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are initially effective in a significant proportion of patients,7,8 there is evidence to suggest that treatment with both of these agents causes an increase in NF-κB activation which has been linked to drug resistance and treatment failure.9,10 Therefore, direct inhibition of NF-κB could potentially resensitize tumor cells, thus highlight- ing this transcription factor as a potential therapeutic tar- get.11-13
Pyrrolo[2,1-c][1,4]benzodiazepines (PBD) are naturally occurring molecules produced by Streptomyces bacteria whose family members include anthramycin (Figure 1) and tomaymycin.14,15 PBD are a class of sequence-specific covalent DNA minor groove binding agents that are selective for GC-rich sequences, and have been evaluated as potential chemotherapeutic agents in clinical trials.16,17 More recently, members of the PBD family have been developed as cytotoxic payloads for attachment to anti- bodies to form antibody-drug conjugates, and a number of these are currently undergoing clinical evaluation for the treatment of leukemia and lung cancer.18
This study identified three lead compounds (DC-1-192, DC-1-92 and DC-1-170) (Figure 1) from a library screen of 87 novel synthetic C8-linked benzofused PBD monomer- ic hybrids based on their in vitro cytotoxicity. The com- pounds were then further evaluated for their biological properties, including differential toxicity, in malignant and age-matched normal B and T cells. In terms of their mechanism of action, PBD monomers can recognize and bind to specific sequences of DNA and therefore have the potential to act as competitive inhibitors of transcription factors. Previous research has shown that PBD monomers such as GWL-78 preferentially inhibit the transcription factor NF-Y,19 while PBD monomers such as the DC-81- indole hybrid20 and KMR-28-39 are potent NF-κB inhibitors.21 The aim of this study was to determine the biological properties of these novel C8-linked benzofused PBD monomers by investigating their cytotoxic profiles in multiple myeloma cell lines, primary CLL cells and age- matched normal B- and T-lymphocytes. We went on to investigate their ability to inhibit NF-κB and whether they could potentiate the effects of the targeted agents bortezomib and ibrutinib, currently used in the treatment of myeloma and CLL, respectively.
Novel PBD inhibit NF-κB in hematologic cancers Methods
Detailed methods can be found in the Online Supplementary Appendix.
Cell lines, primary chronic lymphocytic leukemia cells and normal lymphocytes
Primary CLL cell lines (n=46) and age-matched normal B and T cells were obtained with informed consent in accordance with the ethical approval granted by South East Wales Research Ethics Committee (02/4806). In addition, five multiple myeloma cell lines, JJN3, U266, OPM2, MM.1S and H929, were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen. The provenance of the cell lines was verified by multiplex polymerase chain reaction of minisatellite markers; all were certified mycoplasma-free.
Measurement of in vitro apoptosis
Apoptosis was assessed using annexin V and propidium
iodide labeling. Samples were analyzed using an Accuri C6 flow cytometer with CFlow software (BD Biosciences).
Enzyme-linked immunosorbent assay for NF-κB subunits
Nuclear levels of p65, p50, p52 and RelB DNA binding were assessed in JJN3 and U266 cells treated for 4 h with DC-1-92, DC-1-170 (0nM 20 nM) and DC-1-192 (0nM 5 nM).
Synergy with bortezomib and ibrutinib
Figure 1. The structures of anthramycin and three structurally-related C8-linked benzofused PBD hybrids. Anthramycin (the first PBD to be isolated from a Streptomyces species), and the three synthetic PBD, DC-1-192, DC-1-92 and DC-1-170, identified as lead compounds in this study.
The synergy between the PBD monomers and bortezomib or ibrutinib was determined in JJN3 cells and primary CLL cells, respectively. Fixed molar ratios were derived from experimental- ly-determined median lethal dose (LD ) values for each PBD and
50
clinically achievable concentrations of bortezomib and ibrutinib.
RNA Isolation and sequencing
JJN3 cells were treated with 20 nM of either DC-1-170 or DC- 1-192 for 4 h. RNA was extracted using an RNeasy mini-kit (Qiagen) in accordance with the manufacturer’s instructions. Subsequently, 100-900 ng of high-quality total RNA (RNA integrity number >8) was depleted of ribosomal RNA, and sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero GoldTM kit (Illumina Inc.).
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