Multiple myeloma (MM) is a hematologic neoplasm of plasmatic cells that infiltrates the bone marrow and secrete monoclonal immunoglobulins. MM is the second most common hematologic malignancy in the world, and despite the therapeutic advances, the disease is associated with a poor outcome, due to disease relapsing and the development of treatment resistances (1–3).
MM is preceded by a pre-malignant and asymptomatic stage called monoclonal gammopathy of undetermined significance (MGUS) and intermediate stage termed smoldering multiple myeloma (SMM). Then, most patients develop the symptomatic stage known as MM and characterized by the monoclonal protein secretion and the end-organ damage (1, 2). Finally, MM cells can develop the ability of proliferation outside the bone marrow and produce more aggressive stages such as extramedullary multiple myeloma (EMM) or plasma cell leukemia (PCL) (1, 2).
How the asymptomatic MGUS stage become a MM remains unknown, but many factors are involved, being the genetic alterations the most frequent causes of disease progression. Also, cells present in the bone marrow microenvironment play an important role in MM progression. These cells, that include bone marrow stromal cells, endothelial cells, and other hematopoietic cells, produce chemokines and other factors that interact with MM cells favoring their migration to the bone marrow and facilitating their proliferation and survival (4).
Rho GTPases are small GTP-binding proteins with an important role in converting extracellular signals into a large range of cellular responses, including cell adhesion, cell-cycle progression, cell migration, cell morphogenesis, gene expression, and actin cytoskeleton dynamics (5–7). Therefore, Rho proteins have been widely described as important regulators of tumour cell proliferation, survival, and invasion (8). Rho proteins also play an important role in the development of hematological neoplasms since they are involved in chemotaxis and motility in lymphoid lines through ROCK-LIMK pathway (9). Specifically, RhoU, an atypical GTPase Rho protein, has been recently related to MM progression, as its expression increases in MGUS patients, and it is downregulated as the disease progresses (10). Rnd3 protein is also an atypical GTPase that belongs to Rho family and it has been involved in cancer progression and drug resistance in different type of cancers (11). However, the possible role of Rnd3 in hematological neoplasm remains unknown.
The CRISPR/Cas9 gene editing system has been recently used in several cell lines and mouse models to understand the role of various genetic alterations in pathogenesis of hematologic malignancies, as well as discovering new therapeutic targets for future clinical stages (12, 13). The CRISPR system is a bacterial adaptive immune system that requires the endonuclease Cas9 from Streptococcus pyogenes (or analogous proteins from other species) and a single guide of RNA (sgRNA). That guide leads the nuclease activity to complementary sequences in the substrate DNA, usually on the coding region (14). CRISPR/Cas9 is used for genome editing by introducing deletions on the protein coding ORF (open reading frame) using homology repair (HR) or nonhomologous end joining (NHEJ) repair. These deletions can lead to frame shifts that result in a loss of function of the encoded protein (15). CRISPR/Cas9 is a permanent technique that allows the generation of knockout cell lines by genome editing, but it has variable tolerance for mismatches between its sgRNA and the target DNA sequence, so off-targets effects are common with this technology (16). Nowadays, there are variants of this technique which need a catalytically inactivated Cas9, known as a dead Cas9 (dCas9). This dCas9 can be used to activate or repress gene expression when it is associated to a sgRNA directed to specific gene promoter region. If the dCas9 is fused to a transcriptional activator such as VP64, the gene expression is activated, and it is known as CRISPR activation. However, dCas9 can be fused to a transcriptional repressor, most known as Krüppel-associated box (KRAB), that can induce DNA methylation and decrease the accessibility of chromatin at the enhancer and promoter regions and, therefore, represses gene expression at transcriptional level. This technology is called CRISPR interference (CRISPRi) (17, 18). CRISPRi is frequently used to perform genetic screens in mammalian cells (18) and also is used to cell engineering and regenerative medicine like retinal, muscle, nerve, or bone degeneration among others (17). Furthermore, CRISPRi can be used to silencing gene expression in specific human cell types, such as neurons or iPSCs (19, 20). This silencing can be constitutive or inducible, but frequently it is stable when it is achieved by lentiviral transduction.
In this work, we describe and analyze the efficacy of CRISPRi technology when it is used to silencing Rnd3 expression in MM cell lines by lentiviral transduction.