Activina: A Key Factor Mediating Chemoresistance in B-Cell Type Acute Lymphoblastic Leukemia

B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common cancer in children. Multidrug high-dose chemotherapy has increased the 5-year event free survival rate to more than 80%. However, the prognosis of refractory/relapsed (r/r) ALL remains dismal. Increasing evidence indicates that treatment failure could be linked to the protective role of leukemic bone marrow (BM) microenvironment. The identification of the molecules involved in the crosstalk between leukemic cells and the BM niche would be crucial to establish niche-targeted therapies to be combined with conventional drugs.

Recently, we identified ActivinA (ActA) as a new potentially targetable leukemia-promoting factor, which is significantly overexpressed in the BM plasma of B-ALL patients. We demonstrated that ActA is able to potentiate the migratory and invasive ability of B-ALL cells in vitro and enhance both BM engraftment and metastatic potential of leukemic cells in a xenograft mouse model. Literature data indicate that ActA exerts a pro-tumoral role in several solid tumors, by promoting cancer cell migration, proliferation and chemoresistance.

The aim of this work was to ascertain whether ActivinA could mediate resistance to standard chemotherapy agents routinely used in pediatric B-ALL, such as Vincristine (VCR), Dexamethasone (Dex) and Asparaginase (ASNase).

To investigate the role of ActA on B-ALL cell chemoresistance, we stimulated or not 697 cells with ActA for 24h. Cells were then treated with increasing concentrations of Dex, ASNase and VCR for 72h to obtain a dose-response curve. Cell viability was evaluated by flow cytometry and used to calculate the inhibitory dose 50 (IC₅₀). Interestingly, the IC₅₀ of ActA stimulated 697 cells for Dex and ASNase was in both cases about ten-fold higher than NS controls. In the case of VCR-treated cells, we did not detect any difference in IC₅₀. These data suggest that ActA is able to convey resistance to Dex and ASNase-induced cell death, while sparing VCR-based leukemic cell killing, possibly due to different mechanisms of action. Validation experiments, with selected doses of Dex and ASNase (7 µg/mL and 1 U/mL, respectively), confirmed the ability of ActA to induce chemoresistance. In detail, the median viability of ActA stimulated 697 cells after 3 days of treatment with Dex was 64.15% (range=51.95-73.75%). This percentage was significantly higher compared to NS cells (median viability=41.95%, range=31.95-60.93%, Wilcoxon test: p<0.0001). Alongside, the viability of ActA stimulated cells (median=53.40%, range=38.70-75.00%) was also significantly increased (median=31.40%, range=22.45-54.25%, p=0.001) after ASNase treatment.

To understand the molecular mechanisms underlying Acta-mediated chemoprotection, we firstly evaluated in our experimental setting the percentage of 697 cells expressing the active form of Caspase-3, by flow cytometry. Our results highlight that the activation of Caspase-3 was significantly reduced in ActA pre-stimulated cells treated with Dex (mean fold change (FC) ActA/NS=0.48±0.22, p=0.0003, One-sample t test) and ASNase (mean FC=0.49±0.062, p<0.0001), compared to NS cells.

Then, we focused on the impact of ActA on anti- and pro-apoptotic factors playing a key role in drug-mediated apoptosis. By western blot, we demonstrated that Bcl-2 was significantly increased upon Dex treatment in ActA pre-stimulated cells (median FC ActA+Dex/NS untreated cells=0.63, range=0.49-1.12, Wilcoxon test: p<0.05) compared to NS cells (median FC NS+Dex/NS untreated cells=0.43, range=0.26-0.74). In contrast, we did not observe any difference with ASNase. Furthermore, we demonstrated that ActA was able to decrease the drug-induced cleavage activation of pro-apoptotic factors BAX and BAK.

Finally, we investigated by confocal microscopy the levels of intracellular Reactive Oxygen Species (ROS), well-known key elements of the drug-induced oxidative stress, resulting in the activation of apoptosis. Interestingly, upon Dex treatment we observed a reduction of ROS levels of 59.7%±4.6% (mean±sd) in ActA-stimulated cells compared to NS. In the case of ASNase treatment the mean reduction was 46.9%±8.8%.

Overall, our data suggest that ActA plays a pivotal role in B-ALL cell chemoprotection and that its targeting may be a promising strategy to be used in combination with standard chemotherapy in r/r ALL patients.

Rizzari:Servier: Consultancy, Honoraria, Research Funding. Biondi:Novartis: Honoraria; Amgen: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board.