CCL5 Mediates Target-Kinase Independent Resistance to FLT3 Inhibitors in FLT3-ITD-Positive AML

FLT3-ITD tyrosine kinase inhibitors (TKI) show limited clinical activity in acute myeloid leukemia (AML) due to emerging resistance. However, only in a small number of cases, secondary FLT3 mutations can be identified that mediate the resistance. Therefore, other mechanisms must be active. We hypothesized that soluble factors such as cytokines might protect AML cells from TKI mediated cell death and thus contribute to treatment resistance.For this analysis, we generated PKC412- and sorafenib-resistant MOLM-13 cell lines as an in vitro model to study resistance to FLT3-ITD TKIs in human AML. In two out of five PKC412-resistant cell lines, cytokine arrays, ELISAs and quantitative PCR revealed an up to 5-fold overexpression of CCL5/RANTES in cell supernatants as well as on mRNA-level compared to PKC412 sensitive MOLM-13 cells. Notably, via MTS-assays and flow cytometry analysis, we were able to show that both short- and longterm CCL5-treatment mediated resistance against PKC412 to TKI sensitive MOLM-13 cells. In resistant MOLM-13 cell lines with high CCL5-release, CCL5-treatment downregulated the main CCL5-receptor CCR5, and these cell lines showed the lowest chemotaxis index among PKC412-resistant cell lines when migrating against CCL5 as well as CXCL12/SDF-1α. In addition, regulation of CXCR4, which is present in a complex together with CCR5 seems to be also dependant from the CCL5-level, since in the PKC412-resistant cell lines with high CCL5-release, we found CXCR4 to be downregulated on protein level. Moreover, in an ELISA we could show, that these cells secrete almost no SDF-1α, which is the known CXCR4 ligand. Finally, we analyzed isolatedblasts from patients with FLT3-mutated AML before and after treatment with FLT3 inhibitors for changes in CCL5 mRNA expression. Interestingly, isolated AML blasts revealed increased CCL5 transcripts after PKC412-treatment in three cases tested compared to controls. These observations suggest that CCL5 may be an important factor mediating resistance to FLT3 inhibitors in FLT3-mutated AML and could possibly serve as a biomarker to predict resistance to treatment.