Pancreatic adenocarcinoma (PDAC) is an aggressive disease that is typically diagnosed at a late stage. PDAC development occurs over approximately 20 years and the initiating event is often an activating mutation in key cell signalling hub KRAS. Despite extensive research into PDAC and KRAS, the impact of both genetic and non-genetic variability during the earliest steps of carcinogenesis are scantly described. We hypothesise that both forms of heterogeneity might alter the efficiency of PDAC initiation. On the one hand, oncogenic KRAS alleles might rewire cellular signalling and metabolic networks causing phenotypes and adaptation in an allelespecific manner. On the other hand, cell-to-cell variability within mutant clones might permit a population of cells to escape tumour suppressive mechanisms such as senescence and adapt to oncogenic signalling, therefore supporting early cancer development. We have combined cell genetics, single-cell biochemistry and a multiomics approach to investigate both genetic and non-genetic heterogeneity during initiation of PDAC. We generated an isogenic panel of cell lines harbouring the three most common mutations of KRAS in PDAC (G12D, G12R, G12V). This system consisted of inducible expression of KRAS mutants in immortalized but non-transformed pancreatic cells to mimic PDAC initiation. In Chapter 2, we explore the short-term cellular response to KRAS induction and adaptation of cells to oncogenic KRAS. Using western blotting, metabolomics and RNAseq we show how specific KRAS mutant alleles influence the cell signalling, nucleotide metabolism and cell morphology. We show that the G12D and G12V mutants, frequently observed in KRAS-driven cancers, differ significantly from the PDAC-specific G12R mutant. The mutants display allele-specific growth factor signalling, purine metabolism rewiring and an epithelial to mesenchymal transition. In Chapter 3, we looked in greater temporal resolution and at a single-cell level at the immediate signalling and metabolic response to KRAS mutations, focusing mainly on G12D. We used a FRET sensor reporting mitochondrial ATP levels in live cells to study G12D cells at multiple time points. We found an early drop in mitochondrial ATP which was only identified when considering intracellular heterogeneity. This drop was followed by a peak in mitochondrial ATP at a later time point, caused by a combination of purine synthesis and mitochondrial ATP production. We also observed an increase in activated and total levels of mitochondrial dynamics protein DRP1 and the presence of an anti-oxidant signature. The involvement of DRP1 suggests an increase in mitochondrial homeostasis early during adaptation to oncogenic KRAS. Using techniques such as metabolomics, phosphoflow and scRNAseq we were able to further dissect this adaptation process. We also found heterogeneity in cells exploring broader levels of phosphorylated ERK upon G12D induction, which further varied over the studied time course. The recorded variability of pERK levels supports the hypothesis of an oncogenic signalling sweetspot. Taken together, this study sheds light on the process of adaptation to oncogenic KRAS. Adaptation to mutant KRAS occurs over multiple stages even within this phase of early adaptation. These stages include variation in pERK signalling, nucleotide metabolism and likely mitochondrial homeostasis. Our results suggest that both genetic and non-genetic heterogeneity play important roles in these processes and thus, the earliest events of PDAC initiation. Understanding signalling and metabolic rewiring in a KRAS mutant specific manner may help us untangle allele-specific dependencies that could inform patient stratification and disease management. Furthermore, identifying processes by which normal cells can adapt to oncogenic KRAS at a single-cell level reveals the necessary steps for disease initiation, and may provide options for preventative intervention.