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Loss of Pax5 Exploits Sca1-BCR-Ablp190 Susceptibility to Confer the Metabolic Shift Essential for Pb-ALL Alberto Martín-Lorenzo1,2, Franziska Auer3,4, Lai N

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Loss of Pax5 Exploits Sca1-BCR-Ablp190 Susceptibility to Confer the Metabolic Shift Essential for Pb-ALL Alberto Martín-Lorenzo1,2, Franziska Auer3,4, Lai N Published OnlineFirst February 28, 2018; DOI: 10.1158/0008-5472.CAN-17-3262 Cancer Tumor Biology and Immunology Research Loss of Pax5 Exploits Sca1-BCR-ABLp190 Susceptibility to Confer the Metabolic Shift Essential for pB-ALL Alberto Martín-Lorenzo1,2, Franziska Auer3,4, Lai N. Chan3, Idoia García-Ramírez1,2, Ines Gonzalez-Herrero 1,2, Guillermo Rodríguez-Hernandez 1,2, Christoph Bartenhagen5, Martin Dugas5, Michael Gombert4, Sebastian Ginzel4, Oscar Blanco6, Alberto Orfao7, Diego Alonso-Lopez 8, Javier De Las Rivas2,9, Maria B. García-Cenador10, Francisco J. García-Criado10, Markus Muschen€ 3, Isidro Sanchez-García 1,2, Arndt Borkhardt5, Carolina Vicente-Duenas~ 1,2, and Julia Hauer5 Abstract p190 Preleukemic clones carrying BCR-ABL oncogenic lesions are the Pax5-deficient leukemic pro-B cells exhibited a metabolic found in neonatal cord blood, where the majority of preleukemic switch toward increased glucose utilization and energy carriers do not convert into precursor B-cell acute lymphoblastic metabolism. Transcriptome analysis revealed that metabolic leukemia (pB-ALL). However, the critical question of how these genes (IDH1, G6PC3, GAPDH, PGK1, MYC, ENO1, ACO1) were preleukemic cells transform into pB-ALL remains undefined. upregulated in Pax5-deficient leukemic cells, and a similar Here, we model a BCR-ABLp190 preleukemic state and show metabolic signature could be observed in human leukemia. Our that limiting BCR-ABLp190 expression to hematopoietic stem/ studies unveil the first in vivo evidence that the combination progenitor cells (HS/PC) in mice (Sca1-BCR-ABLp190) causes between Sca1-BCR-ABLp190 and metabolic reprogramming pB-ALL at low penetrance, which resembles the human disease. imposed by reduced Pax5 expression is sufficient for pB-ALL pB-ALL blast cells were BCR-ABL–negative and transcriptionally development. These findings might help to prevent conversion similar to pro-B/pre-B cells, suggesting disease onset upon of BCR-ABLp190 preleukemic cells. reduced Pax5 functionality. Consistent with this, double Significance: Loss of Pax5 drives metabolic reprogramming, þ À Sca1-BCR-ABLp190þPax5 / mice developed pB-ALL with shorter which together with Sca1-restricted BCR-ABL expression latencies, 90% incidence, and accumulation of genomic altera- enables leukemic transformation. Cancer Res; 78(10); 2669–79. tions in the remaining wild-type Pax5 allele. Mechanistically, Ó2018 AACR. 1Experimental Therapeutics and Translational Oncology Program, Instituto de Introduction Biología Molecular y Celular del Cancer, CSIC/Universidad de Salamanca, Campus M. de Unamuno s/n, Salamanca, Spain. 2Institute of Biomedical Although preleukemic clones carrying BCR-ABLp190 onco- Research of Salamanca (IBSAL), Salamanca, Spain. 3Department of Systems genic lesions are frequentlyfoundinneonatalcordblood 4 Biology, Beckman Research Institute, Monrovia, California. Department of (1, 2), they often remain silent, because the majority of these Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine Uni- carriers do not develop precursor B-cell acute lymphoblastic versity Dusseldorf, Medical Faculty, Dusseldorf, Germany. 5Institute of Medical Informatics, University of Muenster, Muenster, Germany. 6Departamento de leukemia (pB-ALL). In addition, small fractions of normal B Anatomía Patologica, Universidad de Salamanca, Salamanca, Spain. 7Servicio de cells in healthy adults carry silent BCR-ABL oncogenes (3). Citometría and Departamento de Medicina, Universidad de Salamanca, Sala- These findings suggest that BCR-ABLp190 might promote manca, Spain. 8Bioinformatics Unit, Cancer Research Center (CSIC-USAL) leukemogenesis by creating an aberrant progenitor compart- Salamanca, Spain. 9Bioinformatics and Functional Genomics Research Group, 10 ment that is susceptible to malignant transformation through Cancer Research Center (CSIC-USAL), Salamanca, Spain. Departamento de accumulation of additional secondary hits, which act as drivers Cirugía, Universidad de Salamanca, Salamanca, Spain. p190 of leukemogenesis. Thus, BCR-ABL does not seem to be a Note: Supplementary data for this article are available at Cancer Research dominant oncogene within the natural cellular hematopoietic Online (http://cancerres.aacrjournals.org/). stem/progenitor cell (HS/PC) compartment where the Corrected online July 19, 2018. BCR-ABLp190 oncogenic lesion takes place. This is further A. Martín-Lorenzo, F. Auer, and L.N. Chan are co-first authors of this article. supported by both clinical data showing that BCR-ABLp190- Corresponding Author: Julia Hauer, Heinrich-Heine University Dusseldorf, induced tumorigenesis is not reversible through the unique Moorenstr. 5, Duesseldorf 40225, Germany. Phone: 4902-1181-17680; Fax: inactivation of the gene defect initiating leukemia development 4902-1181-16707; E-mail: [email protected]; M. Muschen,€ (4), and by murine in vivo data showing that suppression of E-mail: [email protected]; I. Sanchez-García, [email protected]; A. Borkhardt, BCR-ABLp190 in leukemic mice carrying a tetracycline-repress- ~ E-mail: [email protected]; and C. Vicente-Duenas, ible BCR-ABLp190 transgene did not rescue the malignant phe- E-mail: [email protected] notype (5). However, the mode of action that BCR-ABLp190 doi: 10.1158/0008-5472.CAN-17-3262 exerts in the HS/PC compartment, remains difficult to demon- Ó2018 American Association for Cancer Research. strate using available models of transgenic-driven BCR-ABL ALL www.aacrjournals.org 2669 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst February 28, 2018; DOI: 10.1158/0008-5472.CAN-17-3262 Martín-Lorenzo et al. (6, 7), because the penetrance of most of the respective pB-ALL mic DNA was removed by DNAase treatment (Roche, 04 716 728 disease models is 100%. Thus, current available models are not 001). Real-time PCR reactions were performed in an Eppendorf able to mimic the human scenario, where the presence of the MasterCycler Realplex machine. BCR-ABL transgene in HS/PCs does not necessarily lead to Commercially available assays for quantitative PCR from disease development, but mainly generates a susceptibility that IDT (Integrated DNA Technologies) were used: Hk2 (Assay ID: is preserved in the cancer-initiating cells. In this work, we Mm.PT.S832698746), Ldha (Assay ID: Mm.PT.58.29860774), explored a novel mode of action of BCR-ABLp190 in HS/PCs, Slc2a3 (Assay ID: Mm.PT.30464830), Idh1 (Assay ID: Mm. p190 utilizing mice with restricted expression of the BCR-ABL PT.58.5996441), Pgk1 (Assay ID: Mm.PT.39a.22214854), and oncogene to the stem/progenitor cell compartment. RplpO (Assay ID: Mm.PT.58.43894205). Probes were specif- ically designed to prevent detection of genomic DNA by PCR. Materials and Methods Measurement of RplpO gene product expression was used as an endogenous control. All samples were run in triplicate. Generation of Sca1-BCR-ABLp190 and Sca1-BCR- þ À The comparative CT Method (DDCt) was used to calculate ABLp190þPax5 / þ/À relative expression of the transcript of interest and a positive The heterozygous Pax5 mice (8) have been described pre- þ/À control. viously. Heterozygous Pax5 mice were bred to Sca1-TK-IRES- BCR-ABLp190 mice to generate compound heterozygotes. The Sca1-TK-IRES-BCR-ABLp190 vector was generated as follows. The Microarray data and enrichment analysis 9 kb EcoRI-EcoRI TK-IRES-BCR-ABLp190 cassette was inserted into Total RNA was isolated in two steps using Trizol (Life Tech- the ClaI site of the pLy6 vector (9), resulting in Sca1-TK-IRES-BCR- nologies), followed by RNeasy Mini Kit (Qiagen) purification ABLp190 vector. The transgene fragment was excised from its vector following the manufacturer's RNA Clean-up protocol with the by restriction digestion with NotI, purified for injection (2 ng/mL) optional On-column DNase treatment. Integrity and quality of and injected into CBAxC57BL/6J fertilized eggs. Transgenic mice the RNA were verified by electrophoresis. Samples were analyzed were identified by Southern analysis of tail snip DNA after EcoRI using Affymetrix Mouse Gene 1.0 ST Arrays. digestion. Human ABL cDNA was used for detection of the The data discussed in this publication have been deposited in transgene. Two founder lines were obtained for the Sca1-BCR- NCBI's Gene Expression Omnibus (11) and are accessible p190 ABL transgene with different positional integration of the through GEO Series accession number GSE85600. transgenic construct in both lines. All animal work has been Differentially expressed genes were tested for enrichment conducted according to relevant national and international guide- using gene set enrichment analysis (GSEA) from MSigDB lines and it has been approved by the Bioethics Committee of (http://www.broad.mit.edu/gsea/; ref. 12). Gene expression University of Salamanca and by the Bioethics Subcommittee of signatures, specifically up or downregulated in human B-ALL Consejo Superior de Investigaciones Cientificas (CSIC). For all gene sets (13, 14), were assessed for their overlap with up or genotypes both male and female mice of a mixed C57BL/6 Â CBA downregulated genes within tumor specimen, using GSEA background were included in the study. We systematically used enrichment analysis. Moreover enrichment analysis for the littermates of the same breeding. Upon signs of disease, mice were specific normal murine B-cell stage signatures (15), BCR-ABL sacrificed and subjected to standard necropsy procedures. All signature (16), BCR-ABL target
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