FLT3 Inhibitor Quizartinib Facilitates Proliferation and CXCL12-Induced Chemotaxis in Quizartinib Resistant FLT3/ITD ⁺ Hematopoietic Cells By Increasing RUNX1

RUNX1 generally functions as a tumor suppressor in the hematopoietic system. However, RUNX1 expression is significantly elevated in human AML cells with FLT3/ITD mutations, promotes leukemogenesis induced by FLT3/ITD (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD ⁺ cells to type-II FLT3 inhibitor quizartinib (Hirade et al IJH 2016). We previously reported that RUNX1 expression is higher in CXCR4-^low FLT3/ITD ⁺ cells compared to Cxcr4-^high FLT3/ITD ⁺ cells, even though Cxcr4 expression is trans-activated by RUNX1. This difference in RUNX1 expression level was associated with divergent response to CXCL12 in FLT3/ITD ⁺ cells harboring different CXCR4 expression levels that were exposed to quizartinib (Fukuda S. et al. ASH 2019). Our data also demonstrated that RUNX1 expression is down-regulated following withdrawal of quizartinib in FLT3/ITD ⁺ cells that became refractory to quizartinib (Hirade et al. IJH 2016), suggesting that RUNX1 expression may be up-regulated by quizartinib in FLT3/ITD ⁺ cells. Since RUNX1 regulates proliferation of FLT3/ITD ⁺ AML cells, the present study investigated association between RUNX1 expression levels and proliferation of quizartinib resistant FLT3/ITD ⁺ cells that are exposed to quizartinib.

In the sensitive FLT3/ITD ⁺ Ba/F3 cells, RUNX1 protein expression was transiently up-regulated but eventually down-regulated by 5 nM quizartinib, coincident with decline in the viable cells. In contrast, RUNX1 expression was up-regulated by quizartinib and remained elevated in the resistant FLT3/ITD ⁺ Ba/F3 cells. Since RUNX1 enhances proliferation of FLT3/ITD ⁺ cells, we next examined whether proliferation FLT3/ITD ⁺ cells that acquired resistance to quizartinib is facilitated by quizaritinib as a result from quizartinib-mediated up-regulation of RUNX1, using the Cxcr4-^low and Cxcr4-^high FLT3/ITD ⁺ cells that acquired resistance to quizartinib. Although CXCL12 barely enhanced the proliferation of refractory FLT3/ITD ⁺ Ba/F3 cells, 5 nM quizartinib significantly increased the proliferation of both Cxcr4-^low and Cxcr4-^high FLT3/ITD ⁺ Ba/F3 cells that acquired resistance to quizartinib compared to those without quizartinib. This increase in the proliferation of Cxcr4-^low and Cxcr4-^high FLT3/ITD ⁺ Ba/F3 cells coincided with the elevation in RUNX1 and CXCR4 protein expression. Moreover, the resistant Cxcr4-^low FLT3/ITD ⁺ Ba/F3 cells proliferated significantly faster than Cxcr4-^high FLT3/ITD ⁺ cells, with concomitant higher expression of RUNX1 in Cxcr4-^low FLT3/ITD ⁺ cells than in Cxcr4-^high FLT3/ITD ⁺ cells. Likewise, type-I FLT3 inhibitor gilteritinib significantly enhanced proliferation of Cxcr4-^low and Cxcr4-^high FLT3/ITD ⁺ Ba/F3 cells that acquired resistance to gilteritinib. Knocking down Runx1 using shRNAs significantly decreased the enhanced proliferation induced by quizartinib in refractory FLT3/ITD ⁺ Ba/F3 cells, coincident with reduction in CXCR4 expression.

Since CXCR4 expression level was elevated by quizartinib in the FLT3/ITD ⁺ cells refractory to quizartinib, we next examined CXCL12-induced migration in quizartinib-resistant FLT3/ITD ⁺ cells following exposure to quzartinib. Pre-incubating the quizartinib resistant Cxcr4-^low or Cxcr4-^high FLT3/ITD ⁺ Ba/F3 cells with 5 nM quizartinib for 72 hours significantly enhanced their migration to 100 ng/ml of Cxcl12 compared to those without quizartinib, coincident with elevation in RUNX1 levels. Surprisingly, migration of CXCR4-^low FLT3/ITD ⁺ cells to CXCL12 was significantly elevated compared to CXCR4-^high cells, with concomitant higher expression of RUNX1 in Cxcr4-^low FLT3/ITD ⁺ cells than in Cxcr4-^high FLT3/ITD ⁺ cells. Silencing Runx1 using shRNAs significantly decreased migration to CXCL12 in refractory Cxcr4-^low FLT3/ITD ⁺ Ba/F3 cells.

These data indicate that the FLT3 inhibitor itself can facilitate the proliferation and migration to CXCL12 in FLT3/ITD ⁺ cells that are refractory to FLT3 inhibitors by up-regulating RUNX1. The results implicate that FLT3 inhibitors may worsen the disease progression in the patients that became refractory to FLT3 inhibitors by facilitating proliferation and migration to CXCL12 of the resistant FLT3/ITD ⁺ AML cells. In this regard, targeting RUNX1 may represent additional strategy to eradicate resistant FLT3/ITD ⁺ AML cells, in which their proliferation and migration are supported by FLT3 inhibitors.