ETV6-RUNX1 (ER), generated from translocation t(12;21), is the most frequent fusion gene in pediatric cancers, exclusively leading to B-Cell Precursor Acute Lymphoblastic Leukemia. Translocation occurs in fetal hematopoietic stem-progenitor cells (HSPC) but it is insufficient for disease. ER, in fact, is an aberrant transcription factor that expands a silent preleukemic clone with enhanced self-renewal and partial B cell differentiation. Secondary hits are thus required to complete transformation. Epidemiological and experimental data indicate that infections/inflammation play an important role in the preleukemia to leukemia transition. We previously demonstrate that TGFβ1, a pleiotropic cytokine produced after inflammation, favored the persistence of ER+Ba/F3 cells and selected putative preleukemic stem cells in ER+umbilical cord blood (UCB) CD34+cells. We also demonstrated that ER+Ba/F3 showed altered expression of adhesion molecules and impaired migration towards CXCL12. Migration, physical interactions and response to soluble factors determine HSPC fate in the Bone Marrow (BM) niche. BM Mesenchymal Stromal Cells (MSC) are non-redundant regulators of HSPC in the niche; in addition, they possess pro- and anti-inflammatory properties, representing a bridge between hemopoiesis and inflammation. Finally, dysfunctions in MSC can induce myelodisplasia and secondary myeloid leukemia, while MSC inflammation cause genotoxicity in HSPC predicting myeloid leukemia evolution in predisposing syndromes. On that basis, we questioned if interaction between ER+cells, MSC and inflammation could favor preleukemic clone persistence and progression.
The murine proB cell line Ba/F3 was transfected to generate an inducible ER-V5tag expressing model (Ford A, Palmi C, 2009). BM-MSC were characterized and cultured for controlled passages. UCB-CD34+cells were immunomagnetically isolated and lentivirally transduced with pRRL-eGVP or pRRL-ER-eGFP constructs. Cells were treated with IL6/IL1β/TNFα inflammatory cytokines.
Gene Expression Profile shows that ER affects pathways involved in inflammatory response, cell cycle, apoptosis and migration in Ba/F3. In particular, ER+ cells overexpress CXCR2, a chemokine receptor also implicated in cancer, (MFI: ER=1378±807 vs ctr=284±167, p<0.05) and highly migrate toward its ligand CXCL1 (% migrated cell/input: ER=21.5±6.7 vs ctr=2.2±1.8, p<0.01). Interestingly, MSC increases CXCL1 secretion after inflammatory stimulation (murine MSC, pg/mL: basal=78±28 vs +infl.ck=30162±4760, p<0.01). In accordance, ER+ Ba/F3 are highly attracted by inflamed MSC supernatants (% migrated cell/input: ER=30.2±9.1 vs ctr=14.3±9.6, p<0.01) in a CXCR2-dependent manner. Coculturing control and ER+ Ba/F3 with MSC and inflammatory cytokines favored the persistence of preleukemic cells in the coculture (% ER+ fold increase: +MSC vs +MSC+infl.ck = 2.62±0.94, p<0.01). The effect is mediated by soluble factors and results from decreased survival in control (% ann-V negative cells: +MSC=68.4±5.7 vs +MSC+infl.ck=48.2±1.3, p<0.05) but not ER+ Ba/F3; cell proliferation was reduced in both, but the effect was stronger on control Ba/F3 (CSFE MFI fold increase +MSC vs +MSC+infl.ck: ER=2.2±0.6, p<0.001; ctr=4.4±1.8, p<0.05). However, CXCL1 is not implicated. Phosphorilation of histone H2AX and AID mRNA levels, which are basally higher in ER+ Ba/F3, further increase in both normal and ER+ Ba/F3 cocultured with MSC and inflammatory cytokines, confirming the genotoxicity of MSC inflammation (γH2AX MFI fold increase +MSC vs +MSC+infl.ck: ER=2.5±1, p<0.05; ctr=2.8±1.2, p<0.01) (AID mRNA fold increase basal vs +MSC+infl.ck: ER=6.3±1.6, p<0.05; ctr=14.6±11). Finally, preliminary data show a higher migration towards inflamed MSC also in ER+ UCB-CD34+cells (% migrated cell/input: ER=21.2±2.4 vs ctr=5.2±0.6, p<0.01).
ER expression increases migration towards inflamed BM-MSC supernatants in murine proB cells. Interestingly, MSC and inflammation create favoring microenvironmental conditions for preleukemic cells persistence and DNA damage accumulation. Preliminary results show that inflamed MSC highly attract human ER-expressing UCB-CD34+as well. Collectively, our data support the importance of ER-driven alterations in hematopoietic/BM stromal cells interactions in the leukemogenic process.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.