– State-of-the-art and rationale. Acute lymphoblastic leukemia (ALL) is a malignant proliferation of lymphoid cells blocked at an early stage of differentiation (1). This malignant disease is the most common childhood cancer and most ALL cases derive from B-cell precursors (B-ALL) (2). Current treatments are based on cytotoxic chemotherapy or, in most severe cases, bone marrow transplantation. Fortunately, new targeted therapies, including CAR-T cells, have been recently developed for the treatment of ALL. Nevertheless, a non-negligible number of patients relapse or develop adverse toxicities. Therefore, the search for novel and effective therapies to enhance patient quality of life and survival remains a major challenge.

Over the last years, epigenetic mechanisms have been extensively studied in the context of cancer initiation and progression (3). In this sense, both aberrant DNA methylation and dysregulation of histone modifiers have been implicated in the pathogenesis of ALL (4). Moreover, recent studies have demonstrated that epigenetics-based silencing is behind the resistance of leukemic blasts to the apoptotic effect of chemotherapy (5).

Given the dynamic and reversible nature of epigenetic marks and their proved significant role in the onset and progression of cancer, epigenetic drugs targeting histones modifiers or hypomethylating agents are showing promising results and offer the possibility of being used as combined therapy to potentiate chemotherapy or immunotherapy (6). Thus, a better understanding of histone modification mechanisms and the identification of novel therapeutic targets will eventually set up the basis for novel discoveries, contribute to guide clinical decision-making and advance personalized medicine for pediatric ALL patients.

– Research aims and scientific approach. Despite the advances in the treatment of B-ALL (1), some pediatric patients develop resistance and relapse. Because of that, further studies are still needed to understand the mechanisms by which tumor cells escape chemotherapy and immunotherapy.

Using available DNA methylation data, the Cancer Epigenetics team has evaluated the methylation status of regulatory regions of 435 genes encoding for histone modifiers and identified several candidate genes susceptible of being dysregulated in cancer. Based on this information, we have defined specific objectives that have been included in the following work packages (WPs):

WP1. Study the methylation status of histone modifiers-related genes in pediatric B-ALL samples. To assess the methylation status of these 435 genes encoding for histone modifier proteins in pediatric B-ALL and identify 1) aberrant methylation patterns in particular genes and 2) patient subgroups with different methylation levels for such genes, we will analyze the DNA methylation profile of B-ALL patients with the Infinium MethylationEPIC microarrays. Additionally, we will use B-ALL cell lines to perform further validation studies.

WP2. Determine the link between gene methylation and patients’ outcome. Clinical and biological data will be collected to evaluate the link between selected altered genes and patient characteristics and outcome. We ambitious to identify biomarkers of disease subgroups, biomarkers of response to therapy and biomarkers of overall outcome. To assess whether the methylation of such genes correlates with patient prognosis, we will perform survival analysis splitting the patients in two groups based on their methylation status.

WP3. Investigate the molecular relevance and impact of altered members of the histone modification machinery in B-ALL models. In order to better understand the role of selected genes and their association with patients’ characteristics/outcome, we will generate overexpression and knockout/knockdown models of the corresponding proteins on B-ALL cell lines. These models will be used to further study functional and molecular changes in cell viability, proliferation, apoptosis and cell cycle, among others.

WP4. Interrogate the use of histone modifiers inhibitors and their combination for the treatment of B-ALL in vitro. We will test the efficiency of histone modifier inhibitors on the previously generated B-ALL models. To do so, we will treat cell lines with increasing concentrations of epigenetic inhibitors (separately or in combination) to calculate their corresponding IC50 values and determine their synergic effect.

To sum up, the final goal of this proposal is to identify the epigenetic alterations that are interfering with the expression of histone modifiers and could explain therapeutic failure in pediatric B-ALL patients.

– Bibliography. 1. Malard F, Mohty M. Lancet. 2020 | 2. Studd JB, Cornish AJ, Hoang PH, Law P, Kinnersley B, Houlston R. Blood Cancer J. 2021 | 3. Hanahan D. Cancer Discov. 2022 | 4. Burke MJ, Bhatla T. Front Pediatr. 2014 | 5. Alsagaby SA. Oncol Rev. 2019 | 6. Bates SE. N Engl J Med. 2020.

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