Several lines of evidence suggest that genomic instability in myeloid malignancies is promoted by increased endogenous DNA damage and error-prone repair that lead to disease progression and resistance to therapy. We recently reported increased levels of Poly-(ADP)-ribose polymerase (PARP) and DNA Ligase IIIα as well as increased activity of a highly error-prone pathway for repair of DNA double-strand breaks in myeloid leukemias. Importantly, these leukemia cells are sensitive to inhibitors of DNA Ligase IIIα and PARP, suggesting their dependence on these factors for survival. Parallel studies have shown that transient exposure to DNA demethylating agents at low nM concentrations reprograms cancer cells, altering heritable gene expression patterns in key cellular pathways, including DNA repair pathways, suggesting that pre-treatment of leukemia cells with demethylating agents may further sensitize them to PARP inhibitors.
Thus, established cell lines from acute myeloid leukemia (AML; MV411, KASUMI-1), myelodysplastic syndrome transformed to AML (MDS; P39) and bone marrow mononuclear cells obtained from AML patient samples (N=7) were exposed to non-cytotoxic doses of DNA methyltransferase inhibitors (DNMTis, decitabine, DAC), followed by four days without drug treatment and subsequent treatment with low doses of PARP inhibitors (PARPis; ABT888, or BMN673) alone or in combination with DNMTis. Clonogenicity, apoptosis, DNA repair efficiency, and activity and expression level of DNA repair and DNA methyltransferase proteins were then studied.
In all the cell lines tested, treatment with DAC (5-10nM) followed by ABT888 (500nM) induced a significant decrease in colony survival compared to control or single treatment. The use of a more potent PARPi, BMN673 (0.1-10nM), confirmed that treatment with DNMTis followed by PARPis induces a robust inhibition of AML and MDS cell line colony forming capacity. Interestingly, the same schedule treatment of decitabine followed by PARPis significantly decreases the clonogenic capacity in 4 out of 7 (57%) of bone marrow mononuclear cells from AML patient tested so far, suggesting that DNMTis and PARPis sequential treatment could be a valuable therapeutic option for AML and MDS patient.
We next initiated studies to elucidate the mechanism by which DAC may sensitize myeloid malignancies to PARPis. As expected, DAC treatment alone was sufficient to decrease DNMT1 expression levels and increase caspase 3 cleavage in AML cell lines, compared to control treated cells. But surprisingly, DAC treatment alone also induced a decrease in PARP protein expression, with a further decrease in cells treated with DAC followed by PARPis, suggesting that both methylation and DNA repair signaling alter PARP1 steady-state levels. Moreover, preliminary results show that the presence of PARP on chromatin is decreased with DAC treatment and further decreased following PARPis.
In conclusion, our results suggest that DNMTis reprogram cells, sensitizing them to PARP inhibition in AML/MDS patient and cell line models, paving the way for testing the therapeutic potential of sequential treatment with these agents in clinical trials. We are exploring one hypothesis that decreased levels of PARP on chromatin following DAC treatment may lead to more effective trapping of PARP1 at sites of DNA damage by PARPis, leading to abrogation of DNA repair. Understanding how these proteins interact may explain the mechanisms underlying the sensitization of epigenetically reprogrammed cells to PARPis, and may define the molecular subsets of AML patients that may respond to this novel therapeutic strategy.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.