Periodic Reporting for period 4 - COMAL (COMAL:COhesin Mutations in Acute Leukemia: from modeling and mechanisms to novel therapeutics)
Periodo di rendicontazione: 2020-03-01 al 2021-02-28
Our project aimed to determine the mechanisms whereby alterations in the structural packaging of DNA and the communication between different DNA sequence elements, potentially over long distances, control gene expression, subsequent protein manufacture and through this, cellular behaviour and the mechanisms whereby these processes are altered and subverted to generate cancer. As an exemplar, we chose the tractable tumour Acute Myeloid Leukaemia (AML), focusing on the subtype with predicted loss-of-function mutation of the Cohesin complex. This complex is a global regulator of interactions between DNA segments over long distances through the looping out of intervening DNA, and by controlling enhancer and promoter interactions, and thought to regulate gene expression. M utations in Cohesin complex members occur frequently in other forms of cancer, suggesting the Cohesin complex alter DNA topology and with it gene expression across multiple cell types. This finding is consistent with the fact that alterations of gene expression are present and drive almost every type of cancer.
Cancer is now the most common cause of death amongst western populations. With our ageing population, deaths from malignant disease are predicted to continue increasing. Even in survivors of cancer, continued morbidity brings a significant burden of patient suffering. Financially, cancer patients are lost to the workforce due to death or an inability to perform their duties and cancer treatments are becoming increasing complicated and expensive. Better knowledge of the causes of cancer and improved therapies are key in detecting or preventing developing cases earlier and in treating established cases better and with lower toxicities.
Our project has been highly successful in meeting all its objectives. We established both a cell line and in vivo system of Cohesin loss/depletion and used these to shed considerable light on the function of Cohesin, in particular during cellular processes that require dynamic gene regulation. We also specifically and mechanistically dissected the role of Cohesin in erythroid differentiation, establishing that Cohesin depletion abrogates induction of dynamic erythroid transcriptional programmes and that Cohesin-dependent dynamic gene expression upon erythroid differentiation is both prespecified and repressed by Etv6 in stem cells. We are currently examining the role of loss of Cohesin function in the maintenance of malignant self-renewal and are determining ways to therapeutically target this. These observations have led us to conclude that Etv6 activity is a potential therapeutic target, to induce differentiation in both AML and MDS, preparing the way for screens to identify an effective compound.
Cancer is now the most common cause of death amongst western populations. With our ageing population, deaths from malignant disease are predicted to continue increasing. Even in survivors of cancer, continued morbidity brings a significant burden of patient suffering. Financially, cancer patients are lost to the workforce due to death or an inability to perform their duties and cancer treatments are becoming increasing complicated and expensive. Better knowledge of the causes of cancer and improved therapies are key in detecting or preventing developing cases earlier and in treating established cases better and with lower toxicities.
Our project has been highly successful in meeting all its objectives. We established both a cell line and in vivo system of Cohesin loss/depletion and used these to shed considerable light on the function of Cohesin, in particular during cellular processes that require dynamic gene regulation. We also specifically and mechanistically dissected the role of Cohesin in erythroid differentiation, establishing that Cohesin depletion abrogates induction of dynamic erythroid transcriptional programmes and that Cohesin-dependent dynamic gene expression upon erythroid differentiation is both prespecified and repressed by Etv6 in stem cells. We are currently examining the role of loss of Cohesin function in the maintenance of malignant self-renewal and are determining ways to therapeutically target this. These observations have led us to conclude that Etv6 activity is a potential therapeutic target, to induce differentiation in both AML and MDS, preparing the way for screens to identify an effective compound.
1 Generation of model systems of Cohesin AML
This has been achieved with the generation of a cell line and in vivo system. Work included generating a murine Smc3 conditional knock out strain and haematopoietic stem and progenitor cell lines with inducible knockdown of multiple cohesin complex family members (Smc3, Smc1a, Rad21 and Stag2).
2 Study of the transcriptional alterations in Cohesin AML
This has been achieved with results published in Sasca et al, Blood, 2019. We comprehensively demonstrated that depletion of Cohesin abrogates induction of dynamic erythroid transcriptional programmes and proposed a mechanism for this induction involving the requirement of Cohesin-dependent eviction of the ETS transcription factor Etv6 to facilitate normal erythroid differentiation. This mechanism is abrogated in human AML and MDS and is recapitulated in our cell line system at the phenotypic and transcriptional level. In unpublished data, we have transcriptional evidence of a different mechanism working in myeloid differentiation on which we are performing further analysis. We are also pursuing mechanisms underlying the increase in self-renewal programmes.
3 Mechanistic study of Cohesin AML
This has been partially achieved and also published in Sasca et al, Blood, 2019. We elucidated a mechanism for Cohesin co-ordinated alterations in critical gene expression programmes relating to context-specific interaction with specific haematopoietic transcription factors; for erythroid differentiation, specifically for Etv6. We found depletion of Cohesin severely impairs erythroid differentiation, particularly at loci pre-bound with Etv6 and loss of Cohesin also augments self-renewal transcriptional programmes. We demonstrated loss of Cohesin-mediated alleviation of Etv6 repression, thus blocking erythroid differentiation, as the first detailed mechanisms of how loss of Cohesin activity contributes to myeloid malignancies. This loss of repression appears to be required for dynamic expression at critical erythroid genes during differentiation. As with Objective 2, work included transcriptomic, genomic and mass spectrometry-based proteomic experiments and bioinformatic analysis.
4 Therapeutic study of Cohesin AML
We identified Etv6 activity as a highly promising potential therapeutic target for Cohesin AML as well as other AML subtypes and MDS. Major work included CRISPR-Cas9 screens which identified the role of genes in Cohesin depleted cells. We had planned to complete the screening of compounds as possible therapeutic agents effective against Etv6 activity. The COVID-19 pandemic however affected our access to our lab and patient cells meaning, that unfortunately, no compound was successfully identified by the end of the grant. This aim remains a target for future research.
Our studies have greatly informed the general function of the Cohesin complex, that appears to be dispensible for homeostatic gene expression but is exquisitely required for dynamic gene expression. We explained its effects on differentiation and the maintenance of the leukaemia stem cell state. We disseminated this work widely at conferences (e.g. American Society of Hematology Annual Meeting 2019) and through publication and expect two further publications. We are currently assessing the therapeutic implications of targets such as ETV6 for future exploitation. All published data has been made freely available.
This has been achieved with the generation of a cell line and in vivo system. Work included generating a murine Smc3 conditional knock out strain and haematopoietic stem and progenitor cell lines with inducible knockdown of multiple cohesin complex family members (Smc3, Smc1a, Rad21 and Stag2).
2 Study of the transcriptional alterations in Cohesin AML
This has been achieved with results published in Sasca et al, Blood, 2019. We comprehensively demonstrated that depletion of Cohesin abrogates induction of dynamic erythroid transcriptional programmes and proposed a mechanism for this induction involving the requirement of Cohesin-dependent eviction of the ETS transcription factor Etv6 to facilitate normal erythroid differentiation. This mechanism is abrogated in human AML and MDS and is recapitulated in our cell line system at the phenotypic and transcriptional level. In unpublished data, we have transcriptional evidence of a different mechanism working in myeloid differentiation on which we are performing further analysis. We are also pursuing mechanisms underlying the increase in self-renewal programmes.
3 Mechanistic study of Cohesin AML
This has been partially achieved and also published in Sasca et al, Blood, 2019. We elucidated a mechanism for Cohesin co-ordinated alterations in critical gene expression programmes relating to context-specific interaction with specific haematopoietic transcription factors; for erythroid differentiation, specifically for Etv6. We found depletion of Cohesin severely impairs erythroid differentiation, particularly at loci pre-bound with Etv6 and loss of Cohesin also augments self-renewal transcriptional programmes. We demonstrated loss of Cohesin-mediated alleviation of Etv6 repression, thus blocking erythroid differentiation, as the first detailed mechanisms of how loss of Cohesin activity contributes to myeloid malignancies. This loss of repression appears to be required for dynamic expression at critical erythroid genes during differentiation. As with Objective 2, work included transcriptomic, genomic and mass spectrometry-based proteomic experiments and bioinformatic analysis.
4 Therapeutic study of Cohesin AML
We identified Etv6 activity as a highly promising potential therapeutic target for Cohesin AML as well as other AML subtypes and MDS. Major work included CRISPR-Cas9 screens which identified the role of genes in Cohesin depleted cells. We had planned to complete the screening of compounds as possible therapeutic agents effective against Etv6 activity. The COVID-19 pandemic however affected our access to our lab and patient cells meaning, that unfortunately, no compound was successfully identified by the end of the grant. This aim remains a target for future research.
Our studies have greatly informed the general function of the Cohesin complex, that appears to be dispensible for homeostatic gene expression but is exquisitely required for dynamic gene expression. We explained its effects on differentiation and the maintenance of the leukaemia stem cell state. We disseminated this work widely at conferences (e.g. American Society of Hematology Annual Meeting 2019) and through publication and expect two further publications. We are currently assessing the therapeutic implications of targets such as ETV6 for future exploitation. All published data has been made freely available.
This was the first study to link the Cohesin complex to dynamic gene expression and differentiation in the haematopoietic system. It has linked loss-of-function mutations in myeloid malignancies with requirements for dynamic gene expression and identified, in Etv6, a potential new therapeutic target for AML and MDS.
Cutting edge techniques developed in this project included Promoter Capture Hi-C (originally developed by P Fraser, Schoenfelder et al, J Vis Exp, 2018) which we optimised for the study of the role of Cohesin in determining 3D DNA topology and promoter-enhancer interactions; and Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME, originally developed by J Carroll and C D’Santos, Mohammed et al, Nat Protoc, 2016) which we used to identify chromatin bound Cohesin complex interactors. A crucial innovation was the 3D integration of multiple datasets based on new bioinformatics approaches that are now being used elsewhere. It led us to optimise a new 3D genomic methodology, ChIA-Drop (Zheng et al, Nature 2019), originally developed for drosophila, for the mammalian genome. This allows significant reduction in the required cells and interrogation of multiple interactions and at quasi single cell resolution.
Cutting edge techniques developed in this project included Promoter Capture Hi-C (originally developed by P Fraser, Schoenfelder et al, J Vis Exp, 2018) which we optimised for the study of the role of Cohesin in determining 3D DNA topology and promoter-enhancer interactions; and Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME, originally developed by J Carroll and C D’Santos, Mohammed et al, Nat Protoc, 2016) which we used to identify chromatin bound Cohesin complex interactors. A crucial innovation was the 3D integration of multiple datasets based on new bioinformatics approaches that are now being used elsewhere. It led us to optimise a new 3D genomic methodology, ChIA-Drop (Zheng et al, Nature 2019), originally developed for drosophila, for the mammalian genome. This allows significant reduction in the required cells and interrogation of multiple interactions and at quasi single cell resolution.