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F. Portale et al.
stimulate the migration of ovarian cancer cells suggests that Ca2+ increase could be achieved also in leukemic cells through the activation of non-canonical phospo-AKT, phospho-ERK and Rac1 signaling. In line with this hypothesis, our GEP data demonstrate an ActivinA- dependent increase in DOCK438 and CORO1A39 in the 697 cell line and primary BCP-ALL cells. This increase posi- tively regulates the CDC42/RAC1 pathway, leading to the generation of phosphatidylinositol-3-phosphate, responsi- ble for calcium release from intracellular stores.40 In agree- ment with this, ActivinA-mediated downmodulation of the Rac-GTPase activating protein ARHGAP25, that phys- iologically counterbalances the Rac-activating effect of nucleotide exchange factors,41 could play a prominent role in shaping calcium levels and modulating actin cytoskele- ton42 also in leukemic cells. Moreover, ActivinA could also regulate the extent and duration of calcium responses through PTPRC downregulation. Indeed, in immature B cells, it has been demonstrated that the lack of the PTPRC product, CD45, induces enhanced levels of intracellular calcium that can last longer than in CD45 expressing cells, upon BCR engagement.43 Further studies will be crucial to better understand the possible molecular mechanisms mediating ActivinA differential activity in order to identi- fy potential selective targets to counteract its action.
The ActivinA-mediated migratory advantage observed on BCP-ALL cells was further confirmed in a xenograft mouse model in which we demonstrated that leukemic cells prestimulated with ActivinA were able to engraft in the BM of NSG mice more rapidly than in their untreated counterparts. Overall, our in vivo data corroborate in vitro findings suggesting the effect of the molecule in favoring leukemia. Future studies testing the efficacy of ActivinA ligand traps on BCP-ALL patient-derived xenografts will be crucial to establish the impact of ActivinA on leukemia propagation.
Recent studies importantly linked ActivinA with the enhancement of cell invasion in several solid cancers (colorectal cancer, prostate cancer, breast cancer, glioblas- toma, non-small cell lung cancer).44-48 In the context of BCP-ALL, relapse represents the most common cause of treatment failure, mainly occurring in the BM either in an isolated form or in combination with other extramedullary sites.49 Besides its key role in regulating homing processes in the BM niche, CXCL12 is thought to be involved in the widespread infiltration of other organs because of its constitutive expression in
extramedullary tissues such as liver, spleen, thymus, lung, kidney, and brain.50 Interestingly, our in vitro obser- vation demonstrated that ActivinA significantly increased the ability of leukemic cells to pass through an extracellular matrix in response to CXCL12. In addition, in vivo injected ActivinA-stimulated leukemic cells were more able than untreated cells to reach extramedullary disease target organs such as the meninges and the brain, suggesting a possible role for ActivinA in the promotion of leukemic cell invasiveness. Notably, ActivinA was able to up-regulate the expression of its type I receptors in leukemic cells, thus creating a self-reinforcing signal- ing cascade. Overall, our data suggest the establishment of a positive feedback loop between BCP-ALL cells and MSCs, which, through the key action of MSC-secreted ActivinA, generates a microenvironment favoring leukemia at the expense of normal hematopoiesis. Indeed, it is conceivable that the abundance of ActivinA, along with the decrease in CXCL12 within the BM niche, could lead to a reduction in the healthy HSC pool in favor of leukemic cells. On the other hand, the leukemic cells could access the BM sanctuaries where they can achieve signals necessary for cell survival and therapy resistance. Indeed, our in vitro findings were confirmed by in vivo studies and provide the biological rationale for designing therapeutical approaches targeting ActivinA in patients with BCP-ALL. Therefore, the direct targeting of ActivinA or its key downstream mediators could repre- sent a valuable therapeutic option to be combined with conventional chemotherapeutic agents for decreasing the frequency of relapse in BCP-ALL.
Acknowledgments
The authors would like to thank the nursing and medical staff of the Fondazione MBBM/San Gerardo Hospital and the Bambino Gesù Hospital. We thank Fondazione Matilde Tettamanti, Comitato Maria Letizia Verga, Comitato Stefano Verri, Fondazione MBBM, the Department of Medicine and Surgery of the University of Milano-Bicocca, Whirlpool and GEICO TAIKI-SHA for their generous support.
Funding
This work was partially supported by Associazione Italiana Ricerca sul Cancro (project number IG 2014 Id.15494, to GD’A). This article was also funded by AIRC Special Program Molecular Clinical Oncology - 5 per mille 2018 (project number 21147 to AB).
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