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Inhibiting PARP1 Splicing Along with Inducing DNA Damage As Potential Breast Cancer Therapy

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Inhibiting PARP1 Splicing Along with Inducing DNA Damage As Potential Breast Cancer Therapy Reem Alsayed 3/26/21 03-545, S21 Professor Ihab Younis Inhibiting PARP1 Splicing along with Inducing DNA Damage as Potential Breast Cancer Therapy Student: Reem Alsayed Spring 2021 Professor: Ihab Younis 1 Reem Alsayed 3/26/21 03-545, S21 Professor Ihab Younis Abstract: Triple negative breast cancer is a deadly cancer and once it has metastasized it is deemed incurable. The need for an effective therapy is rising, and recent therapies include targeting the DNA damage response pathway. PARP1 is one of the first responders to DNA damage, and has been targeted for inhibition along with the stimulation of DNA damage as a treatment for breast cancer. However, such treatments lack in specificity, and only target one or two domains of the PARP1 protein, whereas PARP1 has other functions pertaining to multiple cancer hallmarks such as promoting angiogenesis, metastasis, inflammation, life cycle regulation, and regulation of tumorigenic genes. In this project, we hypothesize that by inhibiting the PARP1 protein production, we will be able to effectively inhibit all cancer hallmarks that are facilitated by PARP1, and we achieve this by inhibiting the splicing of PARP1. Splicing is the removal of intervening sequences (introns) in the pre-mRNA and the joining of the expressed sequences (exons). For PARP1, we blocked intron 22 splicing by introducing an Antisense Morpholino Oligonucleotide (AMO) that blocks the binding of the spliceosome. The results obtained demonstrate that 50uM PARP1 AMO inhibits PARP1 splicing >88%, as well as inhibits protein production. Additionally, the combination of PARP1 AMO and Doxorubicin lead to a loss in cell proliferation. 2 Reem Alsayed 3/26/21 03-545, S21 Professor Ihab Younis Introduction: Breast cancer is the deadliest cancer for women in Qatar (Doenelly et al., 2011), and the second worldwide (Harbeck et al., 2019). The rate of breast cancer incidence globally is rising alarmingly, from having 641,000 patients in 1980 to >1.6million in 2010, and a rising trend is expected (Harbeck et al., 2019). Triple negative TN breast cancer is considered to be a challenging cancer to target due to its lack of HER2, Estrogen, and Progesterone receptors that could have been targets for therapy (Godet & Gikes, 2017). Furthermore, metastasized cancers are considered to be incurable according to the therapies available today (Godet & Gikes, 2017), creating a pressing need for an innovative solution and therapy. Splicing is one of the many pre-mRNA processing steps that help the conversion of pre-mRNA into mRNA (El Marabti & Younis, 2018). It occurs in the nucleus and involves removing the interfering sequences of a pre-mRNA, known as introns, and joining the expressed sequences, known as exons (National Cancer Institute, n.d.). This is a crucial step in preparing the mRNA for export out of the nucleus to be translated (El Marabti & Younis, 2018). Alternative splicing occurs when a variable combination of exons and introns are removed/joined, and this forms different isoforms of the mRNA transcript, in turn forming proteins with different function and structure (El Marabti & Younis, 2018). This process has been known to have significance in cancer, because the cancer cells might modify the ratio of the spliced isoforms in a way that would favor cancer progression (El Marabti & Younis, 2018). Splicing is carried out by snRNPs (small nuclear ribonucleo proteins) called U1, U2, U4, U5, and U6 which form the major spliceosome (El Marabti & Younis, 2018). However, a specific set of 770 introns are spliced by a different machinery called the minor spliceosome, and this consists of U11, U12, U4atac, U5, and U6atac snRNPs. These introns are referred to as minor introns due to their splicing by a machinery that is not as widely used as the major spliceosome (Olthof et al., 2019). Additionally, it is important to mention that minor introns are highly conserved and regulated in cancer, and can serve very important roles (Olthof et al., 2019). PARP1 is overexpressed in breast carcinoma that are BRCA1/2-mutated, triple negative as well as receptor-positive (Wang et al., 2017). PARP1 is poly ADP-ribose polymerase 1, and its main functions include using NAD+ as a substrate to catalyze the synthesis of Poly ADP ribose, as well as transferring this molecule onto other proteins (Wang et al., 2017). The domain responsible for the catalytic ability lies at the C-terminus of the protein (Wang et al., 2017). In addition, this function is an essential part of recognizing damaged DNA, and marking it for repair. As such, PARP1 is the very first response to single and double stranded DNA damage (Wang et al., 2017). Specifically, the single stranded DNA breaks that are recognized by PARP1 are either those occurring due to disintegration of oxidize deoxyribose (DNA sugar damage) or as natural intermediates of base excision and repair (Caldecott, 2008). Marking a damaged segment of DNA for repair would trigger a cascade of proteins that will carry out the end processing, gap filling and ligation of the previously damaged area (Caldecott, 2008). Due to its key function in DNA damage repair, PARP1 plays an essential role in cancer. Generally speaking, cancer cells produce more mutations than healthy cells, which is important for mutagenesis and maintaining and creating adaptive genes (Zhivotovsky & Kroemer, 2004). However, excessive unrepaired mutations lead to death and senescence. Cancer cells avoid this by making sure by balancing their mutation level: enough for mutagenesis to occur but not excessive as not to induce senescence. For this balance to occur, DNA damage repair has to take place in order to prevent cancer cells from creating a level of DNA damage that would lead to their apoptosis (Zhivotovsky & Kroemer, 2004). Therefore many cases of cancer, PARP1 protein is over expressed (Wang et al., 2017). Interestingly, excessive DNA lesions lead to apoptosis despite the presence of PARP1 (Wang et al., 2017). PARP1 manages to repair normal amounts of DNA damage, but with excessive DNA lesions and regular amount of PARP1 expression, PARP1 will not be able to bind every lesion, and signal for repair in a timely manner. Based on this, past research has focused on inducing excessive DNA damage and blocking PARP1 activity in order to trigger apoptosis (Vascotto et al., 2016). The most prevalent PARP1 inhibitors work by blocking the NAD+ binding site, otherwise 3 Reem Alsayed 3/26/21 03-545, S21 Professor Ihab Younis known as the active site, and this inhibits the Poly ADP ribose synthesis catalysis (Caldecott, 2008). However, a problem faced with some PARP1 inhibitors is that they display off-target binding, for example, they could bind to certain kinases (Atolín & Mestres, 2014). Another drawback of PARP1 inhibitors is that they only block the Poly- ADP riposylation catalytic activity, when in reality PARP1 is involved in multiple hallmarks of cancer that are not necessarily related to DNA damage repair or Poly-ADP ribosylation (Wang et al., 2017). In addition to its key role in DNA damage repair, PARP1 has been shown to have additional functions that can aid in tumorigenesis, including regulation of gene transcription, upregulating inflammatory signals, regulating cell cycle, and promoting angiogenesis and metastasis (Wang et al., 2017). Gene transcription regulation does not require the catalytic domain, and the rest of the functions mentioned have not yet been listed under a specific domain. An example for gene regulation is that PARP1 was shown to activate vascular endothelial growth factor receptor 1 (VEGFR1) gene, hypoxia-inducible factor 1α and 2A (HIP1α and HIF2A), melanocyte-lineage survival oncogene (MITF), and it suppresses tumor suppressor genes such as p53 and APC. For inflammation, PARP1 was shown to activate NF-κB, which is an inflammatory signal (Wang et al., 2017). Cell cycle modulation and promoting angiogenesis and metastasis is regulated by PARP1 via over activating ERK, which blocks apoptosis and stimulates VEGF and other pro-angiogenic factors (Wang et al., 2017). Among these functions, chromatin remodeling requires the PAR-binding domain, and can function without the catalytic domain (Pines et al. 2012). Moreover, some genes that are activated by PARP1 are activated by the center (automodification) domain (Simbulan-Rosenthal et al., 2003). Ultimately, this tells us that blocking the catalytic domain alone may not be a sufficient form of treatment if PARP1 continues to facilitate other cancer hallmarks. Hence, PARP1 could be inhibited through the blocking of its protein production, and this might be able to block all PARP1 functions. The splicing of PARP1 could be inhibited in several ways. Firstly, it is important to note that PARP1 pre-mRNA consists of 23 exons, and the last one being the one that codes for the catalytic domain of the protein (UniProt, n.d.). Moreover, intron 22 is a minor intron (MIDB, n.d,), and as mentioned previously, minor introns are highly regulated in cancer. Hence, if the splicing of exon 23 is inhibited, this might lead to the inhibition of its protein production. The first method is to inhibit PARP1 intron 22 splicing by adding an AMO (antisense morpholino oligonucleotide) at the junction of exon 22 and intron 23, and this will block the spliceosome from binding there and splicing the intron. Another method is to introduce the AMO at the site of an RNA binding protein that is in charge of aiding the splicing, so this will also be a way to inhibit the splicing of the intron. Inhibiting PARP1 in the past either through chemical means or by siRNA treatment has resulted in apoptosis, G2 cell cycle arrest, and chromatid breaks (Do & Chen, 2013). In this project, we hypothesize that by inhibiting PARP1 splicing we will inhibit PARP1 protein synthesis and all functions related to PARP1, which is expected to be more efficient and specific than the known chemical inhibitors of PARP1 catalytic domain.
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