Identification of Lncrnas As Therapeutic Targets in Chronic

Identifcation of LncRNAs as Therapeutic Targets in Chronic Lymphocytic Leukemia Simay Dolaner1,2, Harpreet Kaur2, Mohit Mazumder2, Julia Panov2,3, Elia Brodsky2 1Department of Molecular Biology and Genetics, Bahcesehir University, Istanbul, Turkey 2Pine Biotech, New Orleans, USA 3Tauber Bioinformatics Research Center, Haifa, Israel KEYWORDS: Chronic Lymphocytic Leukemia, spontaneous regression, therapeutic targets, LncRNAs, transcriptomics ABSTRACT: Chronic Lymphocytic Leukemia (CLL) is a type of blood cancer that has a very heterogeneous biological background and diverse treatment strategies. However, a small part of this malignancy may disappear without receiving any treatment, known as “spontaneous regression”, which occurs as a result of a poorly investigated mechanism. Exposing the underlying causes of this condition can lead to a novel treatment approach for CLL. In this article, we applied in-silico analysis on total RNA expression data from 24 CLL samples to determine possible regulatory mechanisms of spontaneous regression in CLL. These were frst selected by comparing spontaneous regression with progressive samples of CLL at the transcriptional level using two unsupervised machine learning algorithms, i.e., Principal Component Analysis (PCA) and Hier- archical Clustering. Subsequently, the DESeq2 algorithm was used to scrutinize only statisti- cally signifcant (adjusted p-value < 0.01) RNA transcripts that can diferentiate both conditions. Here, at frst, we have elucidated 870 signifcantly diferentially expressed protein-coding genes that were involved in the biogenesis and processing of RNA. Consequently, these fndings led our study to investigate non-coding RNA, and 33 long non-coding RNAs (lncRNAs) were found to be signifcantly diferentially expressed among two conditions based on diferential gene expression analysis. Further, our analysis in the current study suggested lncRNAs, PTPN22-AS1, PCF11-AS1, SYNGAP1-AS1, PRRT3-AS1, and H1FX-AS1 as potential therapeutic targets to trigger spontaneous regression. Ultimately, the results presented here reveal new insights into spontaneous regression and its relationship with non-coding RNAs, particularly lncRNAs. INTRODUCTION is chronic lymphocytic leukemia (CLL), which is According to the American Cancer Society sta- a lymphoid malignancy due to failed apoptosis tistics, leukemia is the second leading blood and aggressive proliferation of mature B cells cancer, with roughly 60,000 new cases identifed [3]. These cells circulate through the blood as in 2020 [1]. Leukemia has diferent subtypes, non-proliferating cells or arrested cells in the which are classifed in the context of the tumor G0/G1 phase of the cell cycle and may afect origin [2]. The most common variety in adults the function of normal cells in other organs [4]. 16 © 2021 Dolaner, Kaur, Mazumder, Panov, Brodsky. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits the user to copy, distribute, and transmit the work provided that the original authors and source are credited. Columbia Undergraduate Science Journal Vol. 15, 2021 Dolaner et. al. CLL has a highly diverse biological and clinical pathways are identifed using next-generation background for each patient that determines the sequencing of mRNA expression [9]. In spite stage of the disease [5]. Although there are sev- of this progress, nearly 20% of CLL cells do eral stages of CLL classifed according to their not show chromosomal abnormalities or ge- genetic background and B cell number, one of nomic variation. Therefore, researchers re- the most intriguing concepts is known as spon- cently shifted their focus to the non-coding taneous regression, which is the disappearance region of the genome and regulatory RNA of the tumor over time either without any treat- molecules, especially long non-coding RNAs, ment or with treatment that is categorized as which are deregulated in many cancers [10]. insufcient to have an impact on the tumor [6]. Long non-coding RNAs (lncRNAs) Spontaneous regression can be seen are a subgroup of non-coding RNAs which in 1-2% of all CLL patients, and it is a phe- are longer than 200 nucleotides and encom- nomenon that is poorly understood [7]. In this pass thousands of diverse transcripts in hu- process, cells that proliferate uncontrolla- mans [11]. There are approximately 100,000 bly are transmitted to the quiescent state so known lncRNAs, and this quantity is ex- that the tumor disappears partially or com- panding each year with the new studies [12]. pletely with time [6]. Spontaneous regres- LncRNAs play a signifcant role in gene sion is not a common feature of cancer cells regulation, controlling multiple cellular mecha- and is regulated by mechanisms that are not nisms involved in tumor progression. They are well-understood. If such mechanisms can be involved in epigenetic regulation of gene ex- determined, target biological molecules that pression via histone modifcation, DNA meth- have a specifc role in disease progression ylation, or acetylation. Specifcally, these epi- can be identifed and manipulated in vitro. genetic regulations may include recruitment Current strategies aim to inhibit BCR of histone remodeling complexes, interaction signaling, which is crucial for the survival of the with histone methyltransferases and demeth- B-cells, and chemokine signaling that creates ylases to regulate DNA methylation, or his- survival signals and attracts leukemic cells to tone acetyltransferases and deacetylases to communicate with its microenvironment [5]. modulate the acetylation [13]. Furthermore, Moreover, activation of apoptosis pathways lncRNAs may regulate gene expression at the via blocking BCL2 activity, an anti-apoptotic transcriptional level by recruiting transcription protein, which is highly expressed in leukemic factors [14]. Moreover, lncRNAs can produce cells, is included in the aforementioned strate- hybrids or act as scafolds through interac- gies [8]. Although these types of targeted ther- tion with proteins to regulate expression at apies improve the outcome of the patients, the the post-translational level, including regula- heterogeneity of the leukemia microenviron- tion of phosphorylation and ubiquitination [15]. ment reinforces the necessity of new targets. In tumor development, lncRNAs can As the targeted therapies may have an impact serve as either tumor suppressors, oncogenes, on tumor surroundings and afect the other cells or even both at the same time for some cancer found in the tumor microenvironment, triggering types [16]. Analysis of lncRNA expression pat- spontaneous regression mechanisms may im- terns had led to the identifcation of putative bio- prove the strategies as well as patient outcome. markers such as HOTAIR, H19, and DLEU1/2 In CLL, the most frequent chromo- [17]. Specifcally, HOTAIR serves as an onco- somal abnormalities and somatic mutations gene by inducing invasiveness and metastasis on the protein-coding region of the genome in several cancers via recruiting a demethylase have been distinguished as a result of ge- [13]. Since this lncRNA is highly expressed in nomic sequencing, and disrupted cellular aggressive tumors, it is considered as a bio- 17 Columbia Undergraduate Science Journal Vol. 15, 2021 Dolaner et. al. marker and a possible therapeutic target for from multiple subtypes of chronic lymphocytic many cancers [18]. DLEU1/2 epigenetically leukemia that can be seen in Table I. In our proj- regulate the tumor suppressors and are delet- ect, spontaneous regression and progressive ed in CLL cells, which results in the progression states were chosen for further analysis to inves- of the CLL. This allows it to serve as a biomark- tigate expression variation specifcally involved er in the diagnosis [19]. On the other hand, H19 in the regression mechanism in CLL cells. has a dual role in diferent cancers by stimu- lating distinct mechanisms through transcrip- Table I. Dataset of the BioProject tion factors, which makes it a target that needs to be studied separately for each cancer [13]. Furthermore, lncRNAs are known to play an important role in cell diferentiation and tissue specifcity [20]. However, characteriza- tion may be compelling because lncRNAs are transcribed in diferent loci and localized dis- tinctly. More importantly, lncRNA expression is RNA-seq Raw Data Processing generally tissue-specifc and can be detected Raw reads were used to construct an RNA se- under certain conditions [21]. As the lncRNAs quencing pipeline that contains pre-processing are diferentially expressed, their roles and ac- using Trimmomatic, mapping of reads on refer- tivities can be identifed in disease conditions. ence transcriptome with Bowtie2-t, and quan- Due to the diverse functions of ln- tifcation using RNA-Seq by Expectation-Max- cRNAs, novel studies are concentrated on imization (RSEM) algorithms with T-Bioinfo the identifcation of lncRNAs as therapeu- server (Supplementary Notes 1) as represent- tic targets. As the expression of these mol- ed in Figure 1. Briefy, in order to get gene ex- ecules is tissue and disease-specifc, this pression levels, duplicated sequences which specifcity makes them excellent targets resulted in PCR amplifcation were removed by compared to protein-coding genes [22]. PCR clean considering best coverage. Then, In the scope of this project, the trigger Trimmomatic was used to remove adaptor se- mechanism behind the spontaneous regres- quences and poor-quality data at the end of the

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