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EGFR is the most common genetically altered oncogene in glioblastoma (GBM), but small-molecule EGFR tyrosine kinase inhibitors (TKI) have failed to yield durable clinical benefit. Here, we show that in two novel model systems of acquired resistance to EGFR TKIs, elevated expression of urokinase plasminogen activator (uPA) drives signaling through the MAPK pathway, which results in suppression of the proapoptotic BCL2-family member protein BIM (BCL2L11). In patient-derived GBM cells and genetic GBM models, uPA is shown to suppress BIM levels through ERK1/2 phosphorylation, which can be reversed by siRNA-mediated knockdown of uPA. TKI-resistant GBMs are resensitized to EGFR TKIs by pharmacologic inhibition of MEK or a BH3 mimetic drug to replace BIM function. A link between the uPA–uPAR–ERK1/2 pathway and BIM has not been previously demonstrated in GBM, and involvement of this signaling axis in resistance provides rationale for a new strategy to target EGFR TKI-resistant GBM. Cancer Res; 75(2); 394–404. ©2014 AACR.

As the infectious agent causing human adult T-cell leukemia (ATL), the human T-cell leukemia virus type 1 (HTLV-1) virus spreads in vivo primarily by cell-to-cell transmission. However, the factors that determine its transmission efficiency are not fully understood. The viral genome encodes the HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cases and is known to promote T-cell proliferation. In this study, we investigated the hypothesis that HBZ also influences the survival of T cells. Through analyzing the transcriptional profile of HBZ-expressing cells, we learned that HBZ suppressed transcription of the proapoptotic gene Bim (Bcl2l11) and that HBZ-expressing cells were resistant to activation-induced apoptosis. Mechanistic investigations into how HBZ suppresses Bim expression revealed that HBZ perturbs the localization and function of FoxO3a, a critical transcriptional activator of the genes encoding Bim and also Fas ligand (FasL). By interacting with FoxO3a, HBZ not only attenuated DNA binding by FoxO3a but also sequestered the inactive form of FoxO3a in the nucleus. In a similar manner, HBZ also inhibited FasL transcription induced by T-cell activation. Further study of ATL cells identified other Bim perturbations by HBZ, including at the level of epigenetic alteration, histone modification in the promoter region of the Bim gene. Collectively, our results indicated that HBZ impairs transcription of the Bim and FasL genes by disrupting FoxO3a function, broadening understanding of how HBZ acts to promote proliferation of HTLV-1–infected T cells by blocking their apoptosis. Cancer Res; 74(1); 188–200. ©2013 AACR.

Recent advances in the treatment of non-small cell lung cancer (NSCLC) stem from the paradigm shift of classifying patients into subtypes based upon the presence of distinct molecular drivers. Subsets of patients, such as those with EGFR mutations and ALK translocations, have dramatic responses in their tumors to tyrosine kinase inhibitors (TKIs) that specifically inhibit these oncogenic drivers. While many patients initially response to TKIs, therapeutic resistance is inevitable. For EGFR-mutant NSCLC, there are multiple described mechanisms of resistance to EGFR TKIs, including epithelial-mesenchymal transition (EMT). Previous studies have implicated the AXL kinase and ZEB1, an EMT transcription factor (EMT-TF), in EMT-mediated EGFR TKI resistance. We have previously demonstrated that the EMT-TF, TWIST1, is required for oncogene-driven NSCLC tumorigenesis, including those tumors with EGFR mutations. In this study, we investigated the role of TWIST1 in EMT-mediated resistance to EGFR TKIs. We have demonstrated that genetic or pharmacologic inhibition of TWIST1 resulted in growth inhibition in a panel of EGFR-mutant NSCLC cell lines and apoptosis in a subset of these lines. Interestingly, TWIST1 overexpression in EGFR-mutant NSCLC cell lines led to EGFR TKI resistance. Conversely, knockdown of TWIST1 in an erlotinib resistant EGFR-mutant NSCLC cell line restored erlotinib sensitivity. We found that TWIST1 mediates resistance to EGFR TKIs through suppression of apoptosis possibly through decreasing the expression of the pro-apoptotic Bcl-2 member, BCL2L11 (BIM). We observed that TWIST1 knockdown increased BIM levels, while TWIST1 overexpression decreased BIM expression. Furthermore, TWIST1-mediated resistance was overcome by treatment with the BCL-2/BCL-XL inhibitor, ABT-737. Knockdown of BIM recapitulated the resistance seen following TWIST1 overexpression, suggesting that TWIST1 suppression of BIM is a mechanism through which TWIST1 leads to EGFR TKI resistance. To explore the role of TWIST1 in modulating EGFR inhibitor sensitivity in vivo, we used an inducible EGFR-mutant transgenic mouse model, CCSP-rtTA/tetO-EGFRL858R (CE), which expresses EGFRL858R in the lung and a EGFR-mutant/Twist1 transgenic model, CCSP-rtTA/tetO-EGFRL858R/ Twist1- tetO7-luc (CET), which expresses both Twist1 and EGFRL858R in the lung. CET mice had a significantly increased tumor burden, decreased apoptosis and a decreased overall survival compared to CE mice following erlotinib treatment. In summary, we found that TWIST1 overexpression leads to EGFR TKI resistance by suppressing EGFR TKI-induced apoptosis through suppressing BIM expression. Future studies aim to establish the mechanisms of TWIST1 suppression of BIM expression and determine if our TWIST1 inhibitor, harmine, is effective in overcoming EMT-mediated resistance. Citation Format: Zachary A. Yochum, Hailun Wang, Jessica A. Cades, Susheel Khetarpal, Eric H. Huang, Phouc T. Tran, Timothy F. Burns. The EMT transcription factor TWIST1 mediates resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2017-4118

BIM (BCL2L11) is a BH3-only proapoptotic member of the Bcl-2 protein family. BIM upregulation is required for apoptosis induction by EGF receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) in EGFR-mutant forms of non–small cell lung cancer (NSCLC). Notably, a BIM deletion polymorphism occurs naturally in 12.9% of East Asian individuals, impairing the generation of the proapoptotic isoform required for the EGFR-TKIs gefitinib and erlotinib and therefore conferring an inherent drug-resistant phenotype. Indeed, patients with NSCLC, who harbored this host BIM polymorphism, exhibited significantly inferior responses to EGFR-TKI treatment than individuals lacking this polymorphism. In an attempt to correct this response defect in the resistant group, we investigated whether the histone deacetylase (HDAC) inhibitor vorinostat could circumvent EGFR-TKI resistance in EGFR-mutant NSCLC cell lines that also harbored the BIM polymorphism. Consistent with our clinical observations, we found that such cells were much less sensitive to gefitinib-induced apoptosis than EGFR-mutant cells, which did not harbor the polymorphism. Notably, vorinostat increased expression in a dose-dependent manner of the proapoptotic BH3 domain-containing isoform of BIM, which was sufficient to restore gefitinib death sensitivity in the EGFR mutant, EGFR-TKI–resistant cells. In xenograft models, while gefitinib induced marked regression via apoptosis of tumors without the BIM polymorphism, its combination with vorinostat was needed to induce marked regression of tumors with the BIM polymorphism in the same manner. Together, our results show how HDAC inhibition can epigenetically restore BIM function and death sensitivity of EGFR-TKI in cases of EGFR-mutant NSCLC where resistance to EGFR-TKI is associated with a common BIM polymorphism. Cancer Res; 73(8); 2428–34. ©2013 AACR.

Inflammation is critical for estrogen (E2) to undertake apoptosis in E2-deprived breast cancer cells. However, it remains unclear how E2 induces inflammation. Here, we demonstrate that E2 increases lipid metabolism and accumulates unfolded proteins in the endoplasmic reticulum in E2-deprived breast cancer cells. These two major alterations result in different inflammatory responses. E2 rapidly increases lipid metabolism related genes such as fatty acid desaturase 1 (FADS1) and CCAAT/enhancer binding protein beta (CEBPB) to modulate adipose inflammation related factors, e.g. IL6/IL6R after two hours treatment, which regulate the cellular proliferation or differentiation. Simultaneously, accumulation of the unfolded protein activates unfolded protein response (UPR) sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring protein 1 alpha (IRE1α) within few hours after exposure to E2. Importantly, inhibition of PERK, rather than IRE1α, is able to completely prevent E2 from induction of late event inflammatory genes associated with apoptosis, such as tumor necrosis factor (TNF) superfamily members, BCL2L11 (Bim), and HMOX1,which are activated by E2 after 48 hours. Consistently, nuclear factor-kappa B (NF-κB) is activated when TNFα is maximal after E2 treatment. Further examination revealed that an anti-inflammatory agent, dexamethasone (Dex), activates glucocorticoid receptor (GR) transcriptional activity and blocks E2-induced apoptosis. These effects can be reversed by the knockdown of GR. A notable mechanistic change is that Dex effectively blocks the phosphorylation of PERK and produces equivalent inhibitory effects on the apoptosis-associated inflammatory genes as the PERK inhibitor. However, Dex has an additional potential to activate lipid metabolism and are additive with E2 to elevate FADS1 and adipokine IL6/IL6R. All of these data, for the first time, describe the mechanisms underlying E2-induced inflammation in E2-deprived breast cancer. It provides important information to restrict the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in estrogen deprived breast cancer patients. Citation Format: Ping Fan, Fadeke A. Agboke, Amit Kumar Tyagi, V. Craig Jordan. Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2863.

Glucocorticoids (GCs) are fundamental components of most chemotherapies employed against lymphoid malignancies because they cause apoptosis and cell cycle arrest. In this study we investigate the possible role of a novel splice variant of human BCL2L11/Bim, termed BB, for GC-induced apoptosis. First evidence for BB was obtained by Affymetrix-based whole genome comparative expression profiling, where a corresponding probe set was found to be induced in 8 of the 13 ALL children and 1 adult during systemic GC monotherapy (Schmidt et al., 2006). The postulated BB mRNA consists of the 5’ portion of Bim (up to and including the BH3-containing exon 8) and the 3’ portion of Bam, a cDNA originally discovered in a multiple myeloma cDNA library (Claudio et al., 2002). Thus, it encodes a putative protein consisting of the N-terminal region of Bim (including its BH3 domain) and 40 C-terminal amino acids derived from Bam and not present in any known Bim transcript. BB and Bim proteins have similar molecular masses and could not be distinguished by conventional Western technology when probed by α-Bim antibody as most α-Bim antibodies recognize the N-terminal region which is common to both isoforms. RT-PCR data and protein analyses carried out by using α-Bam antibody (that recognizes the Bam specific C-terminal region) on GC-treated CEM-C7H2 cells supported the existence of the postulated BB mRNA and protein. Subcellular localization analysis of BB suggested that BB may be directed to mitochondria, ascribed probably to its BH3-domain interaction with other BCL2 proteins and not by its unique C-terminus which in contrast to the Bim C-terminus doesn't localize to mitochondria. The functional analyses with conditional overexpression in ALL in vitro model revealed that overexpressed BB is a killer protein with potency similar to that of Bim and thus may contribute to the anti-leukemic effects of GCs and perhaps other apoptotic responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1029.