Cxcr4 ^low FLT3/ITD⁺ Cells Exhibit Enhanced Resistance to the FLT3 Inhibitor Quizartinib Compared to Cxcr4 ^high FLT3/ITD⁺ Cells By Elevating Runx1 Expression

The Runx1 transcriptional factor is significantly elevated in human AML cells with FLT3/ITD mutations (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD⁺ 32D cells to the type II FLT3 inhibitor quizartinib (Hirade et al. IJH 2016). While Cxcr4 levels are significantly downregulated in human FLT3/ITD⁺ AML cells and FLT3/ITD⁺ Ba/F3 cells compared to FLT3/ITD^- cells (Onishi et al. JBC 2014), Runx1 transactivates Cxcr4 expression (Jacob et al. Blood 2010), indicating that Cxcr4 is inversely regulated by FLT3/ITD and Runx1. Studies have indicated that Cxcl12 in the microenvironment protects FLT3/ITD⁺AML cells from chemotherapeutic insults through Cxcr4 (Zheng et al. Blood 2009). Herein, we investigated the interaction between Cxcl12/Cxcr4 signaling and Runx1 on the refractory activity of FLT3/ITD⁺ cells against quizartinib.

Runx1 shRNAs downregulated Cxcr4 levels in FLT3/ITD⁺ Ba/F3 cells, indicating that Runx1 enhances the expression of Cxcr4. However, a comparison of the intracellular Runx1 levels in FLT3/ITD⁺ Ba/F3 cells expressing different surface Cxcr4 levels that were sorted by FACS based on Cxcr4 expression demonstrated that the Runx1 protein levels were the highest in FLT3/ITD⁺ Ba/F3 cells harboring the lowest Cxcr4 compared to those expressing higher Cxcr4 levels, and were inversely correlated with the Cxcr4 levels (r=-0.78, P<0.05, N=6). Similarly, Runx1 mRNA levels were negatively correlated with the Cxcr4 mRNA levels in human FLT3/ITD⁺ AML cells (r=-0.34, P<0.01, N=78, GSE 1159), which was not observed in FLT3/ITD^- AML cells (N=187). Since Cxcr4 expression is regulated by FLT3/ITD and Runx1, we next compared the response to quizartinib of FLT3/ITD⁺ Ba/F3 cells with different surface expression levels of Cxcr4 in the presence or absence of Cxcl12. No significant difference in proliferation was observed in cells cultured without quizartinib. By contrast, the number of viable cells incubated with quizartinib (2 or 5nM) was significantly higher in Cxcr4 ^dim cells compared to Cxcr4 ^intermediate and Cxcr4 ^high cells. Cxcl12 dose-dependently inhibited the reduction of viable cells induced by 5nM quizartinib in Cxcr4 ^dim FLT3/ITD⁺ Ba/F3 cells (N=5, P<0.05). Similarly, 100ng/ml of Cxcl12 increased the number of Cxcr4 ^dim FLT3/ITD⁺ Ba/F3 cells that became refractory quizartinib (N=3, P<0.05). Likewise, Cxcl12 dose dependently prevented the decline of the number of human FLT3/ITD⁺ MV4-11 and MOLM13 AML cells induced by quizartinib (N=3, P<0.05). The prosurvival effect by Cxcl12 in Cxcr4 ^dim FLT3/ITD⁺ Ba/F3 cells was coincident with the Cxcl12-mediated elevation of Runx1 mRNA; however, Runx1 elevation after stimulation with 100ng/ml Cxcl12 was higher in Cxcr4 ^dim cells compared to Cxcr4 ^high cells. Runx1 shRNA significantly decreased proliferation and partially abrogated the anti-apoptotic effect of Cxcl12 against quizartinib in Cxcr4 ^dim FLT3/ITD⁺ Ba/F3 cells that were sensitive and refractory to quizartinib. ShRNA-mediated Runx1 knockdown also diminished chemotaxis towards Cxcl12 in quizartinib refractory Cxcr4 ^dim FLT3/ITD⁺ Ba/F3 cells (N=3, P<0.05), concomitant with a reduction in Cxcr4 expression. By contrast, similar cyto-protective effects of 10-100ng/ml Cxcl12 against 5nM quizartinib were not observed in Cxcr4 ^high FLT3/ITD⁺ cells. Cxcl12 rather enhanced the proapoptotic effect of 5nM quizartinib in Cxcr4 ^high cells (N=5, P<0.05). These data suggest that Cxcr4 ^dim FLT3/ITD⁺ cells are more refractory to quizartinib than Cxcr4 ^high cells, which is mediated by the elevation of Runx1.

Studies have indicated that Cxcr4 levels are downregulated and Runx1 levels are upregulated by FLT3/ITD, even though Runx1 enhances Cxcr4 expression. Our data indicate that Cxcr4 ^dim FLT3/ITD⁺ cells are more refractory to quizartinib in the presence or absence of Cxcl12 concomitant with higher Runx1 levels, compared to Cxcr4 ^high cells. Antagonizing Runx1 partially abrogated the refractory against quizartinib in Cxcr4 ^dim cells in the presence or absence of Cxcl12. These data implicate that Cxcr4 ^dim FLT3/ITD⁺ cells display increased FLT3/ITD activity compared to Cxcr4 ^high cells as a consequence of elevated Runx1 expression. Runx1 also enhanced chemotaxis of quizartinib resistant FLT3/ITD⁺ cells to Cxcl12. Targeting Runx1 represents an additional strategy to overcome FLT3 inhibitor refractory in FLT3/ITD⁺AML cells that are protected in the microenvironment.