Figure 6.

AXL is widely associated with positive regulation of autophagy signatures in human NSCLC transcriptome samples, and bemcentinib treatment induces programmed cell death displaying hallmarks of immunogenic cell death with the release of DAMPs (A–D) The correlation between AXL mRNA (fragments per kilobase of transcripts per million mapped reads; log₂, × axis) and four individually generated autophagy gene signatures (enrichment score, y axis) in human patient samples from TCGA lung adenocarcinoma cohort (n = 517). Correlation Rho and p value was computed by Spearman correlation coefficient rank test. Correlation between AXL (FKPM log₂) (x-axis) and enrichment score (y-axis). (A) Positive regulation of autophagy gene expression signatures from the Human Autophagy Database (http://autophagy.lu/index.html), (B) MSigDB v6.1.39 (C) Kumar Autophagy Network enrichment score,⁴⁰ (D) the Autophagy database.⁴¹ (E) Release of extracellular ATP after 24 hours treatment as indicated was measured by ATP luciferase assay for HCC827 parental cells and erlotinib-resistant clone ER3. ER10 cells responded similar to ER3 cells (data not shown). Fold change values of extracellular ATP normalized against vehicle control (DMSO) is shown for a representative experiment (n = 4). One-way analysis of variance was followed by Dunnett’s multiple comparisons test comparing every treatment against vehicle (DMSO) control (* p < 0.05, ****p < 0.0001). (F) Release of HMGB1 after 72 hours treatment as indicated was measured by ELISA for HCC827 parental and ER3 cells. Parental cells were treated with erlotinib (8 nM) with or without bemcentinib (0.8 μM), and ER3 cells were treated with erlotinib (1 μM) ± bemcentinib (0.8 μM). ER10 cells responded similar to ER3 cells (data not shown). Samples were normalized against the corresponding resazurin measurement for each well, and further normalized against the vehicle control (DMSO) for each cell line. Data are shown for a representative experiment (n = 4). One-way analysis of variance was followed by Dunnett’s multiple comparisons test comparing every treatment against vehicle (DMSO) control (****p < 0.0001). (G) Flow cytometric assessment of extracellular calreticulin (CALR). Histograms of ER10 cells treated for 7 days as indicated are shown, and MFI for all cell lines from a representative experiment is shown in the corresponding table (n = 4). HCC827 cells were treated with 0.8 μM bemcentinib, and ER3 and ER10 cells with 1.2 μM bemcentinib. (H) Alteration in the expression of major histocompatibility complex 1 assessed by flow cytometry after 7 days treatment with erlotinib (10 μM) with or without bemcentinib (0.8 μM for HCC827 parental cells, 1.2 μM for ER-cells). Histogram shown for ER3 cells. Median fluorescence intensity values for all cell lines are given in the corresponding table. ATP, adenosine triphosphate; CALR, calreticulin; HMGB1, high-mobility group box 1; MFI, median fluorescence intensity; MSigDB, Gene Set Enrichment Analysis Molecular Signatures Database; TCGA, The Cancer Genome Atlas.