Non–small cell lung cancer (NSCLC) is a heterogeneous disease, with multiple different oncogenic driver mutations representing potential therapeutic targets [
1‐
3]. Approximately 25% of NSCLC patients present
KRAS mutations, which confer poor prognosis and high risk of disease recurrence [
4,
5]. Currently, there are no successful treatment strategies that target KRAS mutant tumors [
6‐
8]. Oncogenic KRAS has been shown to be a key factor in promoting metabolic rewiring, although the specific metabolic actors may differ depending on tumour type and genetic context [
9‐
12]. In NSCLC, abnormal activation of KRAS enhances glucose metabolism to fuel oxidative phosphorylation and increases glutamine metabolism, the latter feeding mitochondria and maintaining the redox balance through glutathione biosynthesis [
13‐
16].
Approximately half of NSCLC patients with activating
KRAS lesions have also deletions or inactivating mutations in the serine/threonine kinase 11 gene (
LKB1/STK11) [
17‐
20]. Alterations in LKB1 are spread all along the gene and comprise deletions, insertions, frameshift, nonsense and missense mutations. As recently reported,
STK11/LKB1 mutations were in their overwhelming majority predicted to be deleterious for protein function [
20]. LKB1 is a tumor suppressor that phosphorylates and activates several downstream targets to regulate signal transduction, energy sensing and cell polarity [
21,
22]. It has a pivotal role in metabolic reprogramming and nutrient sensing, mainly through its ability to activate AMP-activated protein kinase (AMPK) [
19,
23‐
26]. Inactivated
LKB1 is found in a wide range of human cancers including those of the pancreas, cervix and lung [
27,
28].
The role of
KRAS mutations and their potential association with other common genetic lung cancer lesions (
LKB1,
TP53) has recently been investigated in different cohorts of human lung adenocarcinomas using transcriptional, mutational, copy-number and proteomic data. These studies highlighted how
LKB1 inactivation is significantly associated with
KRAS mutations compared to
TP53 deletion and that co-occurrence of
KRAS mutation with inactivation of
LKB1,
TP53 or
CDKN2A/B genes generates different tumor subsets with distinct biology, immune profiles, and therapeutic vulnerabilities [
29]. The co-occurrence of
KRAS mutation and
LKB1 loss has been demonstrated to confer poor prognosis on advanced NSCLC patients mainly due to an increase in metastatic burden [
30]. These co-occurring lesions also engendered resistance against anticancer drugs in preclinical murine models of lung adenocarcinoma [
31]. Studies in genetically engineered mice have shown that the simultaneous presence of
KRASG12D mutation and deletion of
LKB1 in the lungs dramatically increases tumor burden and metastasis [
31]. While many efforts have been made to understand the impact of individual genetic alterations, such as those in
KRAS or
LKB1, on cellular metabolism, very little is known about any influence on metabolism of the simultaneous presence of these two genetic alterations. The oncogenic cooperation between the KRAS
G12D mutant and loss of LKB1 expression was firstly characterized in pancreatic cancer, where it disturbed one carbon metabolism and incited epigenetic modifications thus supporting cancer growth [
32]. In NSCLC, co-occurrence of mutant KRAS and LKB1 loss has been shown to impact on the urea cycle enzyme CPS1 providing an alternative pool of carbamoyl phosphate to maintain pyrimidine availability thus imposing a growth advantage on lung cancer cells [
33]. Since both
KRAS mutations and
LKB1 inactivating alterations affect cellular metabolism, it seems propitious to discern metabolic effects induced by the single oncogenic events from those elicited by their co-occurrence, with the ultimate aim to potentially exploit metabolic dependencies for novel therapeutic modalities. With these considerations in mind, we knocked-out the
LKB1 gene in well-characterized NSCLC cell clones harbouring KRAS wild type (WT) or mutant G12C proteins [
16,
34]. We obtained an isogenic system in which
KRAS mutation and
LKB1 inactivation were individually or concomitantly present. The effects of the genetic lesions individually or together on cell metabolism were investigated in these isogenic NSCLC cells by means of an integrated survey of proteomics, stable and dynamic metabolomics and functional in vitro strategies.