Identification and Characterization of 6-Benzylthioinosine as a Novel Myeloid Differentiation-Inducing Compound.
Authors: Wald David N. , Vermaat Hanna , Kang Zizhen , Gerson Stanton L. , Bunting Kevin D. , Tse William 
Published: November 2019

Acute myeloid leukemia (AML) is the most common form of leukemia in adults and despite advances in treatment the 5 year survival is less than 20–50% in adults and significantly lower in the elderly. The remarkable success in treating one relatively uncommon subset of AML, APL, with all trans-retinoic acid (ATRA) illustrates the great promise for agents with greater efficacy and less toxicity. Utilizing ATRA, the presumed cure of 75–85% of patients is possible. ATRA’s remarkable success stems from the fact that AML is a disease characterized by the arrest of differentiation of immature myeloid cells. ATRA overcomes this block in differentiation by forcing leukemic cells to mature. Unfortunately, ATRA is not clinically useful for patients with AML that do not have the APL subtype. Through a compound library screen, we have recently identified several novel leukemia differentiation-inducing compounds structurally unrelated to previously described differentiation-inducers including a particularly promising compound, 6-benzylthioinosine (6BT). 6BT is a nucleoside analogue that induces monocytic differentiation of multiple AML cell lines as well as primary leukemic patient samples as measured by immunophenotyping, morphology, and NBT reduction. 6BT can induce terminal differentiation of leukemic cells as evidenced by complete prevention of colony formation in soft agar assays after pretreatment with low doses of 6BT (5μM) for 72 hours. In addition 6BT has potent in vivo activity. 6BT completely prevents subcutaneous tumor formation in myeloid leukemia mouse xenograft models in nude mice using HL-60 or MV4-11 leukemic cell lines (n=5) and significantly inhibits myeloid leukemia tumor growth in an HL-60 established tumor xenograft model (n=5). In the established tumor model at the end of the 4 week study period, tumors were significantly smaller after 6BT treatment as compared to vehicle treated mice (0.16g +/− 0.05g vs 0.73g +/− 0.28g). Evidence of 6BT-mediated in vivo differentiation exists as 92% of the tumor cells expressed the mature myeloid cell surface marker CD11b as compared to only 16% in the vehicle control after 4 weeks. Early studies suggest 6BT partially depletes purine nucleotide stores leading to growth inhibition and subsequent myeloid differentiation. Treatment of HL-60 cells with 6BT (5μM) leads to rapid depletion of approximately 75% of ATP stores within 24 hours. The depletion of ATP is not due to the differentiation process itself as after 6BT treatment HL-60 cells contain 24.6% +/− 2.8 of the ATP levels of vehicle treated cells compared to 91.2% +/− 7.2 in ATRA (1μM) treated cells. Preliminary studies also indicate that 6BT exhibits relatively low toxicity with an LD50 at 5 days greater than 100μM in human umbilical vein endothelial cells, mouse embryonic fibroblasts, and primary human lymphocytes. The low toxicity is likely related to its unique chemical structure that prevents its uptake into most mammalian cells as it acts as an inhibitor of one of the main nucleoside transporters, ent1. It is known that leukemic cells can express nucleoside transporters that are not widely expressed by other cell types. As differentiation therapy has already been demonstrated in the case of ATRA to significantly improve the prognosis of patients with AML, further characterization of 6BT could lead to a novel AML therapy that is more efficacious, less toxic, and better tolerated especially for elderly patients.