Epigenetics in a spectrum of myeloid diseases and its exploitation for therapy

Mutations in genes encoding chromatin regulators are early events contributing to de-veloping asymptomatic clonal hematopoiesis of indeterminate potential and its frequent progression to myeloid diseases with increasing severity. We focus on the subset of myeloid diseases encompassing myelodysplastic syndromes and their transformation to secondary acute myeloid leukemia. We introduce the major concepts of chromatin regulation that provide the basis of epigenetic regulation. In greater detail, we discuss those chromatin regulators that are frequently mutated in myelodysplastic syndromes. We discuss their role in the epigenetic regulation of normal hematopoiesis and the consequence of their mutation. Finally, we provide an update on the drugs interfering with chromatin regulation approved or in development for myelodysplastic syndromes and acute myeloid leukemia

Divergent leukaemia subclones as cellular models for testing vulnerabilities associated with gains in chromosomes 7, 8 or 18

Haematopoietic malignancies are frequently characterized by karyotypic abnormalities. The development of targeted drugs has been pioneered with compounds against gene products of fusion genes caused by chromosomal translocations. While polysomies are equally frequent as translocations, for many of them we are lacking therapeutic approaches aimed at synthetic lethality. Here, we report two new cell lines, named MBU-7 and MBU-8, that differ in complete trisomy of chromosome18, a partial trisomy of chromosome 7 and a tetrasomy of the p-arm of chromosome 8, but otherwise share the same mutational pattern and complex karyotype. Both cell lines are divergent clones of U-937 cells and have the morphology and immunoprofile of monocytic cells. The distinct karyotypic differences between MBU-7 and MBU-8 are associated with a difference in the specific response to nucleoside analogues. Taken together, we propose the MBU-7 and MBU-8 cell lines described here as suitable in vitro models for screening and testing vulnerabilities that are associated with the disease-relevant polysomies of chromosome 7, 8 and 18

Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome

Background: Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. Results: MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. Conclusions: Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies

Role of histone variant macroH2A and other chromatin regulators in genome regulation and response to drugs

The relevance of epigenetics is increasingly recognized in haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Variants of histones, such as macroH2As, and chromatin remodellers, such as the CHRAC complex, are key players in these epigenetic regulations. On the other hand, non-coding DNA regions, including long interspersed nuclear elements (LINEs), also show increasing relevance, notably in cancer. Here, we studied the relationship between LINEs and macroH2As in a hepatocarcinoma cell line. Computational analysis and experimental work led to conflicting results regarding a potential repressive role of macroH2As on LINEs. In addition, we studied the role of macroH2As as well as other chromatin regulators in the response to the epigenetic treatments Azacitidine and Decitabine in MDS/AML cell lines and a cohort of patient's samples. We were able to show that reduced expression of macroH2A1 and CHRAC complex component, BAZ1A, sensitized MDS/AML cell lines to epigenetic treatments. Furthermore, in patient sample, CHRAC complex components were less expressed in ten-eleven translocase 2 (TET2) mutants and in non-responders to Azacitidine.Cada vez se reconoce más la importancia de la epigenética en enfermedades hematopoyéticas como los síndromes mielodisplásicos (SMD) y la leucemia mieloide aguda (LMA). Las variantes de histonas, como las macroH2A, y los remodeladores de la cromatina, como el complejo CHRAC, son actores claves en estas regulaciones epigenéticas. Por otra parte, las regiones de ADN no codificante, incluidos los elementos nucleares intercalados largos (LINEs), también muestran una relevancia creciente, especialmente en el cáncer. Aquí estudiamos la relación entre los LINEs y las macroH2As en una línea celular de hepatocarcinoma. El análisis computacional y el trabajo experimental condujeron a resultados contradictorios respecto a un potencial papel represivo de las macroH2As sobre los LINEs. Además, estudiamos el papel de las macroH2As, así como de otros reguladores de la cromatina, en la respuesta a los tratamientos epigenéticos Azacitidina y Decitabina en líneas celulares de SMD/LMA y en una cohorte de muestras de pacientes. Pudimos demostrar que la reducción de la expresión de macroH2A1 y del componente del complejo CHRAC, BAZ1A, sensibilizó a las líneas celulares de SMD/LMA a los tratamientos epigenéticos. Además, en la muestra de pacientes, los componentes del complejo CHRAC se expresaban menos en los mutantes de la \textit{ten-eleven} translocasa 2 (TET2) y en los que no respondían a la Azacitidina

Histone Modifications and Their Targeting in Lymphoid Malignancies

Altres ajuts: Deutsche José Carreras Leukämie Stiftung (DJCLS) grant 14R/2018; Fundació La Marató de TV3 257/C/2019In a wide range of lymphoid neoplasms, the process of malignant transformation is associated with somatic mutations in B cells that affect the epigenetic machinery. Consequential alterations in histone modifications contribute to disease-specific changes in the transcriptional program. Affected genes commonly play important roles in cell cycle regulation, apoptosis-inducing signal transduction, and DNA damage response, thus facilitating the emergence of malignant traits that impair immune surveillance and favor the emergence of different B-cell lymphoma subtypes. In the last two decades, the field has made a major effort to develop therapies that target these epigenetic alterations. In this review, we discuss which epigenetic alterations occur in B-cell non-Hodgkin lymphoma. Furthermore, we aim to present in a close to comprehensive manner the current state-of-the-art in the preclinical and clinical development of epigenetic drugs. We focus on therapeutic strategies interfering with histone methylation and acetylation as these are most advanced in being deployed from the bench-to-bedside and have the greatest potential to improve the prognosis of lymphoma patients

Different Gene Sets Are Associated With Azacitidine Response In Vitro Versus in Myelodysplastic Syndrome Patients

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by dysplasia, ineffective hematopoiesis, and predisposition to secondary acute myeloid leukemias (sAML). Azacitidine (AZA) is the standard care for high-risk MDS patients not eligible for allogenic bone marrow transplantation. However, only half of the patients respond to AZA and eventually all patients relapse. Response-predicting biomarkers and combinatorial drugs targets enhancing therapy response and its duration are needed. Here, we have taken a dual approach. First, we have evaluated genes encoding chromatin regulators for their capacity to modulate AZA response. We were able to validate several genes, whose genetic inhibition affected the cellular AZA response, including 4 genes encoding components of Imitation SWItch chromatin remodeling complex pointing toward a specific function and co-vulnerability. Second, we have used a classical cohort analysis approach measuring the expression of a gene panel in bone marrow samples from 36 MDS patients subsequently receiving AZA. The gene panel included the identified AZA modulators, genes known to be involved in AZA metabolism and previously identified candidate modulators. In addition to confirming a number of previously made observations, we were able to identify several new associations, such as NSUN3 that correlated with increased overall survival. Taken together, we have identified a number of genes associated with AZA response in vitro and in patients. These groups of genes are largely nonoverlapping suggesting that different gene sets need to be exploited for the development of combinatorial drug targets and response-predicting biomarkers

Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis

The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis. © 2021. The Author(s)

Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome

Background: Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. Results: MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. Conclusions: Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies

Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis

This project was supported by the FEDER/Ministerio de Ciencia e Innovación - Agencia Estatal de Investigación through the grant PIE16/00011 RESPONSE (to M.B.), the European Research Council ERC-StG-336860 (to J.Z.), the grant Juan de la Cierva- Formación FJCI-2014-22983 (to J.D.), the grant Sara Borrell CD17/00084 (to J.D.), the Marie Skłodowska Curie Training network "ChroMe" H2020-MSCA-ITN-2015-675610 (to M.M.), the FPI predoctoral fellowship BES-2016-077251 (to M.M.L.P.), the SFB 1243 DFG (to K.S.G.), the Austrian Science Fund SFB-F4710 (to J.Z.) and the Deutsche José Carreras Leukämie Stiftung DJCLS 14R/2018 (to K.G. and M.B.). Research in the Buschbeck lab is further supported by the following grants: MINECO grant RTI2018-094005-B-I00 (to M.B.), the Marie Skłodowska Curie Training network 'INTERCEPT-MDS' H2020-MSCA-ITN-2015-953407 (to M.B. and K.S.G.); AGAUR 2017-SGR-305 (to M.B.) and Fundació La Marató de TV3 257/C/2019 (to M.B.). Research at IMP is supported by Boehringer Ingelheim, the Austrian Research Promotion Agency (Headquarter grant FFG-852936) and the Austrian Academy of Sciences. Research at the IJC is generously supported by the 'La Caixa' Foundation, the Fundació Internacional Josep Carreras, Celgene Spain and the CERCA Programme/Generalitat de Catalunya. A.G. received funds by "Agencia Estatal de Investigación" (AEI) through the Plan Nacional "Excelencia" grant number SAF2017-84301-P, by the "Associación Española Contra el Cancer" (AECC) grant number LABAE20040GENT and by the Agency for Management of University and Research Grants (AGAUR) of the Catalan Government grant 2017SGR01743. Proteomic analyses were performed in the IJC Proteomic Unit, which are part of Proteored PRB3 and are supported by grant PT17/0019 from the PE I + D + i 2013-2016, funded by ISCIII and ERDF. We thank Kaoru Tohyama for providing MDS-L cells, members of the Buschbeck lab, the RESPONSE network (PIE16/00011), Blanca Xicoy and Francesc Solé for valuable discussions. We thank the IJC Biobanking unit for sample preparation and storage, Bernat Cucurull from the IJC Proteomics unit for the ClickIT sample preparation, Marco Fernandez for advice and training in flow cytometry and all other staff of IJC and IGTP core facilities for excellent support.The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis

MacroH2As regulate enhancer-promoter contacts affecting enhancer activity and sensitivity to inflammatory cytokines

MacroH2A histone variants have a function in gene regulation that is poorly understood at the molecular level. We report that macroH2A1.2 and macroH2A2 modulate the transcriptional ground state of cancer cells and how they respond to inflammatory cytokines. Removal of macroH2A1.2 and macroH2A2 in hepatoblastoma cells affects the contact frequency of promoters and distal enhancers coinciding with changes in enhancer activity or preceding them in response to the cytokine tumor necrosis factor alpha. Although macroH2As regulate genes in both directions, they globally facilitate the nuclear factor κB (NF-κB)-mediated response. In contrast, macroH2As suppress the response to the pro-inflammatory cytokine interferon gamma. MacroH2A2 has a stronger contribution to gene repression than macroH2A1.2. Taken together, our results suggest that macroH2As have a role in regulating the response of cancer cells to inflammatory signals on the level of chromatin structure. This is likely relevant for the interaction of cancer cells with immune cells of their microenvironment.This research project was supported by the national grants RTI2018-094005-B-I00 and BFU2015-66559-P from FEDER / Ministerio de Ciencia e Innovación - Agencia Estatal de Investigación (to M.B.); PI09/00751 , PI10/02082 , and PI13/02340 from the Instituto de Salud Carlos III (to C.A.); the MECD fellowship FPU14/06542 (to D.C.); AGAUR 2019 FI_B01024 and 2022 FI_B00528 fellowships (to J.C.-R. and A.D.R.-A., respectively); and predoctoral fellowships BES-2016-077251 (to M.-M.L.P.) and PRE2019-088529 (to O.M.). Research in the M.B. lab is further supported by the following grants: the Marie Skłodowska Curie Training network “ INTERCEPT-MDS ” H2020-MSCA-ITN-2020-953407 (to M.B.); MINECO-ISCIII PIE16/00011 (to M.B.); the Deutsche José Carreras Leukämie Stiftung DJCLS 14R/2018 (to M.B.); AGAUR 2017-SGR-305 (to M.B.); and Fundació La Marató de TV3 257/C/2019 (to M.B.). C.A.’s research has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement nos. 668596 ( ChiLTERN ) and 826121 ( iPC ) as well as from CIBERehd ( CB06/04/0033 ) and AGAUR ( 2017-SGR-490 ). C.A. was supported by Ramón y Cajal ( RYC-2010-07249 ) of the Ministry of Science and Innovation of Spain. Research at the IJC is supported by the “ La Caixa” Foundation , the Fundació Internacional Josep Carreras , Celgene Spain , and the CERCA Programme / Generalitat de Catalunya