The Splenic Microenvironment Is a Novel Site of Relapse Following ABT-199 Treatment

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with a poor prognosis and a high risk for relapse. While the leukaemia initially develops in the thymus it spreads in the blood to the bone marrow, lymph nodes and often the spleen. Interestingly, splenomegaly was previously associated with a poor prognosis in leukemic patients. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic. However, one issue with targeted agents, like ABT-199, is the development of resistance to treatment. Our aim was to determine potential in vivosites of resistance/relapse following ABT-199 treatment using a xenograft model of ETP-ALL.

We confirmed that the ETP-ALL LOUCY cell line is BCL-2 dependent and then labelled it with luciferase to enable visualisation of the leukaemia in vivo. Following establishment of the leukaemia in NOD/SCID gamma mice, as assessed by hCD45+, the mice were randomised to receive vehicle control or 50 mg/kg ABT-199 by oral gavage daily for two weeks. While the mice were initially sensitive to ABT-199, the leukaemia started to progress while on treatment. Interestingly, there appeared to be a selective redistribution of the leukaemia to the spleen following ABT-199 treatment. Indeed, LOUCY cells isolated from the spleen of the mice had a reduced BCL-2 dependence, as assessed by BH3 profiling. The reduced BCL-2 dependence correlated with reduced BCL-2 expression at both the mRNA and protein level.

Next, we confirmed that human splenic fibroblasts (HSF) co-cultured with the LOUCY cell line in vitro also altered BCL-2 dependence and expression using BH3 profiling and Western blotting. To identify potential splenic cytokines involved in the regulation of BCL-2 protein expression in ETP-ALL we performed a screening cytokine array. Upon co-culture of the LOUCY cells with HSF there was an increased expression of IL-6, this was confirmed using ELISA. Using an IL-6 receptor antibody we confirmed that blocking IL-6 receptor reversed the change in BCL-2 dependence in the presence of the splenic microenvironment.

Lastly, we confirmed in a T-ALL patient-derived xenograft, that is BCL-2 dependent, that the splenic microenvironment alters the mitochondrial apoptotic threshold. Currently, there are reports in the literature of ETP-ALL patients being treated with ABT-199. While there have been numerous studies lately describing cell autonomous events leading to ABT-199 resistance, our novel finding that the splenic microenvironment is a site of relapse is potentially of great clinical importance for BCL-2 dependent leukemia's.