DNA methylation profiling of myelodysplastic syndromes and clinical response to azacitidine: A multicentre retrospective study.

Real-world data have revealed that a substantial portion of patients with myelodysplastic syndromes (MDS) does not respond to epigenetic therapy with hypomethylating agents (HMAs). The cellular and molecular reasons for this resistance to the demethylating agent and biomarkers that would be able to predict the treatment refractoriness are largely unknown. In this study, we shed light on this enigma by characterizing the epigenomic profiles of patients with MDS treated with azacitidine. Our approach provides a comprehensive view of the evolving DNA methylation architecture of the disease and holds great potential for advancing our understanding of MDS treatment responses to HMAs.

Single-cell Multiomics Analysis of Myelodysplastic Syndromes and Clinical Response to Hypomethylating Therapy.

Alterations in epigenetic marks, such as DNA methylation, represent a hallmark of cancer that has been successfully exploited for therapy in myeloid malignancies. Hypomethylating agents (HMA), such as azacitidine, have become standard-of-care therapy to treat myelodysplastic syndromes (MDS), myeloid neoplasms that can evolve into acute myeloid leukemia. However, our capacity to identify who will respond to HMAs, and the duration of response, remains limited. To shed light on this question, we have leveraged the unprecedented analytic power of single-cell technologies to simultaneously map the genome and immunoproteome of MDS samples throughout clinical evolution. We were able to chart the architecture and evolution of molecular clones in precious paired bone marrow MDS samples at diagnosis and posttreatment to show that a combined imbalance of specific cell lineages with diverse mutational profiles is associated with the clinical response of patients with MDS to hypomethylating therapy. SIGNIFICANCE: MDS are myeloid clonal hemopathies with a low 5-year survival rate, and approximately half of the cases do not respond to standard HMA therapy. Our innovative single-cell multiomics approach offers valuable biological insights and potential biomarkers associated with the demethylating agent efficacy. It also identifies vulnerabilities that can be targeted using personalized combinations of small drugs and antibodies.

The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase.

BACKGROUND: Dynamic chromatin remodeling is associated with changes in the epigenetic pattern of histone acetylations and methylations required for processes based on dynamic chromatin remodeling and implicated in different nuclear functions. These histone epigenetic modifications need to be coordinated, a role that may be mediated by chromatin kinases such as VRK1, which phosphorylates histones H3 and H2A. METHODS: The effect of VRK1 depletion and VRK1 inhibitor, VRK-IN-1, on the acetylation and methylation of histone H3 in K4, K9 and K27 was determined under different conditions, arrested or proliferating cells, in A549 lung adenocarcinoma and U2OS osteosarcoma cells. RESULTS: Chromatin organization is determined by the phosphorylation pattern of histones mediated by different types of enzymes. We have studied how the VRK1 chromatin kinase can alter the epigenetic posttranslational modifications of histones by using siRNA, a specific inhibitor of this kinase (VRK-IN-1), and of histone acetyl and methyl transferases, as well as histone deacetylase and demethylase. Loss of VRK1 implicated a switch in the state of H3K9 posttranslational modifications. VRK1 depletion/inhibition causes a loss of H3K9 acetylation and facilitates its methylation. This effect is similar to that of the KAT inhibitor C646, and to KDM inhibitors as iadademstat (ORY-1001) or JMJD2 inhibitor. Alternatively, HDAC inhibitors (selisistat, panobinostat, vorinostat) and KMT inhibitors (tazemetostat, chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause increase of H3K9ac and a decrease of H3K9me3. VRK1 stably interacts with members of these four enzyme families. However, VRK1 can only play a role on these epigenetic modifications by indirect mechanisms in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1. CONCLUSIONS: The chromatin kinase VRK1 regulates the epigenetic patterns of histone H3 acetylation and methylation in lysines 4, 9 and 27. VRK1 is a master regulator of chromatin organization associated with its specific functions, such as transcription or DNA repair.

Lysine Methyltransferase Inhibitors Impair H4K20me2 and 53BP1 Foci in Response to DNA Damage in Sarcomas, a Synthetic Lethality Strategy.

BACKGROUND: Chromatin is dynamically remodeled to adapt to all DNA-related processes, including DNA damage responses (DDR). This adaptation requires DNA and histone epigenetic modifications, which are mediated by several types of enzymes; among them are lysine methyltransferases (KMTs). METHODS: KMT inhibitors, chaetocin and tazemetostat (TZM), were used to study their role in the DDR induced by ionizing radiation or doxorubicin in two human sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis. RESULTS: Chaetocin and tazemetostat treatments caused a significant increase of H4K16 acetylation, associated with chromatin relaxation, and increased DNA damage, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic marks prevents DNA repair by the NHEJ pathway and leads to cell death. CONCLUSION: KMT inhibitors could function as sensitizers to DNA damage-based therapies and be used in novel synthetic lethality strategies for sarcoma treatment.

Single-cell technologies and analyses in hematopoiesis and hematological malignancies.

In recent years, single-cell technologies have emerged as breakthrough techniques that enable the characterization of hematopoietic cell populations of normal and malignant tissue samples and will be combined in the near future with bulk technologies, currently used in clinical practice, to improve diagnosis, prognosis, and the search for novel molecular targets. These single-cell methods have the advantage of not masking cell-to-cell variation features and involve the study of genetic, epigenetic, transcriptional, and proteomic landscapes from a single-cell perspective. Latest advances in this field have enabled the development of novel strategies that significantly increase both sensitivity and high throughput. In this review, we emphasize emerging techniques aimed at assessing individual or multiomic parameters at single-cell resolution and analyze how these technologies have helped us understand hematopoietic variability and identify unknown and/or rare subpopulations. We also summarize the impact of these single-cell profiling strategies on the characterization of cell diversity within the tumor and the clonal evolution of multiple hematological malignancies in samples from untreated and treated patients, which provide valuable information for diagnosis, prognosis, and future treatments and explain why current therapies may fail. However, despite these improvements, new challenges lie ahead.

The human VRK1 chromatin kinase in cancer biology.

VRK1 is a nuclear Ser-Thr chromatin kinase that does not mutate in cancer, and is overexpressed in many types of tumors and associated with a poor prognosis. Chromatin VRK1 phosphorylates several transcription factors, including p53, histones and proteins implicated in DNA damage response pathways. In the context of cell proliferation, VRK1 regulates entry in cell cycle, chromatin condensation in G2/M, Golgi fragmentation, Cajal body dynamics and nuclear envelope assembly in mitosis. This kinase also controls the initial chromatin relaxation associated with histone acetylation, and the non-homologous-end joining (NHEJ) DNA repair pathway, which involves sequential steps such as γH2AX, NBS1 and 53BP1 foci formation, all phosphorylated by VRK1, in response to ionizing radiation or chemotherapy. In addition, VRK1 can be an alternative target for therapies based on synthetic lethality strategies. Therefore, VRK1 roles on proliferation have a pro-tumorigenic effect. Functions regulating chromatin stability and DNA damage responses have a protective anti-tumor role in normal cells, but in tumor cells can also facilitate resistance to genotoxic treatments.

VRK1 Phosphorylates Tip60/KAT5 and Is Required for H4K16 Acetylation in Response to DNA Damage.

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM-/- cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).

Olaparib and ionizing radiation trigger a cooperative DNA-damage repair response that is impaired by depletion of the VRK1 chromatin kinase.

BACKGROUND: The VRK1 chromatin kinase regulates the organization of locally altered chromatin induced by DNA damage. The combination of ionizing radiation with inhibitors of DNA damage responses increases the accumulation of DNA damage in cancer cells, which facilitates their antitumor effect, a process regulated by VRK1. METHODS: Tumor cell lines with different genetic backgrounds were treated with olaparib to determine their effect on the activation of DNA repair pathways induced by ionizing radiation. The effect of combining olaparib with depletion of the chromatin kinase VRK1 was studied in the context of double-strand breaks repair pathway after treatment with ionizing radiation. The initiation and progression of DDR were studied by specific histone acetylation, as a marker of local chromatin relaxation, and formation of γH2AX and 53BP1 foci. RESULTS: In this work, we have studied the effect that VRK1 by itself or in collaboration with olaparib, an inhibitor of PARP, has on the DNA oxidative damage induced by irradiation in order to identify its potential as a new drug target. The combination of olaparib and ionizing radiation increases DNA damage permitting a significant reduction of their respective doses to achieve a similar amount of DNA damage detected by γH2AX and 53BP1 foci. Different treatment combinations of olaparib and ionizing radiation permitted to reach the maximum level of DNA damage at lower doses of both treatments. Furthermore, we have studied the effect that depletion of the VRK1 chromatin kinase, a regulator of DDR, has on this response. VRK1 knockdown impaired all steps in the DDR induced by these treatments, which were detected by a reduction of sequential markers such as H4K16 ac, γH2AX, NBS1 and 53BP1. Moreover, this effect of VRK1 is independent of TP53 or ATM, two genes frequently mutated in cancer. CONCLUSION: The protective DNA damage response induced by ionizing radiation is impaired by the combination of olaparib with depletion of VRK1, and can be used to reduce doses of radiation and their associated toxicity. Proteins implicated in DNA damage responses are suitable targets for development of new therapeutic strategies and their combination can be an alternative form of synthetic lethality.

Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic target?

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.

VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis.

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3-Thr3ph-survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage.

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.