Abstract 2975: CXCR4 blockade of T cell acute lymphoblastic leukemia causes systemic disease in an NSG model allowing ruxolitinib and venetoclax to synergistically treat cancer burden
Authors: Walker Kirsti L. , Kabakov Sabrina A. , Zhu Fen , Olson Sydney L. , Rui Lixin , Capitini Christian M. 
Published: August 2019

Abstract
T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy that accounts for 25% of adult and 15% of pediatric acute lymphoblastic leukemia (ALL) cases. Relapsed or refractory T-ALL is difficult to salvage with chemotherapy, which causes long-term toxicity, and is often fatal. The JAK/STAT and BCL-2 pathways are upregulated in T-ALL and promote increased T-ALL proliferation and survival. Currently, targeted therapies of the JAK/STAT and BCL-2 pathways have not been investigated in combination. I propose that dual inhibition of the JAK/STAT and BCL-2 pathways, with ruxolitinib and venetoclax respectively, will lead to maximal T-ALL cell death. Jurkat cells were treated with single doses of ruxolitinib (0.156µM - 5µM) or venetoclax (1.56nM - 50nM) in vitro, and analyzed by trypan blue exclusion, MTT and flow cytometry at 24, 48 and 72h post-treatment. Results demonstrate decreased proliferation by MTT, decreased viability by trypan blue exclusion, and increased apoptosis by flow cytometry for the three highest doses of ruxolitinib (1.25µM, 2.5µM and 5µM) and venetoclax (12.5nM, 25nM and 50nM). A synergistic effect was achieved for all three assays at 48 and 72h when cells were treated with a combination dose of ruxolitinib (1.25µM) and venetoclax (25nM; CI&lt;1). This optimal in vitro combined dose significantly decreased proliferation (p&lt;0.0001) and viability (p&lt;0.0001) of Jurkat cells compared to vehicle and single drug treatment groups. Jurkat-GFP cells were injected intravenously into NSG mice to mimic a systemic in vivo xenograft model of T-ALL, and followed for clinical scores and survival. End organs were analyzed for GFP+CD45+ cells by flow cytometry and GFP+ cells by immunohistochemistry (IHC). Compared to single treatments of ruxolitinib and venetoclax, all mice treated with ruxolitinib and/or venetoclax combination therapy developed hind-limb paralysis and died of CNS disease in the spinal cord and brain as shown by IHC. This suggests for the first time that ruxolitinib or venetoclax cannot penetrate the blood brain barrier (BBB). LC-MS-MS studies were performed to confirm that ruxolitinib and venetoclax cannot penetrate the BBB. Previously published data suggests that T-ALL exploits the CXCR4-CXCL12 chemokine pathway to relapse into the CNS. Jurkat cells were analyzed by flow cytometry and showed high expression of the CXCR4 surface receptor while NSG brain tissue showed presence of CXCL12 mRNA and protein by in situ hybridization (ISH) and western blot analysis. Antibody blockade of CXCR4 in vivo prevented the migration of Jurkat cells into the CNS and suggests that disruption of the CXCR4-CXCL12 pathway will result in a systemic model of T-ALL that could allow for ruxolitinib and venetoclax to eliminate T-ALL at primary sites of disease.
Citation Format: Kirsti L. Walker, Sabrina A. Kabakov, Fen Zhu, Sydney L. Olson, Lixin Rui, Christian M. Capitini. CXCR4 blockade of T cell acute lymphoblastic leukemia causes systemic disease in an NSG model allowing ruxolitinib and venetoclax to synergistically treat cancer burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2975.