete responders according to the RECIST criteria. Still, there is a small population of remaining non-proliferative cells that survive in the absence of both targets being able to evolve and spawn new cancer growth. In vitro characterization of those cells confirmed the heterogenous mechanisms a cell can acquire in order to sustain tumor growth independently of CDK4 and RAF1. Both, epigenetic modifications as well as activation of alternative signaling pathways such as PI3K, are found to be responsible of CDK4 and RAF1 independent cell growth. In fact, selective treatment with 5-azacytidine or PI3K inhibitors halts proliferation of CDK4 and RAF1 resistant cells. Further studies to understand the complex crosstalk between the cell cycle and MAP Kinase signaling will shed light on future novel treatments against KRAS mutant tumors.