MAPK modulation and its implications in KRAS mutant lung adenocarcinoma

Resumen

In lung adenocarcinoma (LUAD), virtually all genetic alterations driving tumor progression are directly linked to the RAS-MAPK pathway, upregulating its activity to provide tumor cells with a proliferative advantage. However, excessive MAPK signaling is detrimental. Through mechanisms that are far from being fully decoded, the tumor must undergo a process of selection for a set of both positive and negative regulators to keep balance. Although different mechanisms of MAPK regulation have been demonstrated, both in vitro and in mouse cancer models, its clinical impact is currently unknown. The aim of this thesis has been to shed light on the MAPK regulation network, studying how it is established by KRAS driven tumors in order to maximize their malignancy. Ultimately, we sought to better understand how this modulation is achieved by identifying additional, unknown regulators and developing in vivo and in vitro models to characterize their contribution to RAS-MAPK pathway. First, in order to investigate how MAPK signaling levels impact clinical outcome, we have refined a previously published transcriptional signature of genes whose expression, due to their implication in feedback loops, is dependent on MAPK activity. Further in vitro results confirm that this signature is extremely robust and is able to reflect the different levels of KRAS-MAPK activation. We applied our 4-gene signature to compute MAPK activity in KRAS mutant tumors from TCGA patient transcriptomic and clinical data. Strikingly, high MAPK tumors had a better prognosis compared to those with lower pathway output, implying moderate MAPK activation could confer greater aggressiveness. In parallel, highly active lesions are associated with various stress phenotypes, including increased genomic instability. Then, we analyzed the genomic landscape of the high and low MAPK groups. DUSP4 is a dual specificity phosphatase that originally was considered part of this MAPK-dependent signature, as it forms a transcriptional negative feedback loop with ERK. Paradoxically, this phosphatase¿s expression exhibited anti-correlation with pathway activation. Genomic analysis revealed a particular susceptibility to copy number variations (CNVs) at the DUSP4 locus. Low MAPK patients, with poor survival, showed increased copy number gains of DUSP4, while high MAPK patients, with better prognosis, exhibited more frequent copy number losses since early stages of the disease. Our working hypothesis postulates that DUSP4 loss initially confers a proliferative advantage but this turns into a deleterious effect during later tumor stages likely due to sustained MAPK activity. Using in vivo and in vitro models, we have validated that prompt deletion of DUSP4 grants a proliferative advantage in a KRAS driven context, however chronic ablation of this negative regulator is detrimental and limits tumor development. DUSP4 knockout induces an increase in MAPK activity, inducing stress phenotypes including genomic and oxidative damage. In addition, loss of DUSP4 in KRAS driven cell lines confers increased resistance to MEK inhibition, suggesting that DUSP4, and the observed genomic alterations surrounding this gene, might play a role in modulating MAPK inhibitor responses. In an attempt to model DUSP4 alterations in vivo, we took advantage of the inducible mutant KRAS mouse strain (KrasLSLG12V). We infected these mice with a dual viral system expressing Cre recombinase for KRAS activation and CRISPR/Cas9-mediated DUSP4 knockout, with the objective of studying how DUSP4 loss affects tumor initiation and progression in this context, similar to the one observed in the clinic. We confirmed that DUSP4 loss synergizes with KRAS activation and induces early neoplasias, confirming that loss of function alterations could be positively selected at initial stages. Despite this initial advantage, tumors lacking DUSP4 are less likely to progress into advanced lesions, indicating that a functional DUSP4, meaning functioning MAPK regulation, raises the odds of generating malignant tumors in advanced timepoints of these mouse models. All these results suggest that mechanisms that govern MAPK modulation and limit excessive hyper-signaling are crucial for tumor promotion. In order to better understand this process, we ought to identify additional modulators of the pathway. Combining an in vitro CRISPR screening with a set of differentially expressed genes between High and Low MAPK patients, we have identified several candidates coding for phosphatases PP1, PP2, PP4 and their regulatory subunits as potential novel pathway regulators. In this thesis we have also characterized, as putative regulators, TTC1, a membrane docker protein, and CSK, a negative regulator of the SRC family. In summary, we have shown that RAS-MAPK intricate regulation plays a key role in oncogenesis, as MAPK activation levels dictate patient outcome in KRAS mutant LUAD. This thesis has also contributed to the current knowledge on MAPK modulators, bringing back DUSP4 onto the table due to its unique dual behavior in patients. The characterization of novel candidates and phosphatases will continue to provide molecular insights of the fine tuning of the MAPK signaling cascade, which, down the road, will contribute to the development of innovative therapeutic strategies.