824P - FLT3-ITD induces immune escape in AML via up-regulating CD47 expression and decreased phagpcytic ability of macrophages

Background

FLT3-ITD is one of the most common genetic abnormalities in acute myeloid leukemia (AML) and suggests a poor prognosis. The impact of FLT3-ITD on the immune microenvirenment requires further elucidation. Studies have shown that FLT3-ITD can significantly change the level of immune cells in the bone marrow in mice.

Methods

Flow cytometry was used to detect the CD47 mean fluorescent intensity (MFI). The FLT3 wildtype (FLT3-OE, FLT3 over expression) or ITD mutation (FLT3-ITD) stable overexpression K562/HEL cells were constructed by lentiviral transfections. The TRANSFAC database was used to predict the transcription factor binding sites of the CD47 promoter. We analyzed the location of CD47 gene on the chromosome, the sequence of promoter and first non-coding exon of HOXB5 via Jaspar online tool. The AML mouse model was constructed by injection of FLT3-ITD cells.

Results

LDH-induced macrophage killing was less in FLT3-ITD cell lines than FLT3-WT cell lines. Expression of CD47 can help protect tumor cells from attack by macrophages. The CD47 MFI of the FLT3-ITD group was higher than that of the control group. The CD47 MFI of the FLT3-ITD group was significantly higher than that of FLT3-OE group and FLT3 normal control (FLT3-NC) group. Flow cytometry results showed that FLT3-ITD impaired the activity of red-stained macrophages to phagocytizing green-stained K652/HEL cells. Using the TRANSFAC database, we found higher relative expression of CD47 gene in FLT3-ITD group than other groups and that HOXB5 may regulate the expression of CD47 at transcriptional level. Integrated analysis of Jasper online tool, dual luciferase reporter gene assay and ChIP experiment suggest that HOXB5 directly activate CD47. Compared with other groups, combination of CD47 inhibitor and FLT3-ITD inhibitor Quizartinib (AC220) significantly enhanced phagocytic activity of macrophages. The mouse model of AML further showed that combination of CD47 inhibitor and Quizartinib significantly reduced tumor burden in spleen and bone marrow.

Conclusions

Our data show that FLT3-ITD can induce immune escape to macrophages by upregulating CD47. Combination therapy with CD47 inhibitor and FLT3-ITD inhibitor can be promising for the treatment of FLT3-ITD AML.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Sun Yat-sen University Start-Up Funding.

Disclosure

All authors have declared no conflicts of interest.