CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression

© The Author(s) 2021.BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI15/01471, PI18/01500, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa”, “Consejería de Educación, Junta de Castilla y León” (SA271P18), “Proyectos de Investigación del SACYL”, Spain GRS 2062/A/19, GRS 1847/A/18, GRS1653/A17,“Fundación Memoria Don Samuel Solórzano Barruso” (FS/23-2018), by grants (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Universidad de Salamanca (Programa XIII), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233) and SYNtherapy “Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukemia” (ERAPERMED2018-275); ISCIII (AC18/00093), co-funded by ERDF/ESF, “Investing in your future”. M.Q.Á. and A.E.R.V. are supported with a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”); M.H.S. holds a Sara Borrell postdoctoral contract (CD19/00222) from the Instituto de Salud Carlos III (ISCIII). C.P.C. was supported by an “Ayuda predoctoral en Oncología” (AECC) and is a recipient of a PFIS grant (FI19/00191) from Instituto de Salud Carlos III; PFIS grant and Sara Borrell postdoctoral contrat are co-founded by Fondo Social Europeo (FSE) “El Fondo Social Europeo invierte en tu futuro”; J.L.O. and R.B.S. are supported by a grant from the University of Salamanca (“Contrato postdoctoral programa II”)

CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition

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.This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI15/01471, PI18/01500, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa”, “Consejería de Educación, Junta de Castilla y León” (SA271P18), “Proyectos de Investigación del SACYL”, Spain GRS 1847/A/18, GRS1653/A17,“Fundación Memoria Don Samuel Solórzano Barruso” (FS/23-2018), by grants (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233) and SYNtherapy “Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukemia” (ERAPERMED2018-275); ISCIII (AC18/00093). MQÁ is fully supported by an “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL-EDU/529/2017 PhD scholarship); MHS was supported by a grant from FEHH/Janssen (“Sociedad Española de Hematología y Hemoterapia”) and now holds a Sara Borrell postdoctoral contract (CD19/00222) from Instituto de Salud Carlos III (ISCIII), co-funded by Fondo Social Europeo (FSE) “El Fondo Social Europeo invierte en tu futuro”; AERV is supported with a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”); MG is supported by a Marie Curie Action International Outgoing Fellowship (PIOF-2013-624924); EtH is a Special Fellow of the Leukemia and Lymphoma Society (LLS) and a Scholar of the American Society of Hematology (ASH) and JLO is supported by a grant from the University of Salamanca (“Contrato postdoctoral programa II”).Peer reviewe

Distributional extension of Peltophorus polymitus (Boheman 1845) at Durango, Mexico

Consumption of mezcal in Mexico and its international exportation is increasing. Potential pests of agave, the plant distilled for this alcoholic beverage, need to be studied. Durango is the second state in mezcal production in Mexico. It is important to contribute to the knowledge of potential pests of agaves. Peltophorus polymitus (Boheman 1845) has been historically registered as pest at three municipalities in the State of Durango. However, little is known of the biology or ecology of P. polymitus, a potential pest of the agave crop. The objective of this work was to contribute to the knowledge of the distribution of the weevil of agave (P. polymitus) in Durango. Here, the distribution of P. polymitus was extended to 13 additional municipalities in an altitudinal range from 1,104-2,094 m above sea level, across all the ecoregions of the State.</p

Comparative study of the effects of black or white hail nets on the fruit quality of ‘Golden Delicious’ apples

Introduction. The use of hail nets to protect apples during development and maturation on the tree is very common in Mexico. This practice can cause changes in fruit quality and aroma composition. The effects of the hail net color on the quality and aroma volatile production of apple cultivated in Chihuahua, Mexico, were evaluated. Materials and methods. ‘Golden Delicious’ apple trees were covered with white or black hail nets. Apple samples were harvested weekly from August to early October, and analyzed for weight, axial and equatorial diameters, color (°Hue), firmness, total soluble solids (TSS), acidity, ethylene production (EC), and aroma volatile composition. The photosynthetically available radiation (PAR) was measured every ten days under each hail net and outside. Results and discussion. Apple quality was affected by hail net color. Black hail nets delayed maturation and quality development of apples by one week when compared with white hail nets. The PAR values were 18% lower under black nets than under white nets. Quality parameters at commercial harvest (162 days after full bloom) showed that white-net apples presented 7% lower firmness, 11.1% less acidity, 8.3% higher TSS and a more developed yellow color, when compared with black-net apples. In addition, white-net apples presented higher contents of the main aroma compounds (in μg L-1) 1-hexanol (8.09 vs. 4.38), 2-methyl-1-butanol (6.24 vs. 2.65), and 2-methyl-butyl acetate (0.47 vs. 0.16). At the same maturity stage (beginning of the climacteric rise), no difference was found between white- and black-net apples in TSS, acidity, firmness and aromatic compounds. Conclusion. Hail net functionality goes beyond protecting orchards from hail damage; hail net color affects the apple maturation rate, quality and aroma volatile production

ETV6/ RUNX1 Fusion Gene Abrogation Decreases the Oncogenicity of Tumour Cells in a Preclinical Model of Acute Lymphoblastic Leukaemia

© 2020 by the authors.[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.This work was financially supported in part by a grant from the Consejería de Educación, Junta de Castilla y León, Fondos FEDER (SA085U16, SA271P18), and the Regional Council of Castilla y León SACYL, (GRS 1847/A/18, GRS 2062/A/19), SYNtherapy. Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukaemia (ERAPERMED2018-275); ISCIII (AC18/00093), co-funded by ERDF/ESF, “Investing in your future”, Fundación Castellano Leonesa de Hematología y Hemoterapia (FUCALHH 2017), Proyectos de investigación en Biomedicina, gestión sanitaria y atención sociosanitaria del IBSAL (IBY17/00006), Fundación Memoria Don Samuel Solórzano Barruso, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233).JMHS is supported by a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”), and JLO is supported by a grant from the University of Salamanca (“Contrato postdoctoral programa II 2017-18”)., and AM by a grant from the Junta Provincial de Salamanca of the Asociación Española Contra el Cáncer (AECC)

The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia

CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line. CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.This work was supported in part by a grant from the Consejería de Educación, Junta de Castilla y León, Fondos FEDER (SA085U16 to JMHR and JCYL-EDU/346/2013 PhD scholarship to MHS), the ISCIII-FEDER Spanish Cancer Network (RD12/0036/0069) and the Fondo de Investigaciones Sanitarias (FIS) of the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) “Una manera de hacer Europa” (grant PI15/01471).Peer Reviewe

Destrucción in vitro del oncogen BCR-ABL p210 mediante nucleasas de edición genómica CRISPR/Cas9

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.La caracterización molecular de las alteraciones génicas implicadas en la transformación celular así como el desarrollo de nuevas herramientas de edición genómica cambiarán radicalmente el panorama terapéutico de algunas neoplasias en los próximos años, especialmente el de aquellas asociadas a mutaciones “drivers” bien caracterizadas, como sucede en la leucemia mieloide crónica (LMC). Esta enfermedad se caracteriza por la presencia de la translocación t(9;22)(q34;q11) que genera el oncogén de fusión BCR/ABLp210 y que produce una proteína con actividad tirosinquinasa (TK) constitutiva. Esta actividad TK es crucial y se considera indispensable en el mecanismo de transformación celular al que conduce. El diseño de fármacos inhibidores de TK específicas ha constituido uno de los mayores logros terapéuticos en la medicina actual. Sin embargo, su administración es continua y prolongada en el tiempo, la acción terapéutica se limita a la anulación del efecto a nivel proteico y en ningún caso hay reparación o anulación del daño a nivel genético, lo que en ocasiones conlleva a la aparición de resistencias. Afortunadamente, la aparición de las nuevas herramientas de edición genómica, tipo CRSIPR-Cas9, solventaría todos estos inconvenientes se podrían corregir/eliminar mutaciones a nivel genómico lo que abre un nuevo panorama terapéutico. Este estudio explora el uso de esta tecnología CRSIPR-Cas9 en un modelo celular tumoral específico del oncogén p210 y demuestra su eficacia y utilidad como posible nueva herramienta terapéutica para la LMC.Financiación: HUS272413, PI15/01471 y Junta de Castilla y León.Peer reviewe