Periodic Reporting for period 1 - SirT-IMLEU (Mechanisms of Sirtuin-dependent regulation of immunity and leukemogenesis)
Reporting period: 2020-12-01 to 2022-11-30
Yearly, roughly 53.000 people worldwide are diagnosed with acute lymphoblastic leukemia (ALL), a type of cancer characterized by the development of large numbers of immature lymphocytes. ALL originated from B cell precursors (B-ALL) is the most common pediatric cancer.
This MSCA Action, entitled “Mechanisms of Sirtuin-dependent regulation of immunity and leukemogenesis”, aimed to identify novel molecular mechanisms required for the development of immunity and to further understand how their deregulation participates in the formation of acute lymphoblastic leukemia of B-ALL. Specifically, the functional relationship between Sirtuins (a family of proteins critical for the function of the genome) and the master regulator PAX5 (key transcription factor required to produce B lymphocytes) have been studied.
The main goal of the project was to define the role of SIRT7 in B cell development and leukemia. Our main hypothesis was that SIRT7-dependent regulation of PAX5 played a fundamental role in B cell development and that this interaction was required to sustain B cell fate. A multidisciplinary state-of-the-art approach was designed, including molecular and cellular biology, genomics and mouse models to unravel the molecular mechanisms of SIRT7 in immune regulation. Three different specific aims were defined: 1: Characterization of the functional relationship between SIRT7 and PAX5 in leukemic cells. 2: To analyze the interplay of PAX5 and SIRT7 in gene transcriptional regulation. 3: To determine the contribution of SIRT7 in the maintenance of B cell identity and leukemogenesis.
This project has provided significant advances in our understanding of the molecular biology of B-ALL, identifying a new regulator of B cell development, SIRT7, and defining for the first time mechanisms of PAX5 protein stabilization, thus opening new paths for therapeutic interventions. SIRT7 activating compounds could serve as a novel strategy to restore PAX5 protein levels and reduce tumor progression. This action represents the first attempt to perform such approach. Furthermore, a novel role of SIRT7 in B lymphocyte differentiation has been uncovered, shedding shed light into novel regulatory mechanisms of PAX5 activity, with major implications for treatments for leukemia patients.
This MSCA Action, entitled “Mechanisms of Sirtuin-dependent regulation of immunity and leukemogenesis”, aimed to identify novel molecular mechanisms required for the development of immunity and to further understand how their deregulation participates in the formation of acute lymphoblastic leukemia of B-ALL. Specifically, the functional relationship between Sirtuins (a family of proteins critical for the function of the genome) and the master regulator PAX5 (key transcription factor required to produce B lymphocytes) have been studied.
The main goal of the project was to define the role of SIRT7 in B cell development and leukemia. Our main hypothesis was that SIRT7-dependent regulation of PAX5 played a fundamental role in B cell development and that this interaction was required to sustain B cell fate. A multidisciplinary state-of-the-art approach was designed, including molecular and cellular biology, genomics and mouse models to unravel the molecular mechanisms of SIRT7 in immune regulation. Three different specific aims were defined: 1: Characterization of the functional relationship between SIRT7 and PAX5 in leukemic cells. 2: To analyze the interplay of PAX5 and SIRT7 in gene transcriptional regulation. 3: To determine the contribution of SIRT7 in the maintenance of B cell identity and leukemogenesis.
This project has provided significant advances in our understanding of the molecular biology of B-ALL, identifying a new regulator of B cell development, SIRT7, and defining for the first time mechanisms of PAX5 protein stabilization, thus opening new paths for therapeutic interventions. SIRT7 activating compounds could serve as a novel strategy to restore PAX5 protein levels and reduce tumor progression. This action represents the first attempt to perform such approach. Furthermore, a novel role of SIRT7 in B lymphocyte differentiation has been uncovered, shedding shed light into novel regulatory mechanisms of PAX5 activity, with major implications for treatments for leukemia patients.
In aim 1 (WP2), we performed different types of experiments in leukemia cells with or without SIRT7, monitoring the degradation dynamics of PAX5. The lack of SIRT7 resulted in reduced PAX5 stability as it got degraded twice faster compared to normal cells. We also observed (via western blotting), that PAX5 was hyperacetylated in the absence of SIRT7. Protein acetylation is a reversible modification that can alter protein properties. Because SIRT7 has the capacity to remove acetylation moieties from proteins, our results suggested a functional link between PAX5 acetylation, PAX5 protein turnover and SIRT7. Indeed, acetylated PAX5 resulted in reduced protein stability. Thus, the biochemical interplay between SIRT7 and PAX5 was fully characterized, indicating that SIR7 deacetylates PAX5 to enhance its stability.
In aim 2 (WP3), the functional interplay between SIRT7 and PAX5 in the regulation of gene expression was studied. Firstly, we obtained normal preB cells (this is the cell type most affected by the lack of SIRT7) or lymphocytes lacking SIRT7 and analyzed global gene expression (RNA-sequencing). After Principal component analysis (PCA), we found that lymphocytes with and without SIRT7 had separated PCA dimensions, reinforcing the idea that SIRT7 played an important role in this immune population. After applying a Gene Set Enrichment Analysis, we found that lymphocytes lacking SIRT7 expressed genes of other cell lineages, including T lymphocytes, suggesting that they had impaired cellular identity. Cells lacking PAX5 also presented a similar phenotype and were also consistent with the identified biochemical interplay between PAX5 and SIRT7 (WP2).
PAX5 regulates gene expression by binding and recruiting specific set of proteins called chromatin modifiers, which are able to acetylate chromatin (DNA + histones) to modulate gene expression. As SIRT7 is a chromatin modifier, the interplay between SIRT7 and PAX5 in gene transcriptional regulation was further analyzed. The acetylation levels of histone H3K36 (H3K36ac) were massively increased in cells lacking SIRT7. In addition, we found a major overlap between H3K36ac and the sites were PAX5 binds in the genome, reinforcing the interplay between SIRT7 and PAX5.
In aim 3 (WP4), mouse model transplants were used to analyze the repopulation capacity and cell fate of B lymphocytes lacking SIRT7. Results indicated that the absence of SIRT7 led to a reduced reconstituting capacity of the B cells compartment, which is consistent with the critical role of SIRT7 in the preB cells.
These results have and will be published in a top peer-reviewed scientific journals (June 2023).
Results have also been disseminated in the Scientific community, being orally presented at three different meetings: 1- In the XII Jornades de Recerca BCFI (June 2021), at the Autonomous University of Barcelona (UAB): Title: “Sirtuins in genome integrity and cellular differentiation pathways”. 2- In the FASEB meeting on Reversible Protein Acetylation in Health and Disease (August 2022, Puerto Rico): Title: “SIRT7 regulates PAX5 function and promotes B cell development”. 3- In the Minerva Workshop on Metabolism and Ageing (November 2022, Tel aviv) Title: “SIRT7 regulates B cell generation and aging”.
The grantee has participated in different outreach activities to raise awareness not only about this project but also about the Marie Sklodowska Curie (MSC) programme. She has been interviewed twice at the IJC about her experience as an MSC fellow, her research and future prospects. She also contributed in the MSC Postdoctoral Fellowship info day at the IJC (April 2016), explaining her career path and research interests.
Different outreach activities targeting non-scientific audiences such as school teachers and students from primary and high schools were also done, aiming at raising science awareness among young students and to encourage girls to engage in STEM careers. In November 2021, I also participated in the event “Science is wonderful” organized by the EC.
In aim 2 (WP3), the functional interplay between SIRT7 and PAX5 in the regulation of gene expression was studied. Firstly, we obtained normal preB cells (this is the cell type most affected by the lack of SIRT7) or lymphocytes lacking SIRT7 and analyzed global gene expression (RNA-sequencing). After Principal component analysis (PCA), we found that lymphocytes with and without SIRT7 had separated PCA dimensions, reinforcing the idea that SIRT7 played an important role in this immune population. After applying a Gene Set Enrichment Analysis, we found that lymphocytes lacking SIRT7 expressed genes of other cell lineages, including T lymphocytes, suggesting that they had impaired cellular identity. Cells lacking PAX5 also presented a similar phenotype and were also consistent with the identified biochemical interplay between PAX5 and SIRT7 (WP2).
PAX5 regulates gene expression by binding and recruiting specific set of proteins called chromatin modifiers, which are able to acetylate chromatin (DNA + histones) to modulate gene expression. As SIRT7 is a chromatin modifier, the interplay between SIRT7 and PAX5 in gene transcriptional regulation was further analyzed. The acetylation levels of histone H3K36 (H3K36ac) were massively increased in cells lacking SIRT7. In addition, we found a major overlap between H3K36ac and the sites were PAX5 binds in the genome, reinforcing the interplay between SIRT7 and PAX5.
In aim 3 (WP4), mouse model transplants were used to analyze the repopulation capacity and cell fate of B lymphocytes lacking SIRT7. Results indicated that the absence of SIRT7 led to a reduced reconstituting capacity of the B cells compartment, which is consistent with the critical role of SIRT7 in the preB cells.
These results have and will be published in a top peer-reviewed scientific journals (June 2023).
Results have also been disseminated in the Scientific community, being orally presented at three different meetings: 1- In the XII Jornades de Recerca BCFI (June 2021), at the Autonomous University of Barcelona (UAB): Title: “Sirtuins in genome integrity and cellular differentiation pathways”. 2- In the FASEB meeting on Reversible Protein Acetylation in Health and Disease (August 2022, Puerto Rico): Title: “SIRT7 regulates PAX5 function and promotes B cell development”. 3- In the Minerva Workshop on Metabolism and Ageing (November 2022, Tel aviv) Title: “SIRT7 regulates B cell generation and aging”.
The grantee has participated in different outreach activities to raise awareness not only about this project but also about the Marie Sklodowska Curie (MSC) programme. She has been interviewed twice at the IJC about her experience as an MSC fellow, her research and future prospects. She also contributed in the MSC Postdoctoral Fellowship info day at the IJC (April 2016), explaining her career path and research interests.
Different outreach activities targeting non-scientific audiences such as school teachers and students from primary and high schools were also done, aiming at raising science awareness among young students and to encourage girls to engage in STEM careers. In November 2021, I also participated in the event “Science is wonderful” organized by the EC.
The project has characterized the function of a novel regulator of lymphocyte maturation, the sirtuin SIRT7, and its relationship to leukemia formation, opening new paths to identify potential targets for therapeutic intervention. The functional relationship between SIRT7 and PAX5 has been defined, showing that SIRT7 is a critical regulator of PAX5 protein abundancy inside the cells, and that SIRT7 activating compounds could serve as a novel strategy to restore PAX5 protein levels in B-ALL, reducing their tumorigenic capacity. Developing Sirtuin targeting compounds will be certainly the approach to test. The dissemination and outreach activities have helped to maximize social impact and raise awareness within society and the scientific community about the importance of this action.
All in all, the results obtained have led to significant advances in our understanding of the molecular biology of B-ALL, the most common type of childhood cancer, and will open novel paths for diagnostic and treatment.
All in all, the results obtained have led to significant advances in our understanding of the molecular biology of B-ALL, the most common type of childhood cancer, and will open novel paths for diagnostic and treatment.