RUNX2 Regulates Cell Migration in T-Cell Lineage Acute Lymphoblastic Leukemia
Authors: Sharma Nitesh Devinarayan , Nickl Christian C. , Kang Huining , Ornatowski Wojciech , Winter Stuart S. , Loh Mignon L. , Hunger Stephen P. , Matlawska-Wasowska Ksenia 
Published: November 2019

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy accounting for 10-15% of pediatric ALLs. While cure rates have recently improved, event free survival is <10% in patients with relapsed disease. We recently showed that Runt related transcription factor 2 (RUNX2) is upregulated in high-risk T-ALL including patients harboring KMT2A-R (p< 0.0001), ETP phenotype (p< 0.0001) and those who failed induction therapy (p <0.005) (Sharma et al., AACR Annual Meeting 2018; #4129). We also demonstrated that RUNX2 positively regulates T-ALL survival via Akt/β-catenin/survivin axis and observed a significant increase in survival of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice engrafted with shRNA-RUNX2 depleted T-ALL cells (median survival 52 days) compared to animals engrafted with negative control cells (median survival 38 days; p< 0.0001). Because RUNX2 is involved in invasiveness and metastasis of many solid tumors, we hypothesized that RUNX2 might modulate the migration and dissemination of T-ALL. To test the regulatory effects of RUNX2 on T-ALL migration we used lentiviral transduction to induce or knock down RUNX2 expression in T-ALL cell lines (n = 4) and/or primary patient samples (n = 3), and/or BaF3 cells expressing KMT2A-R (KMT2A-MLLT4 or KMT2A-MLLT1). While induced expression of RUNX2 had no effect on T-ALL cell growth and cell cycle, we discovered that RUNX2 positively regulated the expression of CXCR4 receptor and migration of T-ALL cells towards CXCL12 chemokine (p< 0.0005). Because cell migration is overall an energy-demanding process, we used the Agilent Seahorse XF Analyzer to study leukemic cell metabolism. We found that RUNX2 upregulation leads to increased oxygen consumption rate and ATP production (p< 0.0001) as well as the increase in basal and compensatory glycolysis (p< 0.005), suggesting that RUNX2 potentiates metabolic activity of T-ALL cells. We next utilized flow-based Mito-Tracker Red CMXRos and Reactive Oxygen Species (ROS) assays to examine the impact of RUNX2 on mitochondrial activity. Forced expression of RUNX2 increased mitochondrial membrane potential corresponding to decreased ROS levels. RUNX2 positively regulated the expression of critical regulators of mitochondrial fission (P-DRP1 Ser616, DRP1, MFF and FIS1), fusion (OPA1 and Mfn1, Mfn2) and mitochondrial biogenesis (PGC1α and NRF1). In converse experiments, RUNX2 depletion reduced the oxidative phosphorylation, glycolytic flux and mitochondrial activities in the tested T-ALL cells suggesting that RUNX2 displays unique metabolic signatures that might enhance leukemia aggressiveness. Disruption of mitochondrial fission with Midivi-1 inhibitor (1 μΜ, 16 hr) reduced RUNX2-mediated T-ALL migration towards CXCL12 (P< 0.0001) suggesting that RUNX2 regulates mitochondrial dynamics to fosters T-ALL cell migration in vitro. Similarly, treatment with 2-deoxy-D-glucose (2DG), a glucose analog reduced the levels of migrating T-ALL cells (p< 0.005) indicating the roles of metabolic reprograming in RUNX2-mediated T-ALL cell migration. Our results suggest that RUNX2 regulates mitochondrial dynamics and cell metabolism to promote T-ALL migration. We propose deregulation of RUNX2 expression as a novel mechanism governing T-ALL migration and dissemination. Ongoing in vivo studies will reveal the roles of RUNX2 in T-ALL infiltration to extramedullary tissue.

Disclosures

Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Hunger:Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Jazz: Honoraria; Novartis: Consultancy.