Abstract A35: MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia
Authors: Summers Ryan J. , Minson Katherine A. , Wang Xiaodong , Frye Stephen V. , Earp H. Shelton , DeRyckere Deborah , Graham Douglas K. 
Published: February 2019

Background: Early T-precursor acute lymphoblastic leukemia (ETP-ALL) is a subclass of T-cell ALL (T-ALL) accounting for 15% of pediatric T-ALL cases and characterized by an immature phenotype, resistance to therapy, and high rates of induction failure and relapse (Wood B et al., Blood 2009). MERTK receptor tyrosine kinase is not expressed in normal T cells but is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T-cell phenotype (Graham DK et al., Clin Cancer Res 2006), suggesting a role in ETP-ALL. One potential role is regulation of the anti-apoptotic protein B-cell lymphoma-2 (BCL-2). BCL-2 is specifically expressed in double-negative T-cell precursors and is preferentially expressed in ETP-ALL compared to T-ALL (Chonghaile et al., Cancer Discovery 2014). Moreover, ETP-ALL cells are dependent on BCL-2 for survival. Our previous studies demonstrated regulation of BCL-2 and BCL-2 family members downstream of MERTK in B-ALL and acute myeloid leukemia cells (Linger RM et al., Blood 2013; Lee-Sherick AB et al., Oncotarget 2015). This interplay between MERTK and BCL-2 and their association with an immature T-ALL phenotype suggest that combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL.

Methods: Publicly available mRNA expression data were used to assess MERTK and BCL-2 expression in T-ALL cell lines and patient samples. MERTK and BCL-2 protein expression were determined by immunoblot. ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 kinase inhibitor. MERTK protein was immunoprecipitated from cell lysates and phosphorylated and total proteins were assessed by immunoblot. Alternatively, cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Orthotopic xenografts were established in NSGS mice using an ETP-ALL patient sample, and leukemia burden in peripheral blood (%hCD45+) was monitored by flow cytometry. After engraftment (1.86 +/- 0.43% peripheral blasts), mice were treated once daily with 75 mg/kg MRX-2843 or saline vehicle administered orally. Mice were euthanized when symptoms of advanced leukemia were evident and median survival was determined by Kaplan-Meier analysis.

Results: MERTK mRNA was expressed at significantly higher levels in ETP-ALL cell lines and patient samples relative to other T-ALLs. Similarly, MERTK protein was ubiquitously expressed in ETP-ALL cell lines (n=2), ETP-ALL patient samples (n=2), and a near-ETP-ALL patient sample (n=1). In contrast, only 60% of other T-ALL cell lines (n=5) expressed MERTK. BCL-2 mRNA was expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs and BCL-2 protein was expressed in 2 of 2 ETP-ALL cell lines and 2 of 3 ETP-ALL and near-ETP-ALL patient samples. Treatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK in ETP-ALL cells and induced dose-dependent cell death in the ETP-ALL cell lines PEER (43.2% vs 16% in vehicle-treated cultures, p&lt;0.01) and Loucy (36.6% vs 19.2%, p&lt;0.05). Moreover, in a xenograft model of ETP-ALL, treatment with MRX-2843 for 29 days reduced peripheral blood disease burden by 53.9% (p&lt;0.001) and decreased spleen weight by 64% (p&lt;0.001) compared to vehicle-treated controls. Furthermore, mice treated with MRX-2843 survived for a median of 41 days post-treatment compared to 29 days for control mice (n=8/group, p=0.003).

Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843, a dual MERTK/FLT3 kinase inhibitor, has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. In particular, combination therapy targeting MERTK and BCL-2 may be an effective therapeutic option for ETP-ALL.

Citation Format: Ryan J. Summers, Katherine A. Minson, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A35.