International Journal of Molecular Sciences Review Non-Coding RNAs as Cancer Hallmarks in Chronic Lymphocytic Leukemia Linda Fabris 1, Jaroslav Juracek 2 and George Calin 1,* 1 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; [email protected] 2 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; [email protected] * Correspondence: [email protected] Received: 24 July 2020; Accepted: 10 September 2020; Published: 14 September 2020 Abstract: The discovery of non-coding RNAs (ncRNAs) and their role in tumor onset and progression has revolutionized the way scientists and clinicians study cancers. This discovery opened new layers of complexity in understanding the fine-tuned regulation of cellular processes leading to cancer. NcRNAs represent a heterogeneous group of transcripts, ranging from a few base pairs to several kilobases, that are able to regulate gene networks and intracellular pathways by interacting with DNA, transcripts or proteins. Deregulation of ncRNAs impinge on several cellular responses and can play a major role in each single hallmark of cancer. This review will focus on the most important short and long non-coding RNAs in chronic lymphocytic leukemia (CLL), highlighting their implications as potential biomarkers and therapeutic targets as they relate to the well-established hallmarks of cancer. The key molecular events in the onset of CLL will be contextualized, taking into account the role of the “dark matter” of the genome. Keywords: chronic lymphocytic leukemia; lncRNA; miRNA; hallmarks 1. Introduction: Current Status of Chronic Lymphocytic Leukemia Research Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western world, representing more than 30% of all leukemia cases [1]. It is estimated that 21,040 new CLL cases will be diagnosed in 2020 in the United States [1]. CLL is a malignancy characterized by clonal expansion of CD5 + B-cells that show morphologically mature appearance and accumulate in the blood, bone marrow and secondary lymphoid tissues, resulting in lymphocytosis, bone marrow infiltration, lymphadenopathy and splenomegaly [2]. CLL is a clinically heterogeneous disease, and is divided into two main forms, aggressive and indolent. It is classified by whether the CLL cells express an unmutated (aggressive) or mutated (indolent) immunoglobulin heavy-chain variable region (IGVH) gene, reflecting the stage of normal B cell differentiation. The clinical heterogeneity of CLL reflects differences in the biology of the disease and chromosomal alterations (del13q, del11q, trisomy 12 and del17p), and only in part correlate with aggressive behavior and response to therapy; other events such as somatic mutations, epigenetic changes and non-coding RNAs alterations have been reported to influence the outcome of the disease, but are not used in clinical practice yet. While much attention has been given to the mutation of protein-coding genes, almost 20% of CLL patients do not show chromosomal aberrations; therefore, it is not surprising that attention has shifted to analysis of the RNA molecules that lack protein-coding potential and are collectively referred to as non-coding RNAs (ncRNAs). Int. J. Mol. Sci. 2020, 21, 6720; doi:10.3390/ijms21186720 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6720 2 of 18 ncRNAsInt. J. Mol. Sci. are 2020 divided,, 21, x FOR according PEER REVIEW to their size, into two groups: small ncRNAs (<200 nt)2 andof 19 long ncRNAs (lncRNAs). Small ncRNAs are mostly constituted by microRNAs (miRNAs), which have received morencRNAs attention are divided, in the according last decade, to their particularly size, into two in groups: CLL [3 small,4]. However, ncRNAs (<200 the nt) human and long genome ncRNAs (lncRNAs). Small ncRNAs are mostly constituted by microRNAs (miRNAs), which have encodes also numerous lncRNAs, defined as endogenous cellular RNAs of more than 200 nucleotides in received more attention in the last decade, particularly in CLL [3,4]. However, the human genome length, that lack an open reading frame of significant length. Indeed, lncRNAs are a very heterogeneous encodes also numerous lncRNAs, defined as endogenous cellular RNAs of more than 200 nucleotides groupin oflength, RNA that molecules lack an that open cover reading a broad frame spectrum of significant of molecular length. Indeed, and cellular lncRNAs functions are a very and are deregulatedheterogeneous in many group human of RNA cancers molecules [5,6]. that cover a broad spectrum of molecular and cellular Infunctions this review, and are we deregulated will contextualize in many thehuman role cancers of ncRNAs [5,6]. with respect to the well-known hallmarks of cancerIn (Figure this review,1), which we arewill distinctivecontextualize and the have role theof ncRNAs capabilities with torespect enable to tumorthe well-known growth and metastatichallmarks dissemination of cancer (Figure [7,8]. 1), which are distinctive and have the capabilities to enable tumor growth and metastatic dissemination [7,8]. Figure 1. Non-coding RNAs (ncRNAs) as hallmarks of chronic lymphocytic leukemia (CLL). Figure 1. Non-coding RNAs (ncRNAs) as hallmarks of chronic lymphocytic leukemia (CLL). 2. Sustaining Proliferative Signaling and Resisting Cell Death 2. Sustaining Proliferative Signaling and Resisting Cell Death Due to the slow clinical progression of CLL, this disease has been frequently described as the Dueaccumulation, to the slow rather clinical than proliferation, progression of of malignant CLL, this B diseasecells, as hasa result been of frequentlya deregulated described apoptosis. as the accumulation,The correct rather balance than between proliferation, the two of malignantbiological Bprocesses cells, as ais resulta key of component a deregulated for apoptosis.CLL The correctcharacterization. balance between While the the accumulation two biological of processesmalignant isBa cells key componentin the blood, for bone CLL marrow characterization. and Whilelymphoid the accumulation organs is caused of malignant by an impaired B cells inapoptosi the blood,s, a smaller bone marrowand actively and proliferating lymphoid organs population is caused by anstill impaired persists, apoptosis,mostly in lymph a smaller nodes and (LN) actively and in bone proliferating marrow (BM) population [9,10]. miRNAs still persists, and lncRNAs mostly in lymphare nodes regulating (LN) this and complex in bone process marrow at (BM)various [9, 10levels]. miRNAs (Figure 2), and and lncRNAs in the last are decade regulating several this studies complex processprovided at various evidence levels to (Figuresupport2 ),the and role in of the these last RNA decade types several in leukemia studies and provided lymphoma evidence progression. to support the role of these RNA types in leukemia and lymphoma progression. Int. J. Mol. Sci. 2020, 21, 6720 3 of 18 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 19 FigureFigure 2. Sustaining 2. Sustaining proliferative proliferative signalingsignaling and and resisting resisting cell cell death death hallmarks hallmarks in CLL in CLL.. OneOne of the of the best best studied studied is is the the miR-17 miR-17/92/92 polycistronicpolycistronic miRNA miRNA cluster, cluster, which which is overexpressed is overexpressed in in many lymphoid malignancies, including CLL, and comprises miR-17, miR-18a, miR-19a, miR-19b, many lymphoid malignancies, including CLL, and comprises miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a [11]. A transgenic mouse overexpressing miR-17/92 specifically in B-cells miR-20a,demonstrated and miR-92a that this [11 ].cluster A transgenic could act mouseas an oncogene overexpressing in leukemogenesis; miR-17/92 80% specifically of miR-17/92 in B-cells demonstratedtransgenic thatmice this developed cluster coulda B-cell act malignancy as an oncogene characterized in leukemogenesis; by expansion 80% of ofCD19+ miR-17 B cells/92 transgenic [12]. miceEven developed if the amechanism B-cell malignancy that drives characterized miR-17/92 ex bypression expansion remains of CD19poorly+ understood,B cells [12]. studies Even if the mechanismdemonstrated that drivesthat up-regulation miR-17/92 of expression miRNAs belonging remains to poorlythe miR-17 understood,-92 cluster in studies unmutated demonstrated IGHV thatCLL up-regulation is preceded of by miRNAs induction belonging of MYC, providin to the miR-17-92g a link between cluster MYC, in unmutated B-cell receptor IGHV (BCR) CLL is precededactivation by induction and miR-transcription of MYC, providing in CLL [11,13]. a link miR-17/92 between cluster MYC, regulate B-cell receptor cell cycle (BCR) progression activation and and miR-transcriptionproliferation through in CLL many [11, 13targets,]. miR-17 including/92 cluster CDKN1A, regulate CTGF, cell EGR2, cycle E2Fs, progression IKAROS, PTEN, and proliferation STATs, throughTP53INP1, many targets,TRIM8 and including ZBTB4 CDKN1A,[14]. Among CTGF, these EGR2,genes, E2Fs,ZBTB4 IKAROS, and TP53INP1 PTEN, are STATs, involved TP53INP1, in apoptosis regulation through CDKN1A and TP53 [15], while E2F5 is involved in the G1 arrest [16]. TRIM8 and ZBTB4 [14]. Among these genes, ZBTB4 and TP53INP1 are involved in apoptosis regulation Members of the miR-17/92 cluster, together with the let-7 family of tumor suppressors, miR-26a, and throughmiR-34a CDKN1A have also and been TP53 found [15 to], target while Cyclin