CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities

Future Approaches for Treating Chronic Myeloid Leukemia: CRISPR Therapy

© 2021 by the authors.The constitutively active tyrosine-kinase BCR/ABL1 oncogene plays a key role in human chronic myeloid leukemia development and disease maintenance, and determines most of the features of this leukemia. For this reason, tyrosine-kinase inhibitors are the first-line treatment, offering most patients a life expectancy like that of an equivalent healthy person. However, since the oncogene stays intact, lifelong oral medication is essential, even though this triggers adverse effects in many patients. Furthermore, leukemic stem cells remain quiescent and resistance is observed in approximately 25% of patients. Thus, new therapeutic alternatives are still needed. In this scenario, the interruption/deletion of the oncogenic sequence might be an effective therapeutic option. The emergence of CRISPR (clustered regularly interspaced short palindromic repeats) technology can offer a definitive treatment based on its capacity to induce a specific DNA double strand break. Besides, it has the advantage of providing complete and permanent oncogene knockout, while tyrosine kinase inhibitors (TKIs) only ensure that BCR-ABL1 oncoprotein is inactivated during treatment. CRISPR/Cas9 cuts DNA in a sequence-specific manner making it possible to turn oncogenes off in a way that was not previously feasible in humans. This review describes chronic myeloid leukemia (CML) disease and the main advances in the genome-editing field by which it may be treated in the future.This work was supported by the Instituto de Salud Carlos III (ISCIII), grant number PI17/01895 (ISCIII-FEDER), and by the Fundación Memoria de D. Samuel Solorzano Barroso (FS/29-2020) from the University of Salamanca. E.V. is supported by a predoctoral grant from the University of Salamanca-Banco Santander. P.H.C. and L.M. are supported by Nucleus (platform for research supporting) from the University of Salamanca

Structural organization of the guanine nucleotide exchange factor C3G

Resumen del póster presentado al 5th International Iberian Biophysics Congress, celebrado en Porto (Portugal) del 15 al 17 de junio de 2016.C3G is a guanine nucleotide exchange factor (GEF) that activates the small GTPases Rap1 and R-Ras. C3G is involved in multiple cellular functions including adhesion, migration, cytoskeletal remodeling, cell proliferation, differentiation, transformation, and apoptosis. C3G (120 kDa) has a tripartite structure. The N-terminal region (N-C3G) mediates binding to E-cadherin at nascent adherent junctions. The structure of N-C3G is unknown and bears no similarity to other proteins. The central region contains five Pro-rich sequence motifs that mediate the interaction with protein that contain SH3 domains, such as Crk, p130Cas, Grb2, Hck and c-Abl. The central region also mediates binding to the TC-PTP phosphatase and to ß-catenin, which do not contain SH3 domains. The C-terminal catalytic region consists of a REM (Ras exchange motif) and a Cdc25H domain. Deletion of the N-terminal half of C3G increases the GEF activity, suggesting that this N-terminal half acts as a regulatory element. Yet, the mechanisms of autoinhibition of C3G remain unknown. Here we have identified and characterized an intramolecular interaction between the N-C3G and the catalytic region. We have identified residues important for maintaining the close conformation. This head-tail interaction in C3G resembles autoinhibitory conformations of other GEFs of the Cdc25H family, such as Sos1, Epac2, and RasGRP1. Finally, using recombinant N-C3G fragments we show that this region has a high content of α-helical structure. Sequence analysis suggests the presence of amphipathic helices that could fold in a helical bundle.Peer Reviewe

Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas

C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins Crk and CrkL and by tyrosine phosphorylation. Here, we uncovered mechanisms of C3G autoinhibition and activation. Specifically, we found that two intramolecular interactions regulate the activity of C3G. First, an autoinhibitory region (AIR) within the central domain of C3G binds to and blocks the catalytic Cdc25H domain. Second, the binding of the protein's N-terminal domain to its Ras exchanger motif (REM) is required for its GEF activity. CrkL activated C3G by displacing the AIR/Cdc25HD interaction. Two missense mutations in the AIR found in non-Hodgkin's lymphomas, Y554H and M555K, disrupted the autoinhibitory mechanism. Expression of C3G-Y554H or C3G-M555K in Ba/F3 pro-B cells caused constitutive activation of Rap1 and, consequently, the integrin LFA-1. Our findings suggest that sustained Rap1 activation by deregulated C3G might promote progression of lymphomas and that designing therapeutics to target C3G might treat these malignancies.Peer reviewe