Kumar et al. Blood Cancer Journal (2018) 8:92 DOI 10.1038/s41408-018-0129-9 Blood Cancer Journal ARTICLE Open Access Role of apurinic/apyrimidinic nucleases in the regulation of homologous recombination in myeloma: mechanisms and translational significance Subodh Kumar1,2, Srikanth Talluri1,2, Jagannath Pal1,2,3,XiaoliYuan1,2, Renquan Lu1,2,PuruNanjappa1,2, Mehmet K. Samur1,4,NikhilC.Munshi1,2,4 and Masood A. Shammas1,2 Abstract We have previously reported that homologous recombination (HR) is dysregulated in multiple myeloma (MM) and contributes to genomic instability and development of drug resistance. We now demonstrate that base excision repair (BER) associated apurinic/apyrimidinic (AP) nucleases (APEX1 and APEX2) contribute to regulation of HR in MM cells. Transgenic as well as chemical inhibition of APEX1 and/or APEX2 inhibits HR activity in MM cells, whereas the overexpression of either nuclease in normal human cells, increases HR activity. Regulation of HR by AP nucleases could be attributed, at least in part, to their ability to regulate recombinase (RAD51) expression. We also show that both nucleases interact with major HR regulators and that APEX1 is involved in P73-mediated regulation of RAD51 expression in MM cells. Consistent with the role in HR, we also show that AP-knockdown or treatment with inhibitor of AP nuclease activity increases sensitivity of MM cells to melphalan and PARP inhibitor. Importantly, although inhibition 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; of AP nuclease activity increases cytotoxicity, it reduces genomic instability caused by melphalan. In summary, we show that APEX1 and APEX2, major BER proteins, also contribute to regulation of HR in MM. These data provide basis for potential use of AP nuclease inhibitors in combination with chemotherapeutics such as melphalan for synergistic cytotoxicity in MM. Introduction presents a great obstacle to treatment. Understanding Most cancers display a variety of genomic alterations at mechanisms underlying genomic instability is therefore – diagnosis1 6 and acquire new changes with progression of essential to develop effective strategies for prevention and – disease to advanced stages7 10 indicating an instability of treatment of cancer. – genome1,11 17. We have observed a similar pattern of Normal cellular DNA is subject to a large number of change in multiple myeloma (MM), both at diagnosis and chemical and physical modifications18,19 which may include in relapse samples1. The ability to constantly evolve not missing or altered bases, deletions/insertions, DNA strand only enables the MM cells to acquire new characteristics breaks and cross-linking etc. Therefore, multiple DNA for growth, survival, and progression of disease, but also repair mechanisms exist and ensure that these alterations areconstantlyandefficiently repaired in normal cells. Homologous recombination (HR), which depends on Correspondence: Nikhil C. Munshi ([email protected])or Masood A. Shammas ([email protected]) sequence homology/identity, is one of the most precise 1Dana Farber Cancer Institute, Boston, MA 02215, USA DNA repair mechanism20. HR is involved in the repair of a 2 Veterans Administration Boston Healthcare System, West Roxbury, MA 02132, variety of DNA damage types including DNA double- USA Full list of author information is available at the end of the article. stranded breaks, DNA strand crosslinks and single-stranded These authors contributed equally: Subodh Kumar, Srikanth Talluri, Jagannath Pal © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Blood Cancer Journal Kumar et al. Blood Cancer Journal (2018) 8:92 Page 2 of 10 DNA gaps, and plays a vital role in the maintenance of protocol approved by Institutional Review Board, Dana genomic integrity21. However, dysregulation of HR can also Farber Cancer Institute. Plasma cells were isolated by lead to genomic instability22. Consistent with this, we have Magnet Assisted Cell Sorting (MACS, Miltenyi Biotech) previously demonstrated that elevated HR mediates geno- according to the manufacturer’s protocol, and their purity mic instability and progression in MM23.Usinganother (>95%) confirmed by monitoring cell surface expression cancer model system (Barrett’s adenocarcinoma), we of CD45 and CD3850. demonstrated that dysregulated HR disrupts genomic integrity24 and stability25, and transgenic as well as chemical Reagents suppression of HR, significantly reduced both the genomic Anti-APEX1 antibody, catalogue # NB100-101 was pur- instability as well as growth of cancer cells in mice26. chased from Novus Biologicals, Inc., (Littleton, CO). Anti- Identification of mechanisms underlying dysregulation of APEX2 antibody is developed by us. Anti-RAD51 antibody, HR thus may help prevent genomic evolution and provide catalogue # SC-8349 was purchased from Santa Cruz, Dal- novel targets for cancer prevention. las, TX. Antibodies against GAPDH (catalogue # mAb2118), Investigating mechanisms underlying dysregulation of HP1 (catalogue # 261), and γH2A.X (Ser139, catalogue HR, we identified significant role for apurinic/apyrimidinic #2577) were from Cell Signaling Technology, Inc., Danvers, (AP) nucleases (APEX1 and APEX2). APEX1 and APEX2 MA. For immunoprecipitation studies, anti-APEX1 anti- are the key base excision repair (BER) pathway proteins body (Cat. # ab194, Abcam) and anti-p73 antibody (Cat. # involved in the recognition and repair of AP (or abasic) NBP2-24737, Novus Biologicals) were used. – sites27 30 and play a significant role in genome main- tenance. Development of abasic site, one of the most Modulation of AP nuclease expression common DNA damage, can occur either spontaneously or AP nuclease expression was modulated using chemical by removal of a damaged DNA base by specific enzymes, as well as transgenic manipulations. AP nuclease activity DNA glycosylases. The repair of AP sites is extremely was suppressed using APEX1 inhibitor (APE1 Inhibitor efficient. APEX1 and APEX2 are major BER proteins III; API3) (Axon Medchem LLC, Reston, VA), methox- involved in its initiation. APEX1 is the abasic endonuclease yamine (MX) which inhibits AP nuclease activity by implicated in both the BER and 3′-end processing in binding to aldehyde group of abasic site (Sigma Aldrich, mammalian cells29,31,32. It is a multifunctional protein and Saint Louis, MO) and lentivirus based shRNAs targeting is also responsible for repair of alkylation and oxidative APEX1 and APEX2 (Sigma Aldrich, Saint Louis, MO). For DNA damage in a cell. The protein also functions as a overexpression, the plasmids carrying these genes under reduction–oxidation (redox) factor and hence maintains CMV promoter were used. many transcription factors in an active reduced state33,34. APEX1 is a strong endonuclease but also has a weak 3′-5′ AP nuclease activity assay exonuclease activity for proofreading function35,36. APEX2 AP nuclease activity was assessed using a fluorescence on the other end has a weak endonuclease but a strong based assay reported previously51. Briefly, the oligonu- exonuclease activity35. Elevated levels of APEX1 have been cleotides carrying AP site mimic (Tetrahydrofuran; THF) found in cervical37, ovarian38,39, prostate40, germ cell and a fluorescein label on one strand and a quenching tumors41, and correlate with the radiosensitivity of cervical moiety (Dabcyl-Q) on other strand are synthesized com- cancers42. Here we have explored the role of APEX1 and mercially (Eurogentec Ltd., San Diego, CA). The AP APEX2 in dysregulation of HR in MM. nuclease activity cleaves AP site releasing the short fluorescein-labeled fragment, thus resulting in the Materials and methods increase in fluorescence. Cell lines and patient samples MM cell lines RPMI8226 and U266 were purchased Recombination assays from the American Type Tissue Culture Collection Three different functional assays were used to evaluate (Rockville MD), ARP was a gift from Dr. J. Epstein HR in this study. A plasmid based HR activity assay has (University of Arkansas for Medical Sciences) and MM.1S been reported previously25,26. Here, we also used a mod- obtained from Dr. Steven Rosen (Northwestern Uni- ification of this assay in which HR substrate plasmid is versity, Chicago, IL). Cell lines, confirmed to be negative mixed with MM cell lysate under in vitro HR condi- for mycoplasma, were cultured in RPMI1640 medium tions52, and product of HR measured by Q-PCR. This containing 10% fetal bovine serum (HyClone, South modified version of HR assay was validated and was – Logan, UT) as described previously43 49 and maintained consistent with the assay conducted within intact cells in logarithmic growth. (Supplementary Figure 1). We also used a fluorescence Bone