RESUMEN
Combining multiple drugs broadens the window of therapeutic opportunities and is crucial for diseases that are currently lacking fully curative treatments. A powerful emerging tool for selecting effective drugs and combinations is the high-throughput drug screening (HTP). The histone deacetylase inhibitor (HDACi) givinostat (ITF2357) has been shown to act effectively against CRLF2-rearranged pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a subtype characterized by poor outcome and enriched in children with Down Syndrome, very fragile patients with a high susceptibility to treatment-related toxicity. The aim of this study is to investigate possible synergies with givinostat for these difficult-to-treat patients by performing HTP screening with a library of 174 drugs, either approved or in preclinical studies. By applying this approach to the CRLF2-r MHH-CALL-4 cell line, we identified 19 compounds with higher sensitivity in combination with givinostat compared to the single treatments. Next, the synergy between givinostat and the promising candidates was further validated in CRLF2r cell lines with a broad matrix of concentrations. The combinations with trametinib (MEKi) or venetoclax (BCL2i) were found to be the most effective and with the greatest synergy across three metrics (ZIP, HAS, Bliss). Their efficacy was confirmed in primary blasts treated ex vivo at concentration ranges with a safe profile on healthy cells. Finally, we described givinostat-induced modifications in gene expression of MAPK and BCL-2 family members, supporting the observed synergistic interactions. Overall, our study represents a model of drug repurposing strategy using HTP screening for identifying synergistic, efficient, and safe drug combinations.
RESUMEN
Although a great cure rate has been achieved for pediatric BCP-ALL, approximately 15% of patients do not respond to conventional chemotherapy and experience disease relapse. A major effort to improve the cure rates by treatment intensification would result in an undesirable increase in treatment-related toxicity and mortality, raising the need to identify novel therapeutic approaches. High-throughput (HTP) drug screening enables the profiling of patients' responses in vitro and allows the repurposing of compounds currently used for other diseases, which can be immediately available for clinical application. The aim of this study was to apply HTP drug screening to identify potentially effective compounds for the treatment of pediatric BCP-ALL patients with poor prognosis, such as patients with Down Syndrome (DS) or carrying rearrangements involving PAX5 or KMT2A/MLL genes. Patient-derived Xenografts (PDX) samples from 34 BCP-ALL patients (9 DS CRLF2r, 15 PAX5r, 10 MLLr), 7 human BCP-ALL cell lines and 14 hematopoietic healthy donor samples were screened on a semi-automated HTP drug screening platform using a 174 compound library (FDA/EMA-approved or in preclinical studies). We identified 9 compounds active against BCP-ALL (ABT-199/venetoclax, AUY922/luminespib, dexamethasone, EC144, JQ1, NVP-HSP990, paclitaxel, PF-04929113 and vincristine), but sparing normal cells. Ex vivo validations confirmed that the BCL2 inhibitor venetoclax exerts an anti-leukemic effect against all three ALL subgroups at nanomolar concentrations. Overall, this study points out the benefit of HTP screening application for drug repurposing to allow the identification of effective and clinically translatable therapeutic agents for difficult-to-treat childhood BCP-ALL subgroups.
Asunto(s)
Reposicionamiento de Medicamentos , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Niño , Ensayos Analíticos de Alto Rendimiento , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéuticoRESUMEN
Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.