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Inhibition of KRAS oncogenic activity by interfering with new KRAS interactors

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Universitat de Barcelona. Departament de Biomedicina

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[eng] INTRODUCTION:

The Kirsten rat sarcoma oncogene (KRAS) homologue stands out as a prominent oncogene, distinguished by its notably elevated mutation rate compared to other oncogenes. This genetic anomaly is intricately linked to several highly lethal cancer types such as pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC) (L. Huang et al., 2021). 
The KRAS gene is a member of the rat sarcoma viral oncogene family (RAS), alongside two other human isoforms: the Harvey and neuroblastoma rat sarcoma viral oncogenes (HRAS, NRAS). RAS proteins are small GTPases that transit between an active (GTP-bound) and an inactive (GDP-bound) state; and play a crucial role in regulating essential cellular processes by transducing signals originating from cell surface receptors.

HYPOTESIS AND OBJECTIVES:
Our hypothesis inclusively posits that a promising avenue for combating oncogenic KRAS involves the targeted interaction with either the effector binding domain or the allosteric region of KRAS. By selectively binding to these regions, novel compounds, a peptidomimetic (P1.3) and a small molecule (P14B) have the potential to modulate the interaction between KRAS and effector or non-effector proteins, respectively, ultimately triggering apoptotic pathways leading to cell death. Through a comprehensive understanding of the mechanisms by which these compounds operate, we anticipate the refinement of therapeutic approaches and the development of enhanced treatment strategies. This deeper insight is crucial for optimizing clinical trial outcomes and ultimately improving patient prognosis. 

A) Peptidomimetics binding to the effector domain of KRAS. 

The objectives of this project are the following: 
1. Building upon the experimental findings from P1.3 treatment of cells concerning KRAS downstream signalling and the binding dynamics between KRAS and its main effectors, CRAF and PI3K, our objective is to investigate the direct interaction between P1.3 and KRAS-GTP, and specifically, we aim to discern whether treatment with P1.3 influences the levels of KRAS-GTP. 
2. To assess the toxicity profile of P1.3 through experimentation involving athymic mice. This investigation will provide valuable insights into the safety and tolerability of P1.3 in an in vivo model system. 

B) Small compounds binding to the allosteric domain of KRAS. 
The objectives of this project encompass a comprehensive investigation into the effects of P14 and its derivatives in both Colorectal Cancer (CRC) cells and Pancreatic Ductal Adenocarcinoma (PDAC) cells. To achieve this, we delineate the following steps: 
	1. Exploration of P14 and P14B effects in cultured cell lines: a. Assess the impact of P14 and P14B treatment on KRAS downstream signalling in CRC and PDAC cells. 
	b. Investigate the effects of various P14 derivatives (P14A, P14B, P14C, P14D, P14E, P14F, P14G, P14H, and P14I) in CRC cells. 
	c. Examine the differential influence of P14B on KRAS downstream signalling in cell lines with wild-type KRAS compared to those with oncogenic KRAS. 
	
	d. Analyse the impact of P14B on the binding affinity between oncogenic KRAS and its main effectors: CRAF, BRAF, and ARAF. 
e. Study the effect of P14B on the survival of CRC cells growing in 2D or 3D (Matrigel and CRC patients-derived organoids). 
2. Characterization of P14B Interactions in vitro: 
	
	a. Conduct surface plasmon resonance analysis to elucidate the potential direct interaction of P14B with oncogenic KRAS. 
	b. Employ in vitro calmodulin (CaM)-pulldown assay to investigate the effect of P14B on the CaM binding to KRAS. 
	

3. Determine the protein interactions of KRAS affected by P14B treatment utilizing the proximity-dependent biotinylation technology (UltraID).

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Tesis Doctorals - Departament - Biomedicina

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Inhibition of KRAS oncogenic activity by interfering with new KRAS interactors