José Luis Ordóñez

Germline heterozygous variants in RUNX1 lead to Familial Platelet Disorder with Myeloid Leukemia Predisposition (FPD/AML). Cellular and/or animal models are helpful to uncovering the role of a variant in disease progression. Twenty-five mice per genotype (RUNX1^WT/WT, RUNX1^WT/L43S, RUNX1^L43S/L43S), previously generated by CRISPR/Cas9, and nine sub-lethally irradiated mice per genotype were investigated. Peripheral blood (PB), bone marrow (BM), and spleen samples were analyzed by flow cytometry and histopathology. Deregulated genes were analyzed by RNA-seq in BM. An aberrant myeloid Mac1⁺Sca1⁺ckit⁻ population in the PB, BM, and spleen of two homozygous and one heterozygous mouse was observed, as well as BM hypercellularity. No Mac1⁺Sca1⁺ckit⁻ cells were detected in any RUNX1^WT/WT mice. Moreover, the spleen of both homozygous mice showed destruction of the white/red pulp and the presence of apoptotic cells. The aberrant population was also detected in four irradiated mice, two heterozygous and two homozygous, in their PB, BM, and spleen. RNA-seq studies showed 698 genes significantly deregulated in the three non-irradiated Mac1⁺Sca1⁺ckit⁻ mice vs. six healthy mice, highlighting the alteration of genes involved in apoptosis and DNA repair. These results indicate that the homozygous form of the variant p.Leu43Ser may contribute to the pathogenesis of aberrant cells.

Renal vasculature, which is highly innervated by sympathetic fibers, contributes to cardiovascular homeostasis. This renal sympathetic outflow is inhibited by 5-HT in normoglycaemic rats. Considering that diabetes induces cardiovascular complications, we aimed to determine whether diabetic state modifies noradrenergic input at renal level and its serotonergic modulation in rats. Alloxan diabetic rats were anaesthetized (pentobarbital; 60 mg/kg i.p.) and prepared for in situ autoperfusion of the left kidney to continuously measure systemic blood pressure (SBP), heart rate (HR), and renal perfusion pressure (RPP). Electrical stimulation of renal sympathetic outflow induces frequency-dependent increases (Δ) in RPP (23.9 ± 2.1, 59.5 ± 1.9, and 80.5 ± 3.5 mm Hg at 2, 4, and 6 Hz, respectively), which were higher than in normoglycaemic rats, without modifying HR or SBP. Intraarterial bolus of 5-HT and 5-CT (5-HT_1/5/7 agonist) reduced electrically induced ΔRPP. Only L-694,247 (5-HT_1D agonist) reproduced 5-CT inhibition on sympathetic-induced vasoconstrictions, whereas it did not modify exogenous noradrenaline-induced ΔRPP. 5-CT inhibition was exclusively abolished by i.v. bolus of LY310762 (5-HT_1D antagonist). An inhibitor of guanylyl cyclase, ODQ (i.v.), completely reversed the L-694,247 inhibitory effect. In conclusion, diabetes induces an enhancement in sympathetic-induced vasopressor responses at the renal level. Prejunctional 5-HT_1D receptors, via the nitric oxide pathway, inhibit noradrenergic-induced vasoconstrictions in diabetic rats.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we have developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR-ABL1-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the on-cogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative expression analyses showed significantly higher level of expression of the BCR-Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study is a proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Background: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high‐risk alterations such as del(11q)/ATM‐mutations and del(17p)/TP53‐mutations have not been established.Methods: We integrated next‐generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response.Results: Targeted sequencing analysis of the co‐occurrence of high‐risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co‐occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9‐edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition.Conclusions: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

Background: The t(12;21)(p13;q22), which fuses ETV6 and RUNX1 genes, is the most common genetic abnormality in children with B-cell precursor acute lymphoblastic leukaemia. The implication of the fusion protein in leukemogenesis seems to be clear. However, its role in the maintenance of the disease continues to be controversial. Methods: Generation of an in vitro ETV6/RUNX1 knock out model using the CRISPR/Cas9 gene editing system. Functional characterization by RNA sequencing, proliferation assays, apoptosis and pharmacologic studies, and generation of edited-cell xenograft model. Results: The expression of ETV6/RUNX1 fusion gene was completely eliminated, thus generating a powerful model on which to study the role of the fusion gene in leukemic cells. The loss of fusion gene expression led to the deregulation of biological processes affecting survival such as apoptosis resistance and cell proliferation capacity. Tumour cells showed higher levels of apoptosis, lower proliferation rate and a greater sensitivity to PI3K inhibitors in vitro along as a decrease in tumour growth in xenografts models after ETV6/RUNX1 fusion gene abrogation. Conclusions: ETV6/RUNX1 fusion protein seems to play an important role in the maintenance of the leukemic phenotype and could thus become a potential therapeutic target.

Background: The t(12;21)(p13;q22), which fuses ETV6 and RUNX1 genes, is the most common genetic abnormality in children with B-cell precursor acute lymphoblastic leukaemia. The implication of the fusion protein in leukaemogenesis seems to be clear. However, its role in the maintenance of the disease continues to be controversial. Aim: To eliminate the expression of the ETV6/RUNX1 fusion gene, in order to elucidate the effect in the leukaemic cells. Methods: Generation of an in vitro ETV6/RUNX1 knock out model using the genetic modification system CRISPR/Cas9. Functional studies and generation of edited-cell xenograft model were carried out. Results: For the first time, the expression of ETV6/RUNX1 fusion gene was completely eliminated, thus generating a powerful model on which to study the role of the fusion gene in leukaemic cells. ETV6/RUNX1 inactivation caused the deregulation of cellular processes that could be participating in the maintenance of the leukaemic phenotype, such as differentiation and lymphoid activation, apoptosis, cell signaling and cell migration. Tumour cells showed higher levels of apoptosis, lower proliferation rate and a greater sensitivity to PI3K inhibitors in vitro along as a decrease in tumour growth in xenografts models after ETV6/RUNX1 fusion gene abrogation. Conclusions: ETV6/RUNX1 fusion protein plays a fundamental role in the maintenance of the leukaemic phenotype, thereby being making the fusion protein a potential therapeutic target.

Ewing Sarcoma is a developmental tumor characterized by balanced chromosomal translocations and formation of new fusion genes, which are the main hallmark of this rare entity. Despite the vast knowledge regarding the molecular aspects of this rare malignancy obtained in the last few years, including the discovery of new therapeutic targets, many questions still remain open. In this review we focus on the research on targeted therapies in this malignancy, and discussed some bottlenecks related to this such as the possible role of pathologists, the availability of samples, the lack of appropriate animal models, and the resources needed to carry out preclinical and clinical research.