Patho-)physiological activation of the IL7-receptor (IL7R) signaling contributes to steroid resistance in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Here, we show that activating IL7R pathway mutations and physiological IL7R signaling activate MAPK-ERK signaling, which provokes steroid resistance by phosphorylation of BIM. By mass spectrometry, we demonstrate that phosphorylated BIM is impaired in binding to BCL2, BCLXL and MCL1, shifting the apoptotic balance toward survival. Treatment with MEK inhibitors abolishes this inactivating phosphorylation of BIM and restores its interaction with anti-apoptotic BCL2-protein family members. Importantly, the MEK inhibitor selumetinib synergizes with steroids in both IL7-dependent and IL7-independent steroid resistant pediatric T-ALL PDX samples. Despite the anti-MAPK-ERK activity of ruxolitinib in IL7-induced signaling and JAK1 mutant cells, ruxolitinib only synergizes with steroid treatment in IL7-dependent steroid resistant PDX samples but not in IL7-independent steroid resistant PDX samples. Our study highlights the central role for MAPK-ERK signaling in steroid resistance in T-ALL patients, and demonstrates the broader application of MEK inhibitors over ruxolitinib to resensitize steroid-resistant T-ALL cells. These findings strongly support the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507) and contributes to the optimization and stratification of newly designed T-ALL treatment regimens.
The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.
SummaryAberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.
Introduction Poor prednisolone response in the induction phase of treatment is a risk-stratification marker in the current Dutch DCOG ALL-11 treatment protocol. Physiological or mutational activation of the IL7-receptor (IL7R) provokes steroid resistance in T-ALL. Moreover, relapsed T-ALL patients that harbor IL7R or RAS mutations are considered as an ultra-high-risk group with extremely poor outcome. Aim We studied the contribution of two key IL7R downstream pathways (STAT5B and MAPK-ERK respectively) to steroid resistance in order to optimize future treatment regimens. Results To date, IL7-dependent steroid resistance has been proposed to depend on IL7-induced JAK-STAT signaling and subsequent BCL2 upregulation. Here, we demonstrate that the transforming STATBN642H mutation does not impair steroid sensitivity despite a paradoxical upregulation of BCL2 and BCLXL following steroid treatment. We therefore hypothesized that other pathways contribute to IL7-dependent steroid resistance in T-ALL. By studying T-ALL PDX samples, we observed that physiological IL7-signaling can activate the MAPK-ERK pathway in IL7-dependent and IL7-independent steroid resistant PDX cells. Similar to mutational-driven MAPK-ERK signaling (e.g. by activating IL7R, JAK1 or NRAS mutations), IL7-induced MAPK-ERK signaling leads to the inactivation of the pro-apoptotic protein BIM by inhibitory phosphorylation of both BIM-EL and BIM-L isoforms. By mass spectrometry, we observe that phosphorylated BIM specifically decreases its physical interaction with Aurora kinase A and Bcl-2 family members BMF, BCL2, BCLXL and MCL1, which alters the apoptotic threshold and effectuates steroid resistance. The JAK1/2-inhibitor ruxolitinib and MEK-inhibitor selumetinib are considered for future basket-treatment protocols in IL7R/JAK-STAT-activated or MAPK-ERK-activated leukemias respectively. We demonstrate that both ruxolitinib and selumetinib prevent IL7-induced MAPK-ERK signaling in T-ALL PDX cells. Moreover, ruxolitinib and selumetinib abolish BIM phosphorylation in mutant JAK1-overexpressing SUPT-1 cells, which restores the binding of BIM to BCL2, BCLXL and MCL1. Only selumetinib restores the function of BIM in wild type or mutant NRAS overexpressing SUPT-1 cells, indicating that ruxolitinib only blocks MAPK-ERK signaling in the context of 'upstream' (e.g. mutant IL7R/JAK1 or IL7-induced) pathway activation. Surprisingly, none of our 46 PDX samples respond to ruxolitinib treatment in the absence of IL7, while the majority of these samples demonstrate a robust response towards MEK-inhibition. IL7-dependent steroid resistant PDX samples do respond to ruxolitinib treatment in the presence of IL7, while IL7 exposed IL7-independent steroid resistant PDX samples remain unresponsive to ruxolitinib. We therefore conclude that the therapeutic effect of ruxolitinib is dependent on an IL7-protective effect, limiting its therapeutic application to a selective subset of T-ALL patients. When we combine prednisolone treatment with selumetinib or ruxolitinib, we observe that ruxolitinib only synergizes with prednisolone in IL7-dependent steroid resistant PDX samples in the presence of IL7. Importantly, combination treatment with prednisolone and selumetinib acts highly synergistic in IL7-dependent and IL7-independent steroid resistant samples in the presence and absence of IL7. Conclusion and clinical relevance In addition to described JAK-STAT and PI3K-AKT signaling, our study demonstrates that IL7-induced signaling activates the MAPK-ERK signaling pathway, which actively contributes to IL7-dependent steroid resistance. We demonstrate the central importance of MAPK-ERK signaling in T-ALL, whereas all T-ALL PDX samples tested highly benefit from combined prednisolone and selumetinib treatment. Ruxolitinib only acts synergistically with steroids in the context of IL7-dependent steroid resistance. Moreover, our data proposes that this synergistic effect may (in part) depend on the anti-MAPK-ERK effect downstream of JAK1/2-inhibition. Combined, our study strongly supports the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507), and contributes to the optimization and stratification of newly designed T-ALL treatment regimens. Disclosures Bourquin: Servier: Other: Travel Support. Vormoor:AstraZeneca: Research Funding.
In this study, we measured the kinase activity profiles of 32 pre-treatment tumour biopsies of HER2-positive breast cancer patients. The aim of this study was to assess the prognostic potential of kinase activity levels to identify potential mechanisms of resistance and to predict treatment success of HER2-targeted therapy combined with chemotherapy. Indeed, our system-wide kinase activity analysis, based on targeted mass spectrometry measurement of kinase activation loops, allowed us to link kinase activity to treatment response. Overall, high kinase activity in the HER2-pathway was associated with good treatment outcome. Furthermore, we found eleven kinases differentially regulated between treatment outcome groups. Amongst those, well-known players in therapy resistance were found, such as p38a, ERK and FAK, as well as a potential new player in drug resistance, namely MARK. Lastly, we defined an optimal signature of four kinases in a multiple logistic regression diagnostic test for prediction of treatment outcome (AUC=0.926). This kinase signature showed high sensitivity and specificity, indicating its potential as predictive biomarker for treatment success of HER2-targeted therapy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.