Abstract 2300: Overcoming TKI-resistance in NSCLC using SphK2 inhibitors

In 2020, 228,820 lung cancer cases and 135,720 deaths have been anticipated. NSCLC is currently treated with Tyrosine Kinase Inhibitors (TKIs) such as Erolitinib (ER; second generation) and Osimertinib (OR; third generation). However, patients often develop resistance to these drugs overtime. This acquired resistance to TKIs is due to a secondary T790M mutation in the kinase domain which could be responsible for inducing Epithelial Mesenchymal Transition (EMT). EMT is a vital process in development of metastasis and occurs, by downregulation of E-cadherin, and upregulation of proteins like N-cadherin and vimentin. Therefore, it is imperative to find other molecularly targeted therapies to combat TKI resistance. Sphingosine kinase (SphK) is a type of lipid kinase that phosphorylates sphingosine to sphingosine-phosphates. There are two functional SphK isoforms (SphK1 and SphK2) that have been identified and characterized in mammalian cells. As a member of the SphK family, SphK2 activity can be stimulated by a series of cytokines such as EGF and VEGF, and acts as an oncogenic enzyme in tumor cells. Overexpression of SphK2 has been demonstrated in many cancer types and is closely related with cancer progression and the poor survival of patients. Currently minimal studies have been done on lung cancer and SphK2. This study investigated the roles of SphK2 in OR-resistant wild-type EGFR H2170 and H358, EGFR-mutated and TKI-sensitive H3255 cells (L858R mutation), and EGFR mutated TKI-resistant H1975 cells (L858R and T790M mutation). The modulation of EMT biomarkers was determined by qPCR. Key EMT regulators such as vimentin and N-cadherin were upregulated by 2.5 and 1.5-fold, respectively, while E-cadherin was downregulated by 1.5-fold. Immunofluorescence studies with Vimentin showed that H1975OR cells were elongated and have a mesenchymal phenotype compared to the H3255OR and their respective parental counterparts. MTT studies done using inhibitors of SphK2 in NSCLC OR-resistant cells showed a 3.0-fold decrease in cell viability in comparison to a 2.0-fold decrease in their parental counterparts. In wound closure assays at 24 hours the wound closure was 1.5-fold less in cells treated with SphK2 inhibitor as compared to parental cells treated with SphK2 inhibitors. Currently studies are being done to determine expression of SphK2 in late and early stage NSCLC tumors. In conclusion SphK2 was found to be a crucial positive regulator of different modes of cell migration and a key biomarker in combating TKI-resistance in NSCLC. Citation Format: Nabiha Haleema Khan, Neelu Puri. Overcoming TKI-resistance in NSCLC using SphK2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2300.