P3.02-078 Establishing Malignant Pleural Mesothelioma Primary Cell Lines Using the 3D Spheroid Method Produces a Model with Better Tumor Architecture

Abstract

Background: KRAS mutant non-small cell lung cancers (NSCLC) bearing co-mutations in LKB1 (KL tumors) have a distinct biology, molecular vulnerabilities, and therapeutic sensitivities. KL tumors are characterized by upregulation of the NRF2/KEAP1 pathway which may serve as a compensatory mechanism to maintain redox homeostasis in the face of oxidative stress. The NRF2/KEAP1 pathway plays a role in regulating glutathione synthesis from glutamate. We hypothesized that glutaminase inhibition would reduce intracellular glutamate, reduce the ability to tolerate oxidative stress, and enhance radiotherapy sensitivity in KL tumors with KEAP1/NRF2 pathway upregulation. Method: Expression of NRF2 and NRF2 related genes were analyzed by Western blotting in isogenic KRAS-mutant LKB1 deficient/proficient NSCLC cell lines. Intracellular reactive oxygen species (ROS) levels were monitored using 2'7'-dichlorofluorescein (DCFDA) and flow cytometry. Cell apoptosis analysis was determined using PE-conjugated annexin-V/7-AAD staining and carried out by flow cytometry. Cell growth was evaluated by Cell Titer Glo Assays. Ionizing radiation sensitivity was assessed by clonogenic cell survival assay (CSA). Result: LKB1 loss increased ROS accumulation and resulted in upregulation of NRF2 and NRF2 target genes in isogenic KRAS-mutant cell lines. KL cells were significantly more resistant to ROS than cells with KRAS mutation alone, as determined by the treatment with the ROS donor H 2 O 2. NRF knockdown abrogated this resistance to in KL cells. KL cells also were more resistant to radiotherapy. Re-expression of LKB1 or NRF2 pathway suppression (via KEAP1 expression or NRF2 knockdown) enhanced radiotherapy sensitivity as measured by CSA with a dose enhancement ratio (DER) 1.6 and 1.2, respectively, at a surviving fraction of 0.5. The glutaminase inhibitor CB839 enhanced the radio-sensitivity of KL cells with NRF2 pathway activation (via KEAP1 mutation). Re-expression of LKB1 or NRF2 pathway suppression abrogated the CB839-induced radiosensitization. Conclusion: Our results suggest that upregulation of the NRF2/KEAP1 pathway in NSCLC bearing co-mutations in KRAS and LKB1 is critical to maintain redox homeostasis and GLS inhibition can sensitize KL tumors to radiotherapy in an LKB1-and KEAP1/NRF2-dependent manner.

Genomic Deletion of BAP1 and CDKN2A Are Useful Markers for Quality Control of Malignant Pleural Mesothelioma (MPM) Primary Cultures

“…It is therefore important to establish cultured cells from mesothelioma biopsies and pleural effusions to be utilized for studying the cellular, molecular, and genetic levels of this tumor. Over a period of four years (2013–2017), we established fifteen cell lines (defined as successful subculture) from a primary culture [ 27 ] from fourteen human tumor and/or plural effusion samples. In the current study, we utilized these established mesothelioma cell lines to determine biomarker expression in a system that closely parallels the tumor.…”

“…This study aimed to utilize the MPM cell lines that were established between 2013–2017 [ 27 ] to study different types of biomarkers, including protein markers using IHC and genomic markers using qualitative FISH analysis coupled with absolute quantification analysis with droplet digital PCR (ddPCR).…”