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528 The Central Role of MAPK-ERK Signaling in IL7-Dependent and IL7-Independent Steroid Resistance Reveals a Broad Application of MEK-Inhibitors Compared to JAK1/2-Inhibition in T-ALL

Program: Oral and Poster Abstracts
Type: Oral
Session: 605. Molecular Pharmacology, Drug Resistance—Lymphoid and Other Diseases: Molecular pharmacology and drug resistance mechanisms in lymphoproliferative disorders
Hematology Disease Topics & Pathways:
Leukemia, ALL, Biological, Diseases, Therapies, Biological Processes, Lymphoid Malignancies, Clinically relevant, TKI, pathways, signal transduction
Monday, December 7, 2020: 8:00 AM

Jordy C.G. Van Der Zwet, MD1*, Jessica G.C.A.M. Buijs-Gladdines1*, Valentina Cordo', MSc1*, Donna Debets, MSc2*, Willem K. Smits1*, Zhongli Chen, MSc1*, Jelle Dylus3*, Guido Zaman, PhD3*, Maarten Altelaar, PhD2*, Koichi Oshima, MD, PhD4, Beat Bornhauser5*, Jean-Pierre Bourquin, MD, PhD5, Jan Cools, PhD6, Adolfo A. Ferrando, MD, PhD7, Josef Vormoor, MD, PhD1, Rob Pieters, MD, PhD, MSc1, Britta Julia Vormoor, MD8* and Jules P.P. Meijerink, PhD1

1Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands
2Bijvoet Center of Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht, Netherlands
3Netherlands Translational Research Center, Oss, Netherlands
4Columbia University Medical Center, New York, NY
5Department of Pediatric Oncology, Children’s Research Centre, University Children’s Hospital Zurich, Zurich, Switzerland
6Center for Cancer Biology, VIB-KU Leuven, Leuven, Belgium
7Institute for Cancer Genetics, Columbia University, New York, NY
8Princess Máxima Center for Pediatric Oncology, Utrecht, Utrecht, Netherlands

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.

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.

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: Vormoor: AstraZeneca: Research Funding.

*signifies non-member of ASH