Thesis title: In vitro anti-tumor activity of poly(ADP-polymerase) inhibitors as single agents or combined with hypomethylating agents or high-dose ascorbate in Myelodysplastic Syndrome and Arsenic Trioxide-Resistant Acute Promyelocytic Leukemia
Poly(ADP-ribose) polymerase inhibitors (PARPi) are a new class of anticancer drugs approved for DNA repair defective solid tumors that target enzymes involved in modulation of DNA repair, DNA demethylation and regulation of chromatin structure. Here, two different models of hematological malignancies, Myelodysplastic Syndromes (MDS) and Acute Promyelocytic Leukemia (APL), typically characterized by high genomic instability were studied for their susceptibility to the anticancer activity of PARPi, at clinically relevant concentrations. PARPi were tested as monotherapy or in combination with DNA damaging and demethylating agents (i.e., azacytidine/decitabine and high-dose vitamin C in the form of ascorbate), whose effects on DNA lead to PARP1 intervention.
Hypomethylating agents, currently in clinical practice for AML and intermediate/high-risk MDS, are cytidine analogues able to induce DNA damage as a consequence of their random incorporation into nucleic acids and to act as DNA methyltransferases (DNMTs) inhibitors.
Vitamin C is a co-factor of the Ten-Eleven Translocation (TET) enzymes that catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC); the subsequent conversion in cytosine generates DNA lesions that recruit PARP1 and activates the base excision repair (BER) pathway.
In the first part of my research activity, primary cultures of bone marrow mononuclear cells were obtained from patients with MDS (n=28) and in vitro treatment with the PARPi olaparib caused cytotoxic effects in most samples (IC50 2.0–24.8 µM), at concentrations lower than the plasma peak (Cmax) reached in cancer patients. In samples with low olaparib IC50s, the PARPi induced DNA damage and preferentially killed myeloid cells, sparing lymphocytes. Interestingly, olaparib also induced differentiating effects as reveled by morphological and immunophenotypic analysis and by analysis of transcription factors (PU.1 and CEBPA) drivers of granulocytic and monocytic differentiation. The combination of olaparib with the hypomethylating agent decitabine resulted in augmented cytotoxic and differentiating effects, likely due to the altered processing of the aberrantly incorporated cytidine analogue and DNMT1 entrapment in DNA.
In the second part of my research activity, ATO-sensitive and -resistant APL clones were generated from the NB4 APL cell line and analyzed for their susceptibility to five clinically used PARPi (olaparib, niraparib, rucaparib, veliparib and talazoparib). In fact, in 5-10% of APL cases patients fail to respond to first-line treatment with all trans retinoic acid (ATRA)/arsenic trioxide (ATO) or experience disease relapse. The resulting PARPi IC50 values were far below (olaparib and niraparib), within the range (talazoparib) or above (rucaparib and veliparib) the Cmax reported in cancer patients. ATO-resistant APL cells were also susceptible to the hypomethylating agents azacitidine and decitabine and to high-dose vitamin C (ascorbate) as single agents. The combination of these agents with olaparib, niraparib or talazoparib, resulted in synergistic antitumor activity. Interestingly, PARPi/ascorbate co-treatment increased DNA damage and 5hmC-mediated DNA demethylation, triggering the intervention of PARP1 as a member of the BER pathway. Talazoparib was the most effective PARPi in synergizing with ascorbate, in line with its higher PARP1 trapping potency in DNA, and co-treatment enhanced the talazoparib-mediated PARP1 trapping. Thus, in the presence of PARPi, the ascorbate-mediated generation of 5hmC in DNA may result in stalled repair and cell death of ATO-resistant APL cells.
Overall, these findings encourage further studies on PARPi efficacy in MDS and relapsed/ATO-refractory APL patients as single agents and combined with hypomethylating agents or high-dose ascorbate.