Targeting of MDS and AML Stem Cells Via Inhibition of STAT3 By Pyrimethamine

Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional and epigenetic analysis of highly fractionated stem and progenitor populations in individual patients of MDS and identified STAT3 upregulation in MDS HSCs. qRTPCR in an independent set of sorted MDS/AML HSCs (Lineage-negative, CD34+, CD38-) confirmed the significant increase in STAT3 expression in 60% of cases when compared to healthy controls. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of STAT3 in MDS when compared to healthy controls (FDR<0.1) and importantly, found that increased STAT3 expression was predictive of significantly adverse prognosis (log rank P value < 0.01, median survival of 2.6 years compared to 5.8 years for group with lower STAT3) in patients. This further points to a critical role of STAT3 signaling in AML/MDS pathogenesis and progression.

Next, we studied the functional role of STAT3 by using Pyrimethamine as a clinically relevant inhibitor. Pyrimethamine is an FDA approved antifolate compound that was found to be a specific inhibitor of STAT3 activity in an initial screen of 1120 compounds. 3D modeling studies reveal that Pyrimethamine can occupy the SH2 domain of STAT3 protein with high avidity. It shows half-maximal activity (EC₅₀) for STAT3 inhibition at approximately 1.5 μM, well within the plasma concentrations found with clinical use.

We assessed the effect of Pyrimethamine on cellular proliferation of multiple leukemic cell lines (KG-1, KT-1 and CMK), and observed that these were significantly inhibited at 120 hours of exposure in a dose dependent fashion (t test, p value <0.004, Mean + SD of 3 experiments). Pyrimethamine was also able to induce significant apoptosis in AML cell lines at 72 hours at a 10 μM concentration. (P Value <0.05)

In summary, we have found significant demethylation and increased expression of STAT3 in sorted HSCs from AML and MDS patients. High STAT3 expression is a marker of adverse prognosis in a large cohort of MDS patients. In vitro studies show that Pyrimethamine can inhibit STAT3 activation and is able to inhibit proliferation by inducing apoptosis in leukemic cells. A phase II study evaluating Pyrimethamine for the treatment of intermediate/high-risk MDS patients that have relapsed or are refractory to azanucleoside therapy has been initiated.