Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells

Abstract

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

PCNA-interacting peptides reduce Akt phosphorylation and TLR-mediated cytokine secretion suggesting a role of PCNA in cellular signaling

“…For example, in mature non-proliferating neutrophils, PCNA is exclusively located in the cytosol where it serves as a binding platform for procaspases, inhibits their activation and apoptosis, and thereby regulates neutrophil survival [3]. We have shown that high levels of cytosolic PCNA are found in multiple myeloma cells, and that targeting PCNA with APIM-peptide induces rapid caspase activation (caspases 3,7, 8 and 9) followed by apoptosis in multiple myeloma, but not in healthy primary cells [4]. Recently, cytosolic PCNA was shown to bind and stabilize procaspase-9 in a neuroblastoma cell line, and interestingly S-nitrosylation of PCNA blocked this interaction suggesting that the binding is regulated by posttranslational modifications (PTMs) [5].…”

“…We found that overexpression of the APIM-peptide, or treatment of cells with an APIM-containing cell penetrating peptide, reduced growth rate and increased apoptosis when cancer cells were exposed to chemotherapeutics and/or were stressed, but not, or far less, under normal conditions. Furthermore, a peptide with an impaired APIM-sequence, mutAPIM-peptide, had less effect, thus the APIM-PCNA interaction is required for these activities [4,11].…”

“…We have recently shown that APIM-peptide increases the anti-cancer activity of melphalan, mitomycin C, and bleomycin in three different animal models [4,12].…”

A novel cell permeable DNA replication and repair marker

“…S2). 13,31 In particular, it has been reported that both the PL and p21 peptides interfere with DNA replication. 13,19 The results presented here outline a general strategy to design and efficiently deliver these functional peptides using cell-penetrating peptides providing a versatile tool to potentially target proliferative diseases.…”

Selective inhibition T2AA for human PCNA over PCNA homolog in Trypanosoma brucei

“…We did site directed mutagenesis in the TbPCNA and HsPCNA open reading frames, changing methionine-40 to alanine (M40A) in both homologs. M40 is part of the evolutionary conserved central loop that forms the hydrophobic pocket of PCNA with the IDCL and the C-terminus (CarrascoMiranda et al, 2014;Tsurimoto and Stillman, 1991;Warbrick et al, 1995) to mediate hydrophobic interactions with PIP-box proteins (Muller et al, 2013). We discovered that parasites overexpressing either the TbPCNA M40A mutant (Figure 2 To test this hypothesis, we used the small molecule inhibitor T2AA as the chemical tool to broadly inhibit PCNA/PIP-box protein interactions.…”