Small Molecule Inhibition of LARG/RhoA Signaling Blocks Migration of ETV6-RUNX1 Positive B-Cell Precursor Acute Lymphoblastic Leukemia

Background and Objectives

Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognosis associated with ETV6-RUNX1 positive ALL, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. Leukemic cells reside in the bone marrow microenvironment, where they are nurtured and protected against chemotherapy. In this study, we investigated novel ways to disrupt this leukemic niche by targeting signaling pathways contributing to the migration of ETV6-RUNX1 positive leukemic cells.

Results

Gene expression profiling and subsequent pathway analysis of leukemic blasts of 654 ALL patients revealed a significant enrichment of genes involved in regulation of cellular movement and cell morphology in ETV6-RUNX1 positive BCP-ALL patients compared with ETV6-RUNX1 negative BCP-ALL patients (p < 0.001). In correspondence, the same pathways were significantly upregulated in cord blood-derived hematopoietic progenitor cells (CB-CD34⁺) ectopically expressing ETV6-RUNX1 (p < 10E^-06). LARG (ARHGEF12) was identified to be the most important regulator of this pro-migratory signature. This gene encodes for the G-protein-regulated Rho Guanine Exchange Factor 12, a specific activator of the GTPase RhoA. LARG expression was 5.7-fold higher in ETV6-RUNX1 positive BCP-ALL cells than in ETV6-RUNX1 negative BCP-ALL cells (p < 10E^-06). Similarly, LARG was upregulated 5.4-fold in CB-CD34⁺ cells expressing ETV6-RUNX1 compared with empty vector controls (p = 0.03).

To determine the importance of the LARG/RhoA pathway in the induction of this migratory phenotype, we used two recently identified small molecule inhibitors of the LARG/RhoA pathway: Y16, a specific LARG inhibitor, and G04 (Rhosin), a specific RhoA inhibitor. Both inhibitors significantly reduced the migration of ETV6-RUNX1 positive leukemic cells towards a gradient of CXCL12 (75%, p < 0.01) whereas the migration potential of ETV6-RUNX1 negative leukemic cells remained unaffected. Migration of ETV6-RUNX1 positive leukemic cells towards patient derived mesenchymal stromal cells was inhibited to a similar extent (75-85%, p < 0.01). In contrast, LARG/RhoA inhibition in ETV6-RUNX1 negative cells resulted in an induction of migration towards MSCs. While LARG/RhoA inhibitors reduced migration of ETV6-RUNX1 positive cells in a targeted manner, inhibition of CXCR4 by the CXCR4 antagonist AMD3100 (Plerixafor) reduced the migration of both ETV6-RUNX1 positive cells and ETV6-RUNX1 negative BCP-ALL cells.

Next, we studied the additional cellular effects of LARG/RhoA inhibition on leukemic cells. shRNA-mediated silencing of LARG modestly reduced the proliferation rate of both ETV6-RUNX1 positive and ETV6-RUNX1 negative cell lines (p<0.05). This reduced proliferation rate was accompanied by a moderate induction of apoptosis (p < 0.05). Using a multiplexed cell signalling assay, we further show that silencing of LARG moderately reduced the phosphorylation level of CREB (S133, 25%, p<0.05), while the phosphorylation state of other key signaling proteins was unaffected (including PI3K/PKB, MAPK/ERK, JAK/STAT and NFκB). In line with these data, inhibition of LARG/RhoA signaling with small molecule inhibitors did not affect cell survival of primary ETV6-RUNX1 positive BCP-ALL cells or resistance of these cells to Prednisolone or L-Asparaginase.

Conclusion

Together, our data indicate that small molecule inhibition of LARG/RhoA signaling affects the migration potential of ETV6-RUNX1 positive cells. In contrast to inhibition of the CXCR4/CXCL12 axis that reduces the migration of both leukemic and healthy hematopoietic cells, inhibition of the LARG/RhoA signaling pathway appears to specifically inhibit ETV6-RUNX1 positive cell migration. Our results warrant further studies to establish the clinical benefit of disrupting the leukemic niche in ETV6-RUNX1 positive BCP-ALL patients using small molecule inhibitors of LARG/RhoA signaling.