Abstract
Responses to targeted therapies are often dramatic in patients with advanced lung cancers that harbour actionable oncogenic mutations, such as mutations in EGFR or KRAS, however relapse is almost inevitable. Drug tolerant persisters (DTPs) are thought to be a major contributor toward acquired resistance and remain a difficult clinical challenge for patients receiving targeted therapies. While our understanding of the DTP phenotype has expanded in recent years, no strategies to eliminate DTPs in a clinical setting have been successful to date. Long non-coding RNAs (lncRNAs) are known to contribute to many processes thought to underpin DTPs. Despite this, the contribution of lncRNAs to drug tolerance is poorly understood, leaving a gap in our understanding of this elusive phenotype.
In this study, we aimed to address this gap by investigating lncRNAs in DTP models in NSCLC. To address this aim, we first developed a model of DTPs in NSCLC cell lines by treating EGFR- mutant PC9 cell lines with the EFGR inhibitor osimertinib and KRASG12C mutant H358 cell lines with the KRAS inhibitor sotorasib. We compared the expression of markers of the DTP phenotype in our models to published literature, determining that we had developed a model of DTPs comparable to previous findings. Adaptive mutability, a hallmark of DTPs in EGFR- mutant models, had not previously been investigated in a KRAS-mutant DTP model. We found that the KRAS-mutant H358 cell line exhibited a transcriptional response that resembled adaptive mutability when treated with sotorasib.
With a model of DTPs established and characterised in NSCLC cell lines, we investigated lncRNAs in our DTP models using a pre-existing bulk RNA-sequencing dataset. We identified several lncRNAs of interest and validated our findings via RT-qPCR. We then investigated scRNA-seq datasets from previous literature, including a dataset from residual disease in patients. We found the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were upregulated by treatment with targeted therapies in both our DTP models and in scRNA-seq datasets from previous studies. MALAT1 and NEAT1 are reported to be involved in several processes that underpin the DTP phenotype and were therefore selected for further study.
We successfully knocked down MALAT1 and NEAT1 with antisense oligonucleotides (ASOs) and performed proliferation assays, concluding that MALAT1-knockdown enhanced the effect of both osimertinib and sotorasib in PC9 and H358 cell lines respectively. We then performed cell confluency assays under treatment over long time points and identified that MALAT1 knockdown reduced cell confluency for up to 5 weeks in PC9 DTP models treated with osimertinib. Using both RT-qPCR and SplintR RT-qPCR approaches, we determined that inhibition of cell growth was likely to be abated in the previous long-term assays due to ASO dilution or degradation.
We then sought to understand the contribution of MALAT1 in DTPs. MALAT1 and NEAT1 are known to regulate several DNA damage and repair pathways that are known to contribute to the adaptive mutability phenotype in DTPs. We investigated knockdown of MALAT1 and NEAT1 in pathways that contribute to adaptive mutability in DTPs via RT-qPCR, western blotting and flow cytometry. We found that while MALAT1 may regulate homologous recombination in untreated controls, this effect does not appear to be additive or synergistic with drug treatment in DTPs in our models.
To better understand pathways that MALAT1 may regulate in DTPs, we performed bulk RNA- sequencing following ASO-mediated knockdown of MALAT1 in osimertinib treated PC9 DTP models. We compared expression to untransfected osimertinib treated PC9 DTPs over 3-, 7- and 19-day time points. We found that MALAT1 may regulate expression of the IL-6 family of cytokines and signalling downstream of these pathways, including plasminogen activation and Wnt signalling pathways as these pathways were upregulated in MALAT1-KD DTP models.
Overall, the work described in this thesis represents the first evidence for a role for the lncRNA MALAT1 in drug-tolerant persisters in NSCLC. These findings suggest that MALAT1 may be important for the DTP phenotype by regulating the IL-6 cytokine family and could represent a candidate for therapeutic targeting in NSCLC tumours undergoing treatment with targeted therapies.
Overall, the work described in this thesis represents the first evidence for a role for the lncRNA MALAT1 in drug-tolerant persisters in NSCLC. These findings suggest that MALAT1 may be important for the DTP phenotype by regulating the IL-6 cytokine family and could represent a candidate for therapeutic targeting in NSCLC tumours undergoing treatment with targeted therapies.