MI-503 is an efficient and selective Menin-MLL inhibitor with an IC50 of 14.7 nM. In human MLL leukemia cell lines, MI-503 has a significant growth inhibition effect (GI = 250 nM-570 nM).

Menin-MLL:4.7 nM (cell free)

Treatment of murine bone marrow cells (BMC) transformed with the mLL-AF9 oncogene with MI-503 results in substantial growth inhibition (GI50: 0.22 μM). The cell growth inhibitory effect of MI-503 is time-dependent, with a pronounced effect achieved after 7-10 days of treatment.

Following a single intravenous or oral administration, MI-503 rapidly attains high concentrations in peripheral blood and demonstrates significant oral bioavailability (75%). This compound effectively suppresses tumor growth through daily intraperitoneal (i.p.) doses, leading to an over 80% decrease in MV4;11 tumor volume, including complete regression in two mice. Continuous treatment over ten days notably delays the progression of mLL leukemia and substantially lowers the leukemia tumor burden in mice. Additionally, combined use of MI-503 and MI-463 significantly diminishes the expression of Hoxa9 and Meis1, indicating a potent antitumor activity.

Leukemia cells are treated with MI-503 or 0.25% DMSO and cultured at 37 °C for 7 days. Media is changed on day 4, viable cell numbers are restored to the original concentration and MI-503 are re-supplied. MTT cell proliferation assay kit is then employed, and plates are read for absorbance at 570 nm using a microplate reader.

For efficacy studies in MV4;11 subcutaneous xenograft mice model, 5×10^6 cells are injected into the 4-6 week old female BALB/c nude mice. Treatment is started when the tumor size reached ~100 mm^3. Vehicle (25% DMSO, 25% PEG400, 50% PBS) or compounds (MI-463 or MI-503) are administrated once daily at designated doses using i.p. injections.