Abstract A099: Utilising interactomics to uncover oncogenic KRAS signalling networks in the context of the tumor microenvironment
Authors: Haider Nasir A. , Kelly Joanna , Smith Duncan , Jorgensen Claus 
Published: January 2024

Abstract
Extensive genetic analyses of Pancreatic Ductal Adenocarcinoma (PDAC) tumors have identified the GTPase KRAS as a major driver of tumorigenesis, with over 90% of tumors possessing oncogenic KRAS mutations, primarily at the mutational hotspot G12. KRAS G12 mutants are constitutively active, and promote cancer growth by hyperdriving multiple downstream effector pathways via direct physical interactions with proteins such as PI3K and RAF. Despite KRASG12Mut being a promising drug target, direct targeting of KRASG12Mut or downstream pathways has shown limited success, with the emergence of rapid resistance. This resistance is associated with compensatory rewiring of parallel pathways, and the emergence of drug-resistant mutations. Further, the failure to translate these therapies into the clinic maybe due to the inability of many preclinical models to incorporate the contribution of the tumor microenvironment (TME). Approaches to study KRAS signalling in complex multicellular environments have often relied on techniques such as transcriptomics that utilise inference to determine signalling dynamics rather than the direct contribution of KRAS to these outputs. Further, these approaches are often unable to determine cell-type specific signalling. A promising methodology to capture cell-specific KRAS signalling in a heterocellular setting is proximity labelling. This technique involves the engineering of cell lines to express a KRASG12Mut transgene fused to a promiscuous biotin ligase (TurboID). The KRASG12Mut-TurboID fusion is capable of labelling proximal proteins within a radius of ~10 nm with a biotin tag. Biotinylated proteins can be isolated and identified using mass spectrometry. As the labelling radius is narrow, with a short labelling time, this approach is ideal for cell-type specific signalling network analysis. Utilising a novel three-dimensional hydrogel scaffold mimicking the human tumor microenvironment, we are characterising the interactomes of KRASG12Mut across a library of patient derived organoids (hPDOs) grown in co-cultures with stromal cells using proximity labelling. Initial characterisation of KRASG12Mut interactomes in a cohort of 10 hPDOs grown in monoculture has highlighted a high degree of diversity among interactors and pathway activation, independent of the type of KRAS mutation. Specifically, unique patterns of interactions were observed in PI3K, mTOR and MAPK pathway components. Further, co-culture of hPDOs with stromal cells, such as fibroblasts and myeloid cells, yields differential engagement of KRAS interactors when compared to hPDOs grown in monoculture indicative of a role for the TME in shaping KRAS signalling. Current work is ongoing to determine the effect of KRAS targeting therapies on adaptive pathway rewiring using this approach. Interrogation of these interactomes has the potential to uncover resistance mechanisms that emerge in response to KRAS pathway inhibition, and identify novel targets for combinatorial therapy for future studies and clinical trials for pancreatic cancer patients.
Citation Format: Nasir A. Haider, Joanna Kelly, Duncan Smith, Claus Jorgensen. Utilising interactomics to uncover oncogenic KRAS signalling networks in the context of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A099.