Breaking Ground in CLL Research: Novel Mouse Model Unveils New Therapeutic Pathways

Significance

Chronic lymphocytic leukemia (CLL) is a hematological malignancy characterized by the accumulation of clonal CD5+ B lymphocytes in the blood, bone marrow, and secondary lymphoid tissues. Despite advancements in understanding the genetic and molecular mechanisms of CLL, the disease remains incurable, with variable clinical outcomes ranging from indolent to highly aggressive forms. Traditionally, CLL has been associated with various genetic alterations, including mutations in TP53 and ATM genes and deletions in chromosomal regions such as 11q and 17p. However, recent research has identified the overexpression of the RRAS2 gene, rather than mutations, as a significant driver of CLL pathogenesis. The R-RAS2 protein, a member of the RAS family of small GTPases, plays a critical role in cellular signaling, cell proliferation, survival, and differentiation. Unlike the classical RAS proteins, which are frequently mutated in various cancers, R-RAS2 contributes to oncogenesis primarily through overexpression. Current treatments for CLL include chemoimmunotherapy and targeted therapies such as ibrutinib and venetoclax which have significantly improved patient outcome, however, resistance and relapse remain significant challenges. Thus, there is an urgent need to develop better novel therapeutic strategies that can effectively target the molecular mechanisms of CLL. Additionally, understanding the role of R-RAS2 in CLL and developing therapies that target this protein could provide a more effective and durable treatment option. To this end, the research led by Professor Balbino Alarcón and conducted by Postdoctoral fellow Dr. Alejandro Hortal, Ana Villanueva, Irene Arellano, Cristina Prieto, Pilar Mendoza, Xosé Bustelo at the Center for Molecular Biology Severo Ochoa of Madrid addressed the limitations of existing CLL models and therapies and developed a novel mouse model that overexpresses the RRAS2 gene. The new model, Rosa26-RRAS2^fl/flxmb1-Cre, was designed to mimic human CLL more accurately and to provide a reliable platform for preclinical testing of new therapeutic agents. The work is now published in Cancers Journal ¹.

The researchers created the Rosa26-RRAS2^fl/flxmb1-Cre mouse model by inserting the human RRAS2 gene, tagged with an HA epitope, into the Rosa26 locus of C57Bl/6J mice. The gene was flanked by LoxP sites, allowing for Cre recombinase-mediated recombination to induce RRAS2 overexpression specifically in B cells ². This was achieved by crossing the Rosa26-RRAS2^fl/fl mice with mb1-Cre mice, which express Cre recombinase under the mb1 promoter, active in early B cell precursors and mature B cells. The authors showed that the Rosa26-RRAS2^fl/flxmb1-Cre mice exhibited approximately twice the amount of R-RAS2 protein in their spleens compared to control mice. This confirmed the successful generation of the mouse model with specific and significant overexpression of RRAS2 in B cells. They validated the new mouse model by treating the Rosa26-RRAS2^fl/flxmb1-Cre mice with two established CLL drugs, ibrutinib and venetoclax. They divided the mice into three groups and received either vehicle, ibrutinib, or venetoclax. They found ibrutinib and venetoclax reduced splenomegaly in the mice and also both drugs significantly decreased spleen weights, which indicates their efficacy in reducing the disease burden. Notably, venetoclax treatment led to a significant reduction in the number of total CD19+ B cells in the spleen, whereas ibrutinib did not. Additionally, venetoclax reduced the percentage and number of GFP+ CD19+ B cells, indicative of RRAS2 overexpression.

The authors analyzed as well the bone marrow and blood of treated mice to evaluate the impact of drug treatments on leukemic cells and used flow cytometry to assess the presence of leukemic CD19+CD5+ B cells and other B cell populations in these tissues and found that venetoclax treatment resulted in a significant reduction in the number of immature B220^highIgM+ B cells in the bone marrow which suggests a blockade in B cell maturation. This reduction in immature B cells correlated with a decrease in the total CD19+ population in the spleen and indicates that venetoclax was not selective for leukemic cells and affected normal B cell maturation. On the other hand, ibrutinib uniquely reduced the percentage of malignant CD5+ B cells, which demonstrates its specific efficacy in targeting leukemic cells. In contrast, venetoclax did not significantly affect these cells in the blood.

The team further investigated the effects of the treatments on lymphoid organs and found both ibrutinib and venetoclax treatments reduced spleen follicle sizes. This finding is important as it indicates that these treatments effectively reduce the leukemic cell burden in the spleen. Venetoclax was more potent in reducing follicle sizes, which aligns with its impact on the overall B cell population. Indeed, the reduction in follicle size reflects the therapeutic effects observed in human CLL patients treated with these drugs. The researchers used western blotting to quantify R-RAS2 protein levels in the spleens of Rosa26-RRAS2^fl/flxmb1-Cre mice compared to controls and found that Rosa26-RRAS2^fl/flxmb1-Cre mice expressed twice as much R-RAS2 protein.

In conclusion, the newly developed Rosa26-RRAS2^fl/flxmb1-Cre mouse model by Professor Balbino Alarcón and his group is an excellent CLL model with leukemic characteristics appearing early in the mice’s lives which is in contrast with previous models that require long latency periods or additional genetic manipulations. Additionally, it closely mimics human CLL, with the overexpression of the RRAS2 gene without mutations. Moreover, the successful validation with existing therapies establishes the model as a valuable tool for preclinical testing of new therapeutic agents, which will accelerate the transition from bench to bedside.

In a statement to Medicine Innovates, the authors said “based on this study and our previous research, we believe that the overexpression of the GTPase RRAS2, in the absence of activating mutations, is a key driver in the development of the majority of chronic lymphocytic leukemias, affecting both Ig-mutated and unmutated types. Our novel mouse model develops CLL rapidly and serves as a convenient and disease-relevant platform for testing new therapeutic approaches against CLL”.

Breaking Ground in CLL Research: Novel Mouse Model Unveils New Therapeutic Pathways - Medicine Innovates — The sizes of two Control spleens (left) and two spleens of mice overexpressing RRAS2 and have CLL leukemia

About the author

Name: Balbino Alarcon
Position: Group leader-Professor of Research CSIC
Scopus code: N-9648-2016
ORCID number: 0000-0001-7820-1070
e-mail: [email protected]
Webpage

My most important scientific contributions are framed in two areas of research: immunology and cancer. Among my various contributions in immunology, perhaps the most outstanding from an intellectual and subsequently translational point of view, has been the demonstration that the receptor for the antigen of T lymphocytes (TCR) undergoes conformational changes after the binding of antigens and agonistic antibodies (Cell, 2002). This discovery broke the existing dogma that the TCR did not undergo conformational changes, but rather signal transmission occurred through mere mechanisms of aggregation and/or molecular exclusion. This discovery has revolutionized the view of the TCR, perhaps the most important molecule in the functioning of the adaptive immune response. In fact, and just as an example, the TCR enables vaccines to work or for so-called “checkpoint inhibitors” to have anticancer activity. It is also part of the development of cancer therapy known as “CAR-T”.

We based the demonstration of the existence of conformational changes in the TCR on the observation that, as a consequence of antigen stimulation, the TCR exposes a sequence that recruits the adaptor protein Nck. This occurs through an SH3 domain of Nck that has a unique “pocket”. This offered the opportunity to design molecules that interfere with the TCR-Nck binding. Following this idea, we designed, through an “in silico” procedure, a low molecular weight inhibitor to fit into that pocket and prevent the recruitment of Nck to the TCR, and therefore signal transmission. Such inhibitor, which we named AX024, proved to be effective through oral administration in various models of autoimmune diseases: psoriasis, Crohn’s disease/ulcerative colitis, asthma, and multiple sclerosis (Sci. Transl. Med, 2016), all caused to a large extent by uncontrolled activation of T lymphocytes. This inhibitor and its entire family of derivatives were patented (PCT/ES2009/070239 Granted on 31-03-2011 Priority date: 30-06-2008; 201031437. PCT/ES2011/070506 Granted on 03-07-2013, Priority date: 28-09-2010; US Patent 62/607943 Priority date: 20-12-2017) and led to the creation of the company Artax Biopharma Inc based in Boston MA. I am a founding member of Artax, I was its CSO and now I am a member of its Scientific Advisory Board. Artax has improved the initial compound with the lead AX158. This latter compound has completed all regulatory preclinical phases and Phase I. It is currently in Phase II for psoriasis treatment. If the drug meets expectations, it will be the first immunomodulatory drug, not immunosuppressive, acting on the TCR and with a possibility of use in practically all autoimmune diseases. The drug is administered orally, with only a daily dose and unaffected by food intake or not. In conclusion, it is a “First-in-class” drug for many reasons: it modulates TCR signaling and acts by inhibiting a protein-protein interaction, not an enzymatic activity, which distinguishes it from most pharmacological compounds currently in use.

Our interest in characterizing TCR signaling molecules led us to identify not only Nck but also R-RAS2; obtaining novel and impactful results in cancer. R-RAS2 is a GTPase similar to the oncogene K-RAS, which, however, does not undergo activating mutations in practically any type of cancer. Perhaps for this reason, interest in R-RAS2 declined despite being discovered in 1990. Contrary to K-RAS and other similar GTPases, R-RAS2 has a high intrinsic activity of guanosine nucleotide exchange. This peculiarity led us to think that elevated levels of R-RAS2 expression, even in the absence of activating mutations, could cause cancer. Our work, based on the generation of a transgenic mouse that overexpresses the wild-type form of R-RAS2, demonstrated that this hypothesis is correct. In fact, we found that all mice develop chronic lymphocytic leukemia (CLL), a B-cell leukemia that is the most common in the West (Mol Cancer, 2022). This allowed us to translate these observations to humans, analyzing a cohort of patients with CLL. This study showed that more than 80% of patients overexpress non-mutated R-RAS2. Additionally, we found genetic markers in non-coding regions of the RRAS2 gene that are linked to this overexpression. Such markers can be used as a prognostic method as they are linked to worse survival. Currently, we are characterizing RRAS2 as the most frequently implicated gene (68%) in the development of breast cancer, especially in forms known as “triple-negative” and those linked to pregnancy.

In addition to these two lines of research representing the pillars of my research career, we have been involved in the creation of a diagnostic test for immunity to SARS-CoV-2 that was patented and commercialized by a Spanish company during the recent pandemic (EMBO Mol Med, 2021). The method on which this test is based can be used for the evaluation of humoral response to any pathogen.

In summary, I believe that my trajectory shows how research aimed at understanding basic mechanisms can be translated into clinical use. One of the products of this translation, AX158, has reached the Clinic, and another product, genetic markers of RRAS2, is in the process of materializing as a prognostic marker, in addition to pointing to R-RAS2 as a target for future therapies.

My publications have been cited 11,056 times and their h-index is 55.

About the author

Alejandro Hortal

I started working in the biomedical research field in 2014 and, since then, I have been involved in different research projects. During my bachelor final research project, carried out in the group of María Monsalve at the IIBm in Madrid (Spain), I studied the involvement of metabolism in liver pathogenesis and its potential to progress to full cancer development. Following this, during my first Master research project, I worked on the role of the ubiquitine-proteasome system and its relationship with cancer in the group of Daniele Guardavaccaro at the Hubrecht Institute in Utrecht (the Netherlands). For my second Master research project I moved to the group of Marcos Malumbres at the CNIO in Madrid (Spain) where I worked studying the cell cycle MASTL kinase as a potential cancer therapeutic target. Finally, in October 2017 I joined the group of Balbino Alarcón at the CBMSO in Madrid, where I am currently working as a postdoctoral researcher, to do my PhD. In this project, I studied and described the driving role of RRAS2 in the development of chronic lymphocytic leukemia (CLL). As a result of this research, I successfully defended my PhD thesis in September 2023. These results have led to several publications that unravel the relevant role of RRAS2 in different aspects of CLL. Importantly, part of these publications describe a new mouse model with specific overexpression of RRAS2 in B lymphocytes that can be used as a tool to deepen the understanding of CLL as well as to test new therapeutic avenues for this disease.

References

Hortal AM, Villanueva A, Arellano I, Prieto C, Mendoza P, Bustelo XR, Alarcón B. Mice Overexpressing Wild-Type RRAS2 Are a Novel Model for Preclinical Testing of Anti-Chronic Lymphocytic Leukemia Therapies. Cancers (Basel). 2023 Dec 12;15(24):5817. doi: 10.3390/cancers15245817.

Go To Cancers (Basel).

Hortal AM, Oeste CL, Cifuentes C, Alcoceba M, Fernández-Pisonero I, Clavaín L, Tercero R, Mendoza P, Domínguez V, García-Flores M, Pintado B, Abia D, García-Macías C, Navarro-Bailón A, Bustelo XR, González M, Alarcón B. Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia. Mol Cancer. 2022 Feb 4;21(1):35. doi: 10.1186/s12943-022-01496-x.

Go To Mol Cancer.