PF161 THIOREDOXIN SYSTEM INHIBITORS ARE EFFECTIVE AGAINST B CELL ACUTE LYMPHOBLASTIC LEUKEMIA IN VITRO AND IN VIVO
Authors: Fidyt K. , Pastorczak A. , Goral A. , Muchowicz A. , Komorowski L. , Cyran J. , Pal D. , Blair H. , Burdzinska A. , Pedzisz P. , Patkowska E. , Lech‐Maranda E. , Mlynarski W. , Golab J. , Firczuk M. 
Published: June 2019

Background:

B‐cell acute lymphoblastic leukemia (B‐ALL) is a heterogeneous hematological disorder characterized by accumulation of B cell precursors. Although undeniable improvement has been made in the therapy of

B‐ALL (>90% of pediatric patients experience remission), there are still specific subtypes associated with poor outcome. Among such subtypes are B‐ALL with mixed lineage leukemia rearrangements (MLLr ALL) or BCR‐ABL1 fusion protein (BCR‐ABL1+ ALL). Importantly, MLLr ALL does not respond to novel immunotherapies, thus novel treatment options are urgently needed. More and more data highlight disturbed redox homeostasis as a novel target in cancer treatment. Cancer cells upregulate different antioxidant machineries to overcome excessive production of reactive oxygen species (ROS) in result of increased metabolism and uncontrolled proliferation. One of such ROS‐defending systems is the thioredoxin (TXN) system, which is composed of three independent enzymes: thioredoxin reductase (TXNRD), thioredoxin (TXN), and peroxiredoxin (PRDX).

Aims:

To investigate the efficacy of TXN‐family enzymes inhibitors – auranofin (AUR) and adenanthin (ADE) in B‐ALL in vitro and in vivo.

Methods:

Primary material was isolated from pediatric and adult B‐ALL patients at diagnosis. Primograft B‐ALL samples representing BRC‐ABL1+ and MLLr ALL were generated in NOD scid gamma (NSG) mice by tail vein injections of primary B‐ALL cells. The engraftment of leukemic cells was assessed by whole blood staining and flow cytometry. Primary bone marrow derived mesenchymal stem cells (BM‐MSC) were isolated from non‐leukemic donors who underwent hip replacement surgery. Cytostatic/cytotoxic effects of TXN‐family enzymes inhibitors, AUR and ADE were assessed in mono‐ and co‐culture with BM‐MSC by MTT viability assay or by trypan blue exclusion method. The efficacy of AUR in vivo was evaluated in MLLr ALL patient derived xenograft model in NSG mice. For mechanistic studies we used SEM cell line representing MLLr ALL.

Results:

We have previously found increased mRNA and protein levels of particular enzymes of TXN system in B‐ALL cell lines and primary cells, thus we evaluated efficacy of two thioredoxin system inhibitors, AUR and ADE in B‐ALL primary cells. We found that both inhibitors decreased the viability of various B‐ALL cells grown not only in monoculture, but also in a co‐culture with bone marrow derived mesenchymal stem cells (BM‐MSC) of confirmed characteristics (Fig. 1A), which were shown to protect leukemic cells from drugs toxic effects. Next, we compared the effects of both inhibitors against normal peripheral blood mononuclear cells (PBMC) and found that ADE is significantly more toxic than AUR, thus we chose the latter for further studies. Accordingly, we observed that in patient derived xenograft model of MLLr ALL, AUR delayed the progression and prolonged the survival of NSG mice (Fig. 1B). Elucidating the mechanism standing behind AUR toxic effects, we found increased oxidative stress, as we observed elevated ROS levels which were abrogated by ROS scavengers, pyruvate and catalase. In addition, we observed that AUR induced oxidative stress biomarker, heme oxygenase 1 and moderate DNA oxidative damage in primograft B‐ALL cells (Fig. 1C).

Summary/Conclusion:

Above results show that targeting TXN system with their inhibitors can be considered as novel approach in the treatment of B‐ALL. Considering that AUR is an FDA approved drug, it could be repurposed and tested in combination with other targeted drugs in B‐ALL.

image