For patients with T-cell lineage acute lymphoblastic leukemia (T-ALL) who fail induction or relapse, event free survival is <10%. Mixed lineage leukemia gene rearrangements (KMT2A-R) are recurrent translocations found in ~3-5% of T-ALL and are determinants of high-risk disease. However, the roles of downstream targets of KMT2A chimeras such as Runt-related transcription factor 2 (RUNX2) remain uclear. We assessed RUNX2 mRNA levels in 100 pediatric T-ALL samples derived from the COG AALL0434 study. RUNX2 expression was upregulated in patients harboring KMT2A-R (p <0.0005) and those who failed induction therapy (p <0.005). Therefore, we hypothesized that RUNX2 is a critical determinant of high-risk T-ALL. To investigate the relationship between RUNX2 and KMT2A chimeras we utilized the chromatin immunoprecipitation assay to assess primary T-ALL samples with KMT2A-R (n = 3). We found that RUNX2 targets KMT2A-R (p < 0.0005) and conversely, that KMT2A chimeras target the promoter region of RUNX2 (p < 0.0005). We next demonstrated that KMT2A-MLLT4 induces RUNX2 expression. To further test the effects of RUNX2 on KMT2A-R, we performed shRNA mediated silencing of RUNX2 expression in primary T-ALL cells (n =3). RUNX2 silencing decreased KMT2A levels, suggesting the functional cross talk between KMT2A-R and RUNX2. To test the regulatory role of RUNX2 on T-ALL signal transduction we knocked down RUNX2 in T-ALL primary samples and cell lines (Loucy and Jurkat). RUNX2 silencing led to a decrease in cellular proliferation (p < 0.0005) indicating that RUNX2 positively regulates T-ALL cell growth in vitro. We next utilized flow-based assays to examine the effects of RUNX2 on cell death and cell cycle. RUNX2 inactivation induced apoptotic cell death and delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of the cell cycle. Apoptotic cell death was associated with increased expression of cleaved caspase 3, 7 and 9. We next examined the effects of RUNX2 on T-ALL cell survival and found that RUNX2 depletion reduced activation of Akt, active β-catenin and survivin levels but had no effect on p53 and Bcl-2. Similar experiments showed that RUNX2 inactivation leads to decreased expression of c-Myc and Ras. In addition, treatment with the specific allosteric Akt inhibitor, MK2206 (10 μM, 24 hrs) reduced RUNX2, active β-catenin, and survivin protein levels suggesting reciprocal activation between RUNX2 and Akt. To study the role of RUNX2 in leukemia burden, Jurkat cells transduced with RUNX2 shRNA and control plasmid were transplanted intravenously into the NSG mice (5 mice/group). RUNX2 silencing significantly decreased leukemia burden in spleen (p <0.001) and bone marrow (p <0.01) of the tested animals compared to the negative control group. We propose upregulation of RUNX2 expression as a novel mechanism governing high-risk T-ALL. We postulate that the Akt/β-catenin/survivin axis serves as a functional driver of RUNX2-mediated T-ALL survival.

Citation Format: Nitesh Devinarayan Sharma, Christian K. Nickl, Stuart S. Winter, Huining Kang, Ksenia Matlawska Wasowska. RUNX2 as a regulator of high-risk T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4129.