Fig 2: Effects of BAY1143572 on MCL-1 protein and RNA levels in in vitro models of esophageal adenocarcinoma. (A) Cells were treated with BAY1143572 at the indicated doses for 4 h. The phosphorylation of RNAPII and the expression of MCL-1 were examined by Western blotting. (B) Cells were treated with 1 µM BAY1143572 for 4 h after pretreatment with or without MG-123 for 1 h. MCL-1 protein levels were assessed by Western blotting. (C) MCL-1 mRNA levels were measured by quantitative real-time polymerase chain reaction (qPCR) after treatment with the indicated doses of BAY1143572 for 4 h. (D) Chromatin immunoprecipitation (ChIP) was used to assess the binding of HIF-1α to the MCL-1 promoter in FLO-1 and OE33 cells treated with 1 µM BAY1143572 or vehicle only for 4 h. qPCR results show the means of experiments performed in triplicate for each treatment condition. Similar results were observed in two independent experiments.

Fig 3: BCL-XL and MCL-1 but not BCL-2 are highly expressed in SCCHN cell lines and oral cavity tissue microarrays from SCCHN patients.a Western blot analysis of the three major anti-apoptotic BCL-2 family proteins in SCCHN cell lines. Sites of primary tumors are indicated in brackets: OC oral cavity, LX larynx, OP oropharynx. b BH3 profiling with increasing concentrations of BIM peptide revealed that the indicated SCCHN cell lines were primed to undergo apoptosis. c SCCHN cell lines exposed to the indicated BH3 mimetics (100 nM) for 24 h failed to undergo apoptosis, as assessed by phosphatidylserine (PS) externalization. d–f Representative IHC images of oral cavity (OC) normal tissue, primary tumor core and advancing front (AF), stained using antibodies against d BCL-2 e BCL-XL, and f MCL-1 (clone D5V5L) and counterstained with hematoxylin. Scale bars 50 μm. Dot plots depict the relative staining intensities (H-scores) of the specified antibody. Each dot represents the data from an individual patient. The IHC images shown correspond to the yellow circles in the graph. b, c One-way ANOVAs with Dunnett’s multiple comparisons tests, with a single pooled variance. Error bars = mean ± SEM of at least three independent experiments. d–f Normality tests were performed, followed by Kruskal Wallis tests with Dunn’s multiple comparisons tests. Error bars = mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Pharmacological inhibition of MCL1 in PEL and control cell lines and MCL1 expression in PEL tumors. a Calculated IC50 values of S63845 in different cell lines. Based on Kotschy et al.53, Daudi and MEG-01 cells are MCL1-independent cell lines while Raji and KMS-12-BM are MCL1-dependent cell lines. Error bars, SEM; n ≥ 3 biological replicates. b MCL1 staining in tonsilar sections confirms specificity of our staining protocol. Germinal center cells (GC) express high levels of MCL1 while mantle zone cells (MZ) express low to undetectable levels of MCL1. c Example of a histological characterization of tumor sections from patient D (84-year HIV(−) female with EBV(+) tumor) with hematoxylin and eosin staining (H&E), LANA immunohistochemical stain, or MCL1 immunohistochemical stain. d MCL1 immunohistochemical stains for tumor sections from patients A–C. Corresponding scale bars are depicted in lower right corner of each image

Fig 5: Revised working model of the main host oncogenic gene dependencies in PEL cell lines. a Genes involved in NF-κB and Jak/Stat signaling are dispensable in most PEL cell lines and may serve fitness functions. b Addiction to constitutively active mTOR signaling was not captured in this study, due to confounding housekeeping function of mTOR. c Critical PEL-specific oncogene dependencies (PSODs) on IRF4, MDM2, CCND2, CFLAR, and MCL1 (in blue), most of which are druggable using agents shown in red