Background: The SLIT/ROBO axis has been shown to play a key role in many physiological processes such as organogenesis, axon guidance and angiogenesis. Additionally, several studies conducted in solid tumors observed frequent hypermethylation of either Slit or Robo at their promoter site. In the context of leukemia, Golos et al., 2019 showed that the low expression of SLIT2 was associated with lower overall survival in adults with acute myeloid leukemia (AML). Furthermore, it was demonstrated by our group that the knockdown of SLIT2 in Acute Promyelocytic Leukemia (APL) cells leads to an increase of cell proliferation in vitro and a more aggressive course of the disease in vivo (Weinhäuser et al., 2020). Aims: Given the evidence of the relevance of SLIT2 in APL, we opted to transfer our gained knowledge to a more challenging leukemia subset and decided to study the role of SLIT2 in AML. Methods: We first evaluated the methylation pattern of SLIT2 in AML patients compared to healthy donors by analyzing publicly available datasets (GSE58477, normal karyotype blasts: 62, healthy CD34+: 10; GSE63409, LSC: 14, HSC: 5), followed by the assessment of the level of SLIT2 in the bone marrow (BM) plasma of AML patients and healthy donors by ELISA. Additionally, to functionally assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV4-11, and MOLM13) with recombinant SLIT2 (50ng/mL) in vitro. We performed the knockdown of SLIT2 in AML cell lines (THP-1 and OCI-AML3) and evaluated their proliferation capacity as well. Moreover, AML cells were treated with decitabine and recombinant SLIT2. Finally, we evaluated the anti-leukemic effects of SLIT2 in vivo. To do so NSGS mice were transplanted with luciferase-transduced MV4-11 cells and the animals were either treated with vehicle (control group) or recombinant SLIT2 (25 ng/g of body weight) two days after transplant for a period of one week. Results: Our analysis indicated increased methylation at the SLIT2 promoter site in AML patients compared to healthy CD34+. In accordance with our analysis, we detected decreased level of SLIT2 protein in the bone marrow (BM) plasma of AML patients (1.43 ng/mL) when compared to healthy donors (3.51 ng/mL) (p<0.05). The functional role of SLIT2 was determined with the treatment of KASUMI1, MV4-11 and MOLM13 cells with recombinant SLIT2 in vitro. Our results showed that SLIT2 treatment reduced the cell proliferation and colony formation capacity and induced cell cycle retention in the G1 phase for all AML cell lines. Contrarily, the knockdown of SLIT2 promoted increased cell proliferation in THP-1 and OCI-AML3 cell lines. Moreover, we observed increased induction of decitabine-induced apoptosis when MV4-11 and MOLM13 cells were treated in the presence of recombinant SLIT2. Finally, the engraftment and progression of the disease of NSGS mice transplanted with luciferase-transduced MV4-11 cells were monitored by the detection of luciferase bioluminescent signals. Our results showed, that SLIT2 treatment was able to significantly delay the progression of AML in vivo. Mice treated with SLIT2 presented improved overall survival (vehicle 15d: CI 13-16d; SLIT2 19d: 95% CI: 16-22d. P value = 0.0320), decrease leukemic infiltration in the BM and spleen, reduced spleen size (vehicle: 125 mg; SLIT2: 100 mg P value= 0.04), indicating lower disease burden when compared to the control group. Summary/Conclusion: In conclusion, our results suggest that SLIT2 has tumor suppressive functions in AML in vitro and in vivo highlighting the therapeutic potential with low cytotoxicity of SLIT2 in AML.