<p dir="ltr">Lung cancer is the leading cause of cancer-related deaths, and although important therapy advancements have been achieved, ~1.6 million people die from lung cancer annually. Non-small cell lung cancer (NSCLC), which makes up ~85% of lung cancer cases, is mainly treated with radiotherapy, chemotherapies, and targeted agents. Targeted agents are selected based on the mutation spectrum of the tumor. In NSCLC the epidermal growth factor receptor (EGFR) is commonly mutated and, leads to increased proliferation and cell survival. The standard-of-care treatment for patients with activating mutations in EGFR is treatment with tyrosine kinase inhibitors (TKI), such as erlotinib. While tumors initially respond to TKIs, after 1-2 years most patients develop resistance. In ~60% of TKI resistant tumors, resistance is the result of a secondary mutation in EGFR, whereas in the remaining 20%, tumors turn on bypass track-signals to overcome inhibition of the EGFR pathway. However, 15-20% of the cases the mechanisms underlying resistance are unknown. Most studies focus on the gain of function of oncogenes as mediators of resistance; however, little is known about the role that tumor suppressors play in TKI resistance. Hence, we performed a genome-wide CRISPR Cas9 knock-out screen to identify genes that when knocked-out would drive erlotinib resistance, and KMT5C was identified as the top candidate. KMT5C is a histone methyltransferase that trimethylates H4K20 (H4K20me3), enabling the establishment of constitutive and facultative heterochromatin. Data from human samples suggests that the <i>KMT5C</i> transcript is globally downregulated in NSCLC and in tumor samples resistant to the third generation TKI osimertinib. Additionally, loss of the modification H4K20me3, influences prognosis of NSCLC, indicating that loss of KMT5C function is a crucial mechanism in carcinogenesis. Here we describe how loss of KMT5C leads to increased transcription of the oncogene MET, due to a loss in H4K20me3-mediated repression of a long non-coding RNA transcription (LINC01510) upstream of MET. This mechanism was found to be partially responsible in driving TKI resistance in EGFR mutant cells. Historically, KMT5C has been associated with generation of constitutive heterochromatin (cHC); however, recent reports, including our own, indicate that KMT5C also regulates transcription in regions outside of cHC. Our preliminary evidence suggests that deposition of H42K0me3 via KMT5C in regions outside of cHC, is less stable than in cHC regions. This novel finding led us to hypothesize that regulation of KMT5C and H42K0me3 at different regions of heterochromatin is a dynamic process.</p>

<p>Lung cancer is the third most prevalent cancer in the world; however it is the leading cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for ~85% of the lung cancer cases. The current strategies to treat NSCLC patients with frequent causal genetic mutations is through targeted therapeutics. Approximately 10-35% of NSCLC patient tumors have activated mutations in the Epidermal Growth Factor Receptor (EGFR) resulting in uncontrolled cellular proliferation. The standard-of care for such patients is EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), a class of targeted therapeutics that specifically inhibit EGFR activity. One such EGFR-TKI used in this study is erlotinib. Following erlotinib treatment, tumors rapidly regress at first; however, over 50% of patients develop erlotinib resistance within a year post treatment. Development of resistance remains to be the major challenge in treatment of NSCLC using EGFR-TKIs such as erlotinib. </p> <p>In approximately 60% of cases, acquired erlotinib resistance in patients is attributed to a secondary mutation in EGFR, whereas in about 20% of cases, activation of alternative signaling pathways is the reported mechanism. For the remaining 15-20% of <a>cases</a> the mechanism of resistance remains unknown. Therefore, it can be speculated that the common methods used to identify genetic mutations in tumors post erlotinib treatment, such as histologic analysis and genetic screening may fail to identify alterations in epigenetic mediators of erlotinib resistance, also including microRNAs (miRNAs). MiRNAs are short non-coding RNAs that post-transcriptionally negatively regulate their target transcripts. Hence, in this study two comprehensive screens were simultaneously conducted in erlotinib sensitive cells: 1) a genome-wide knock-out screen, conducted with the hypothesis that loss of function of certain genes drive erlotinib resistance, 2) a miRNA overexpression screen, conducted with the hypothesis that certain miRNAs drive the development of erlotinib resistance when overexpressed. The overreaching goal of the study was to identify novel drivers of erlotinib resistance such as microRNAs or other epigenetic factors in NSCLC.</p><p>The findings of this study led to the identification of a tumor suppressive protein and an epigenetic regulator, SUV420H2 (KMT5C) that has never been reported to be involved in erlotinib resistance. On the other hand, the miRNA overexpression screen identified five miRNAs that contribute to erlotinib resistance that were extensively analyzed using multiple bioinformatic tools. It was predicted that the miRNAs mediate erlotinib resistance via multiple pathways, owing to the ability of each miRNA to target multiple transcripts via partial complementarity. Importantly, a correlation between the two screens was identified clearly supporting the use of two simultaneous screens as a reliable technique to determine highly significant miRNA-target interactions. Overall, the findings from this study suggest that epigenetic factors, such as histone modifiers and miRNAs function as critical mediators of erlotinib resistance, possibly belonging to the 15-20% of NSCLC cases with unidentified mechanisms involved in erlotinib resistance.</p><p></p>