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Novel therapeutic strategies to treat pancreatic and lung cancer

Periodic Reporting for period 4 - THERACAN (Novel therapeutic strategies to treat pancreatic and lung cancer)

Reporting period: 2021-07-01 to 2022-12-31

A quarter of all solid tumors harbor a mutation in the KRAS oncogenes. Despite the identification of these mutations over 30 years ago, no selective drugs have been approved to treat these malignancies. Our aim is to identify and validate targets with potential therapeutic value to devise novel strategies to treat these two human cancers with unacceptable low survival rates and unmet medical needs: pancreatic ductal adenocarcinoma and KRAS mutant lung adenocarcinoma. Although these tumor types have distinct pathological and clinical manifestations, they are both driven by KRAS mutations.

Our objectives are (1) to identify those genes activated in the cancer initiating cells responsible for the onset of pancreatic and lung tumors. We reasoned that genes implicated in the initial stages of tumor development will be maintained during tumor evolution and will not be affected by the intra-tumoral heterogeneity generated during tumor progression, (2) to identify and validate genes capable of reprogramming the stroma of pancreatic tumors to hamper its pro-tumoral effects, (3) to define the molecular events that control senescence, a naturally occurring process that serves as a barrier to tumor development, (4) to interrogate the role of known targets with suspected therapeutic value in tumor progression using a new generation of mouse tumor models that allow the temporal separation of tumor development from target ablation or inactivation. These studies will make it possible to design combination therapies capable of effectively eradicating advanced tumors, and (5) to validate these combination therapies using best-in-class inhibitors in state-of-the-art preclinical trial platforms based on mouse models and human tumor samples. The results derived from these studies will guide the design of new clinical trials that should have a positive impact in the treatment of these deadly diseases.
KRAS mutations are responsible for almost one quester of all human solid tumors including the three big killers, colorectal, lung and pancreatic adenocarcinomas. Although KRAS oncogenes were identified in human tumors four decades ago, the first KRAS selective inhibitor was not approved until 2021. In addition, current KRAS inhibitors only block one of the multiple KRAS oncogenic isoforms, KRASG12C, present in a percentage of lung adenocarcinomas, but absent in pancreatic tumors. Moreover, tumor resistance to these KRAS inhibitors has become a significant issue in the clinic resulting in overall survival not significantly better than classical chemotherapy. Considering this scenario, the work reported by Sanclemente et al. (Cancer Cell, 2018) and Blasco et al (Cancer Cell, 2019) represent mayor breakthroughs in the long quest to identify suitable and effective therapeutic strategies against KRAS mutant cancers.

Our experimental approach is based on targeting KRAS signaling pathways instead of KRAS itself. As summarized in our review published in Cancer Cell (Drosten and Barbacid,2020), tampering with the main KRAS signaling pathways, that is, the MAPK and the PI3K pathways is extremely toxic, which has led to disappointing results in the clinic of RAF, MEK, ERK and PI3K inhibitors. However, our discovery that ablation of RAF1 induced significant regression of 2/3 of KRAS/P53-driven lung tumors with acceptable toxicities, a result comparable to those obtained with KRAS inhibitors, opened new avenues to develop effective therapeutic strategies, either by themselves or in combination with forthcoming KRAS inhibitors. Indeed, in the study published in the Proceedings of the National Academy (Esteban-Burgos et al., PNAS, 2020), we showed that combination of RAF1 ablation and expression of a kinase inactive isoform of the cell cycle kinase CDK4 not only resulted in the complete regression of a quarter of all lung tumors, but completely prevented tumor progression. These results illustrate that inhibiting KRAS signaling effectors could be even more efficient than inhibiting KRAS itself.

Even a more relevant breakthrough was provided by our studies describing a therapeutic strategy to fight pancreatic tumors. In this study (Blasco et al., Cancer Cell, 2019) we demonstrated that concomitant ablation of RAF1 and EGFR led to the complete regression of a significant fraction of advance pancreatic tumors. It is worth noting that this was the first time that these aggressive tumors have been shown to regress completely, including the disappearance of their desmoplastic stroma.

The identification of RAF1 as a critical therapeutic target for KRAS mutant tumors raised the possibility of generating selective inhibitors against this protein kinase. However, expression of two independent kinase dead RAF1 isoforms in genetically engineered mouse models of lung adenocarcinoma, revealed that the kinase activity of RAF1 was not involved in tumor progression. Instead, RAF1 appears to contribute to tumor development by means of its inhibitory activity on pro-apoptotic kinases such as ASK1 and MST2 (Sanclemente et al., Cancer Cell, 2021). These observations represent a major breakthrough on two accounts. On the one hand, they explain the failure of RAF kinase inhibitors in clinical trials and have discourage ongoing efforts by the pharma industry to generate additional RAF1 kinase inhibitors. In addition, they opened the door to the design of selective degrons, that is, molecules capable of degrading RAF1, and in consequence to mimick the genetic results obtained upon ablating RAF1 expression, in a pharmacological scenario.

Based on these completely unexpected results, we initiated a project, not included in the original proposal (in 2016 nobody would have thought that the RAF1 kinase would not be involved in tumor progression) aimed at identifying RAF1 degron molecules. To this end, we succeeded in expressing the full-length RAF1 protein in the presence of tow independent chaperones, Hsp90 and CDC37. Then in collaboration with Dr. G Montoya (Univ of Copenhagen) we resolved the structure of RAF1 at a 3.9 A resolution by Cryo-EM technology. These studies revealed structural vulnerabilities that have allowed us to identify a pocket that could house small molecules capable of separating the CDC37 chaperone from RAF1, leading to its rapid degradation. Identification of pharmacologically viable RAF1 degrons are likely to have a significant impact in a not too distant future in our quest to block KRAS mutant tumors in a clinical scenario.
elimination of Raf1 induces regression of Lung adenocarcinmas
EGFR and Raf1 elimination induces complete regression of 50% of mouse pancreatic tumors