Midostaurin Reduces Regulatory T Cell Population in Healthy and Acute Myeloid Leukemia Cells

Background: Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by the rapid growth of abnormally differentiated hematopoietic cells called blasts which accumulate in the blood to cause bone marrow failure. The only curative option available to patients with AML is intensive chemotherapy followed by allogeneic stem cell transplant (Allo-HSCT). FMS-Like Tyrosine Kinase III Internal Tandem Duplication (FLT3 -ITD), a common mutation in patients with AML, is associated with poor prognosis. Although Allo-HSCT has significantly improved the dismal outcome of patients with FLT3 -ITD positive AML, ~50% of patients relapse within two years after transplant. The recognition and elimination of leukemic clones by donor T cells or the graft vs leukemia (GvL) effect contributes significantly to the success of Allo-HSCT. FLT3 inhibitors have been extensively investigated in patients with FLT3 -ITD AML, and have demonstrated promising results in clinical trials. How FLT3 inhibitors affect T cells and whether adding these targeted therapies to Allo-HSCT will have a therapeutic benefits or drawbacks is unknown. Because T cells play a crucial role in the GvL effect, investigating therapeutic approaches to modulate the T cell populations, function, and repertoire to enhance the GvL effect is highly needed. Regulatory T cells (Treg), a subset of CD4⁺ T cells with suppressive functions to maintain self-tolerance, are present at a higher percentage in patients with AML, and are recognized to drive immunosurveillance evasion in leukemic cells.

Here, we hypothesize that current FLT3 inhibitors affect T cell population and function either through their off-target effect on T cell signaling pathways or through induction of leukemic cell apoptosis and presentation of leukemic antigens. In this abstract we report the effect of four FLT3 inhibitors (sorafenib, midostaurin, tandutinib, and quizartenib) on T cell population.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from freshly collected blood samples obtained from healthy donors (N=5). PBMC were stimulated with IL-2 (5ng/ml), IL-7 (10ng/ml), and treated with 1mM of one of the following FLT3 inhibitors (sorafenib, midostaurin, tandutinib, and quizartenib) for 72 hours. We analyzed T cell populations (CD4+, CD8+, and CD25+ cells) by flow cytometry. We also measured Treg marker FOXP3 mRNA expression by quantitative RT-PCR. Similarly, blood samples from patients with AML were examined (N=3). Cell viability were also assessed by alamar blue assay and by flow cytometry.

Results: Healthy PBMCs treated with midostaurin had a significant decrease in CD4+CD25+ T cell population when compared with other treatment groups (N=3, 80% decrease, P=0.0006). sorafenib showed a modest decrease in CD4+CD25+ T cells (N=3, P=0.12). On the other hand, tandutinib and quizartenib did not affect CD4+CD25+ population. No effect was observed on total lymphocytes, CD3, CD8, CD4 T cell populations, when cells were treated with FLT3 inhibitors. In addition, we found that treatment with midostaurin resulted in significant decrease in relative FOXP3 mRNA expression when compared with other treatment groups (N=5, P=0.02). Viability assay confirmed that FLT3 inhibitors did not affect cell viability in healthy PBMC but they significantly decreased the viability of MV4-1 cells (a FLT3 -ITD positive AML cell line) (50-70% decrease P=0.0007). Although the percentages of T cells were very low in AML blood samples obtained at diagnosis, the observed trend was similar to that found in healthy PBMCs. CD4+CD25+ population was reduced compared to controls (N=2, 2-fold, P=0.10). Similarly, treatment of AML primary blasts with midostaurin also reduced FOXP3 mRNA expression (n=2, 50% decrease, P=0.0001)

Conclusions: These results indicate that treatment with midostaurin causes a decrease in regulatory T cell population. Although the mechanism by which midostaurin reduces Tregs is not clear, our data indicates a possible off-target effects of this multi-kinase inhibitor on T cell signaling pathways. We are currently exploring the effect of midostaurin on Tregs function and repertoire. In light of the recent FDA approval of midostaurin use in patients with AML, these results highlight a novel therapeutic advantage of this drug that may be beneficial particularly in the setting of Allo-HSCT.