B-Cell Precursor Acute Lymphoblastic Leukemia Instructs Mesenchymal Stromal Cells to Alter Interferon-Related Gene Expression and Cyto/Chemokine Secretion

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient-derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel options for therapeutic intervention. Therefore, our research is focused on the interaction between BCP-ALL cells and MSCs. We aimed to determine gene expression changes in BCP-ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto- and chemokines are differentially secreted upon contact between BCP-ALL cells and MSCs.

Methods: We performed co-cultures of primary MSCs and BCP-ALL cells, and mono-cultures of MSCs or BCP-ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Moreover, migration experiments using transwells (3.0µm pore size) were performed to determine the level of BCP-ALL migration towards cyto/chemokines of interest.

Results: RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with BCP-ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between BCP-ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples. This suggests that the observed changes are induced by the leukemic cells and that leukemic cells manipulate MSCs. Survival benefit (0.3 - 37.9%) was observed in BCP-ALL cells after co-culture with MSCs compared to BCP-ALL mono-culture. Moreover, pro-inflammatory cytokines, and several migration-related chemokines such as CCL2, CXCL8, and CXCL10/IP-10 were upregulated in 5 out of 15 co-cultures compared to the sum of the separate mono-cultures of BCP-ALL and MSCs. A gradient of CXCL10/IP-10 in transwell experiments showed that this chemokine did not enhance the migration of primary BCP-ALL cells, suggesting that the ALL-induced secretion of this chemokine serves a different role in BCP-ALL. The role of CXCL10/IP-10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies.

Conclusion: Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow stromal cells upon exposure to BCP-ALL. These induced changes may be important for BCP-ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche, and may provide an alternative way to eliminate leukemic cells more efficaciously. Functional studies addressing this concept are currently being executed and the results will be presented during the meeting.