Evaluation of an Actinomycin D/VX-680 aurora kinase inhibitor combination in p53-based cyclotherapy

Abstract

AbstrAct:p53-based cyclotherapy is proving to be a promising approach to palliate undesired effects of chemotherapy in patients with tumours carrying p53 mutations. For example, pre-treatment of cell cultures with Nutlin-3, a highly-selective inhibitor of the p53-mdm2 interaction, has been successfully used as a cytostatic agent to protect normal cells, but not p53-defective cells, from subsequent treatment with mitotic poisons or S-phase specific drugs. Here we sought to evaluate whether low doses of Actinomycin D (LDActD), a clinically-approved drug and potent p53 activator, could substitute Nutlin-3 in p53-based cyclotherapy. We found that pre-treatment with LDActD before adding the aurora kinase inhibitor VX-680 protects normal fibroblasts from polyploidy and nuclear morphology abnormalities induced by VX-680. However, and although to a lower extent than normal fibroblasts, tumour cell lines bearing p53 mutations were also protected by LDActD (but not Nutlin-3) from VX-680-induced polyploidy. We also report that a difference between the response of p53 wild-type cells and p53-defective cells to the LDActD/VX-680 sequential combination is that only the former fail to enter S-phase and therefore accumulate in G1/G0. We propose that drugs that incorporate into DNA during S-phase may perform better as second drugs than mitotic poisons in cyclotherapy approaches using LDActD as a cytostatic agent.

An evaluation of small-molecule p53 activators as chemoprotectants ameliorating adverse effects of anticancer drugs in normal cells

“…S2). These observations, which extend our previous work using LDactD in combination with the Aurora kinase inhibitor VX680, 6 suggest that the performance of nutlin-3 and LDactD in cyclotherapy is comparable. However, Figure 6 shows that, unlike nutlin-3, LDactD shields HCT116-p53ko cells from the cytotoxicity caused by Ara-C and vinca alkaloids.…”

“…30 It has been shown that human epidermal keratinocytes undergo reversible cell cycle arrest when exposed to 1 nM ActD for 24 h. 30 Similarly, HNDFs incubated with 4 nM ActD for 72 h re-entered the cell cycle when cultivated further in fresh medium. 6 In agreement with these observations, Figure 2A shows that HNDFs accumulate in G 1 and G 2 in response to LDactD, while Figure 2B confirms the reversibility of the cell cycle arrest.…”

“…30 LDactD (1-4 nM) has a reversible cytostatic effect on HNDFs ( Fig. 2 and Rao et al 6 ), and, like the other p53 activators tested, it protects HNDFs from cytotoxicity and appearance of aberrant nuclei caused by VNB, VRL, Ara-C and GMTB (Fig. 3).…”

“…[36][37][38] As a DNA-intercalating compound, ActD is highly unselective, cytotoxic and genotoxic at high doses. However, it does not induce DNA damage markers at low nanomolar concentrations, 6,30 and LDactD can mimic nutlin-3 exposure in terms of p53-dependent mRNA expression profiles. 30 It has been shown that human epidermal keratinocytes undergo reversible cell cycle arrest when exposed to 1 nM ActD for 24 h. 30 Similarly, HNDFs incubated with 4 nM ActD for 72 h re-entered the cell cycle when cultivated further in fresh medium.…”

Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides

“…Inhibition of HDM2 by a wide range of antagonists has been shown to lead to increased cellular levels of wild type p53 and cell death [20–23]. In normal cells, HDM2 inhibition can cause reversible arrest, highlighting a possible target for cyclotherapy regimens [24–26]. The prototypical HDM2 antagonist Nutlin and its numerous derivatives comprise a signature chemotope that recapitulates the interaction of 3 key residues in the N-terminal region of p53 (F19, W23, L26) with a hydrophobic pocket in the N-terminal domain of HDM2 [20, 27–29].…”