K-RAS mutations and DNA repair function in NSCLC
At present, after decades platinum compounds are still the cornerstone for the treatment of NSCLC and still in the targeted therapy era they are the most active compounds used in first line treatment. However, the prognosis of patients with advanced NSCLC remains poor and rarely exceeds 10 months, therefore there is the urgent need to develop strategies for these patients and in particular for those insensitive to platinum compounds. KRAS mutations and ERCC1 expression are indicated as two important biomarkers in predicting the outcome of chemotherapy in NSCLC. However despite several evidence they are not yet implemented in the clinical practice in NSCLC. This because they have been studied in retrospective series and with heterogeneous detection criteria. We hypothesized that there could be a correlation between KRAS and the DNA repair efficiency which renders NSCLC tumour cells insensitive to platinum compounds. Our study on one side will prospectively assess the combined role of the two biomarkers and on the other side define the putative molecular mechanisms of interaction between KRAS and the DNA repair machinery trying to identify also specific alterations in the functional DNA repair status that would render KRAS mutated NSCLC patients amenable to alternative conventi onal or molecularly targeted therapies. Finally, retrospectively we will assess the prognostic potential of novel biomarkers related to either KRAS signalling, DNA repair ability, or combinations thereof in the case series/biobank accrued for the prospective validation of KRAS and/or ERCC-1.
We hypothesized that there could be a correlation between KRAS and the DNA repair efficiency which renders NSCLC tumour cells insensitive to platinum compounds.
At preclinical level we found an up-regulation of the gene DNA polymerase beta, both at mRNA and protein level, in KRAS mutated clones obtained from H1299 cell lines. DNA Polymerase Beta (POLβ) is a protein with a key role in BER (Base Excision Repair) pathway. Studying a panel of human lung cancer cells, a direct correlation between cisplatin resistance and POLβ expression was found and clearly demonstrated the direct involvement of POLβ in the cisplatin resistance of KRAS mutated clones. In fact, POLβ inhibition by treatment with palmitic acid restored cisplatin sensitivity in mice transplanted with KRAS-G12C transfected H1299 cells.
At clinical level, 187 patients have been enrolled. All patients signed the written informed consent. POLβ negative expression, evaluated in the Biorare prospective series, was associated with longer survival.
For the NER (Nucleotide Excision Repair), considered the most important DNA repair system for cisplatin, it seems that only the specific isoform 2 of ERCC1, a protein with a pivotal role in NER seems now to be crucial for an effective repair. Due to this, we applied in the series the PLA (Proximity Ligation Assay) technique which allows the identification of ERCC1 protein only in its active form, when associated with XPF protein. Patients with higher “active” ERCC1 and therefore an increased ability to repair DNA damage result in a significantly shorter progression free survival after platinum treatment (HR=2.58, p=0.020).
The results obtained identify suitable biomarkers (POLβ levels and active ERCC1), which effectively help in predicting prognosis and response to treatment in NSCLC.
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This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 964264.