De Moerloose, Barbara

Ghazavi, Farzaneh

Lammens, Tim

Van Vlierberghe, Pieter

Ghent University Academic Bibliography

VOLUME8 JUNE20173118HEMATOTHESISF. Ghazavi, PhD1,2, T. Lammens, PhD1,3, P. Van Vlierberghe, PhD2,3, B. De Moerloose MD, PhD1,3INTRODUCTIONB-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric malignancy and the cure rate of this disease has increased enormously over the last forty years to a current five-year survival of over 90%, attributed to improved chemotherapeutic treatment schedules and risk-stratification.1 Neverthe-less, some patients still experience treatment failure after initial therapy and therefore, identification of novel biomarkers is critical to distinguish these patients at diagnosis, adapt their treatment schedules and to develop novel strategies using new therapeutic targets. Recent developments in whole genome techniques (DNA, RNA, and protein) provided detailed infor- mation concerning the molecular alterations important for tumour emergence, maintenance and progression. Several groups have demonstrated the power of high- resolution, genome-wide approaches to identify new molecular lesions in leukaemia. As such, deletion, ampli-fication, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation were identified in 40% of B-progenitor ALL cases using single nucleotide poly-morphism arrays.2-5 Novel molecular genetic insights  in paediatric B-cell precursor acute lymphoblastic leukaemiaSUMMARYPaediatric B-cell precursor acute lymphoblastic leukaemia arises from recurrent genetic lesions that block precursor B-cell differentiation and drive aberrant proliferation and cell survival. Risk-adapted intensive chemotherapy has been a major breakthrough in reaching the current survival rates of >90% for this ALL subtype. Recent developments in genome-wide genetic analysis have provided a wide range of chromosomal and genomic abnormalities characterising B-cell precursor acute lymphoblastic leukaemia, several of which are associated with patient outcome. This article summarises the results of several studies performed during the PhD thesis of Dr Farzaneh Ghazavi. This research project has led to the identification of a novel molecular lesion predicting poor outcome, a novel targetable pathway in a subgroup of B-cell precursor acute lympho-blastic leukaemia patients and resulted in the identification of an ETV6/RUNX1-specific long non-coding RNA signature providing novel biological insights into ETV6/RUNX1-mediated leukemogenesis.(BELG J HEMATOL 2017;8(3):118-21)1Department of Paediatric Haematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium, 2Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 3Cancer Research Institute Ghent, Ghent, Belgium.Please send all correspondence to: T. Lammens, PhD, Ghent University Hospital, Department of Paediatric Haematology-Oncology and Stem Cell Transplantation, 3K12D, De Pintelaan 185, B-9000 Ghent, Belgium, tel: 0032 9 332 24 80, fax: 0032 9 332 38 75, email: tim.lammens@ugent.be.Conflict of interest: The authors have nothing to disclose and indicate no potential conflict of interest.Keywords: CD200/BTLA deletions, ETV6/RUNX1, long non-coding RNA, PI3K/AKT/mTOR signalling pathway, precursor B acute lymphoblastic leukaemia.Acknowledgment: This PhD thesis was supported in part by a grant from the Belgian Foundation Against Cancer (grant 2010-187), the Cancer Plan – Action 29 from the Belgian Federal Public Service of Health, and Kinderkankerfonds (a non-profit childhood cancer foundation under Belgian law).VOLUME8 JUNE2017119This paper discusses the results obtained through studying the genome, long non-coding repertoire and proteome during the PhD thesis of Dr Farzaneh Ghazavi and provides future perspectives.IDENTIFICATION OF CD200/BTLA DELETIONS IN PAEDIATRIC BCP-ALL TREATED ACCORDING TO THE  EORTC-CLG 58951 PROTOCOLThe advent of high-resolution, microarray-based tech-niques such as array-based comparative genomic hybridi-sation (CGH) has enabled the identification of multiple novel DNA copy number alterations targeting key cel-lular pathways in ALL. Some of the identified genetic lesions are clinically relevant and can be integrated into the risk classification of the ALL patients, as recently reviewed by Ghazavi et al.2-5 Nevertheless; novel prog-nostic markers need to be discovered, as illustrated by the observation that a considerable number of relapses remain molecularly unexplained. To identify new genetic lesions with prognostic relevance in BCP-ALL, we performed copy number profiling on leukemic blasts of BCP-ALL patients treated with the EORTC-CLG 58951 protocol. This analysis revealed a recurrent deletion of a genomic region (~164kb) at chr3q13.2, encompassing the lymphoid signalling mole-cules BTLA (CD272) and CD200 genes, in 10% (7 out of 70) of the initial group and 4.8% (56 out of 1,154) of an extended cohort of BCP-ALL patients.6 Mapping the exact breakpoint of CD200/BTLA deletions in primary leukaemia genomes demonstrated that the deletions mostly affected the whole genomic sequence of both CD200 and BTLA genes and that the deletions were likely mediated by abnormal RAG activity, as evidenced by the RAG recognition sequence motif adjacent to the genomic breakpoints. In line with recent reports that identified aberrant RAG recombinase activity as one of the main mechanisms that drive the generation of cooperative genomic lesions in ETV6/RUNX1-positive BCP-ALL, we found that CD200/BTLA deletions were strongly associated with t(12;21) positive leukaemia (p<0.0001), suggesting a genetic interaction between loss of CD200/BTLA and expression of ETV6/RUNX1 during leukemic transformation.7 The presence of CD200/BTLA deletions did not affect the favourable prognosis of ETV6/RUNX1-positive BCP-ALLs. However, and most notably, CD200/BTLA deletions were associated with inferior event-free survival, both in the complete patient population (HR 2.02; 95% CI 1.23-3.32; p=0.005) as well as in the intermediate genetic risk group (HR 4; 99% CI 1.34-11.93; p<0.001). In addition, multivariate analysis confirmed the independent prognostic value of CD200/BTLA deletion in the whole cohort of BCP-ALLs. Altogether, these data suggest the added value of CD200/BTLA status in the risk stratification algorithm of BCP-ALL patients.RPPA-BASED PROTEIN PROFILING IN ETV6/RUNX1-POSITIVE ACUTE LYMPHOBLASTIC LEUKAEMIA PATIENTS WITH LOW CD200 EXPRESSIONIdentification of altered signalling pathways is of utmost importance in cancer therapy since many of these pathways are targetable and thus a potential bull’s eye for increasing treatment efficiency.8-10 We focused on the further elucidation of the signalling pathways affected by the recurrent deletion of CD200 and BTLA, which was previously identified.6 Importantly, both CD200 and BTLA genes, affected by the deletion, encode lymphoid signalling molecules and have been shown to inhibit activation of B-cell receptor (BCR) and MAPK/PI3K signalling, respectively, which are both essential signalling pathways for B-cell proliferation and maturation.11,12 In addition, activation of BTLA upon interaction with its ligand HVEM, attenuates B-cell proliferation as a result of reduced activation of signalling molecules downstream of BCR. Moreover, crosstalk between CD200 and its receptor CD200R has been shown to mediate the inhibition of Ras/MAPK sig- nalling pathway through recruitment of SHIP and/or RasGAP and subsequently deactivation of signalling molecules such as ERK, JNK, and p38 MAPK.12To further explore the functional consequences of reduced CD200 expression on the activity of downstream signalling pathways, reverse phase protein microarray (RPPA) analyses was applied (Ghazavi et al., in prepara-tion). Using this technique we determined the expres-sion and phosphorylation level of 87 signalling molecules in 72 BCP-ALL patients’ specimens. Interestingly, diffe-rential expression analysis of proteins/phosphoproteins between patients with low and high expression of CD200 revealed significant upregulation and hyper- phosphorylation of proteins involved in PI3K/AKT/mTOR signalling pathway in patients with low CD200 expression. Notably, this pathway is an essential regu-lator of many cellular processes such as protein syn- thesis, proliferation and apoptosis and its constitutive activation in many forms of malignancies attests its pathogenetic importance.13,14 Finally, we evaluated the potential of PI-103, a novel dual inhibitor of PI3Kalpha VOLUME8 JUNE20173120and mTOR to inhibit growth in CD200 low cells.15,16 A 72h treatment of REH cells, a cell line carrying a deletion of CD200, showed that PI-103 treatment sig-nificantly reduces the phosphorylation levels of AKT, c-RAF and eIF4G and affects survival.UNIQUE LONG NON-CODING RNA EXPRESSION SIGNATURE IN ETV6/RUNX1-DRIVEN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKAEMIAMammalian genomes comprise ~ 20000 protein- coding genes, only representing <2% of the total genome sequence, whereas more than 90% of the genome is actively transcribed into non-coding RNA (ncRNA). The latter group of RNAs consists of small structural and regulatory RNAs (including rRNAs, tRNAs, snoRNAs and microRNAs) and long non-coding RNAs (lncRNAs, >200 nucleotides).17,18 More than 10,000 lncRNAs have been annotated so far in the human genome sequence.19,20 Functional studies, although limited, revealed that lncRNAs regulate many cellular processes including chromatin modification, transcriptional and post-trans- criptional regulation, mRNA degradation and trans- lation, and trafficking of molecules across the nuclear membrane.21-26 Different studies have recently shown that aberrant expression of non-coding RNAs such as long non-coding RNAs (lncRNA) might contribute to the development and progression of leukaemia.27,28 Identification of disease-associated lncRNAs would not only provide new insights into our understanding of disease mechanisms but might also reveal new prog-nostic biomarkers or novel therapeutic targets for the treatment of diseases. Recently, we established an ETV6/RUNX1-specific lncRNA signature by performing expression profiling microarray in a set of 64 primary BCP-ALL patients including 25 ETV6/RUNX1-positive, seven TCF3/PBX1- positive, fifteen hyperdiploid and seventeen normal karyotype BCP-ALLs.29 The integration of these lncRNA expression data with RNA sequencing results from a panel of thirteen human BCP-ALL leukaemia cell lines, resulted in the identification of sixteen lncRNAs exclu-sively associated with the presence of the ETV6/RUNX1 fusion protein. Complemented with shRNA-mediated silencing of endogenous ETV6/RUNX1 in an ETV6/RUNX1-positive BCP-ALL cell line, the list of ETV6/RUNX1 associated lncRNAs was narrowed down from sixteen to four lncRNAs including lnc-NKX2-3-1, lnc-TIMM21-5, lnc- ASTN1-1, and lnc-RTN4R-1. Finally, in vitro knockdown using LNA™ GapmeR tech-nology suggested that lnc-TIMM21-5 and lnc-ASTN1-1 are probably not involved in transcriptional regulation but, in contrast, lnc-NKX2-3-1 and lnc-RTN4R-1 perturba-tions resulted in a severe effect on overall transcription. CONCLUSIONAltogether, the data obtained during this PhD thesis have contributed to the identification of a novel prog-nostic factor - CD200/BTLA deletion - in BCP-ALL, the elucidation of the signalling pathways downstream of these molecules and delivered fundamental proof that lncRNAs play an important role in distinct subtypes of BCP-ALL.It will be essential to validate the prognostic power of CD200/BTLA deletions in an independent cohort. Since our results are clinically relevant, the biotech company MRC Holland (www.mrcholland.com) has decided to include this alteration in their MLPA assays used by many centres for diagnostic work-up of paediatric ALL. The identification of hyperactive PI3K/AKT/mTOR signalling pathway in BCP-ALLs with low CD200 expression provides a rationale for future experiments using inhibitors of PI3K/mTOR and translation path-HEMATOTHESISKEY MESSAGES FOR CLINICAL PRACTICE1 CD200/BTLA deletions are associated with poor prognosis in BCP-ALL.2 PI3K/AKT/mTOR signalling pathway might serve as a targetable pathway for treatment of BCP-ALL  patients with low CD200 expression.3 lncRNA expression profiling might reveal new prognostic biomarkers and novel therapeutic targets for the treatment of BCP-ALL patients.VOLUME8 JUNE2017121ways in this subgroup of patients. Although our in vitro experiments look very promising, it will be essential to further confirm these findings in vivo using xenograft models of primary patient material.Finally, the data generated in the last part of the study identified a panel of ETV6/RUNX1-specific lncRNAs that might be implicated in the biology of human BCP-ALL and could serve as novel therapeutic targets for the treatment of this prevalent subtype of human leukaemia. Further functional studies of the identified lncRNAs, including the identification of binding partners, should be performed in order to bring these findings to information impacting on the clinical management of patients.REFERENCES1. Hunger SP, Mullighan CG. Acute Lymphoblastic Leukemia in Children. N Engl J Med 2015;373(16):1541-52.2. Ghazavi F, Lammens T, Van Roy N, et al. Molecular basis and clinical signifi-cance of genetic aberrations in B-cell precursor acute lymphoblastic leukemia. Exp Hematol 2015;43(8):640-53.3. Kuiper RP, Schoenmakers EF, van Reijmersdal SV, et al. High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression. Leukemia 2007;21(6):1258-66.4. Moorman AV, Robinson H, Schwab C, et al. Risk-directed treatment intensifi-cation significantly reduces the risk of relapse among children and adolescents with acute lymphoblastic leukemia and intrachromosomal amplification of chromo-some 21: a comparison of the MRC ALL97/99 and UKALL2003 trials. J Clin Oncol 2013;31(27):3389-96.5. Mullighan CG, Su X, Zhang J, et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med 2009;360(5):470-80. 6. Ghazavi F, Clappier E, Lammens T, et al. CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol. Haematologica 2015;100(10):1311-9.7. Papaemmanuil E, Rapado I, Li Y, et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nat Genet 2014;46(2):116-25. 8. Jones S, Zhang X, Parsons DW, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008; 321(5897):1801-6. 9. Cancer Genome Atlas Research Network. Comprehensive genomic charac-terization defines human glioblastoma genes and core pathways. Nature 2008; 455(7216):1061-8. 10. Accordi B, Espina V, Giordan M, et al. Functional protein network activation mapping reveals new potential molecular drug targets for poor prognosis pediatric BCP-ALL. PLoS One 2010;5(10):e13552. 11. Vendel AC, Calemine-Fenaux J, Izrael-Tomasevic A, et al. B and T Lymphocyte Attenuator Regulates B Cell Receptor Signaling by Targeting Syk and BLNK. J Immunol 2009;182(3):1509-17. 12. Zhang SL, Cherwinski H, Sedgwick JD, et al. Molecular mechanisms of CD200 inhibition of mast cell activation. 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Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009;458(7235):223-7.20. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annu Rev Biochem 2012;81(1):145-66.21. Pandey RR, Mondal T, Mohammad F, et al. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 2008;32(2):232-46.22. Nagano T, Mitchell JA, Sanz LA, et al. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 2008;322(5908): 1717-20.23. Martianov I, Ramadass A, Serra Barros A, et al. Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature 2007;445(7128):666-70.24. Tripathi V, Ellis JD, Shen Z, et al. The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 2010;39(6):925-38.25. Carrieri C, Cimatti L, Biagioli M, et al. Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 2012;491(7424):454-7.26. Bertozzi D, Iurlaro R, Sordet O, et al. Characterization of novel antisense HIF-1α transcripts in human cancers. Cell Cycle 2011;10(18):3189-97.27. Fabbri M, Garzon R, Andreeff M, et al. MicroRNAs and noncoding RNAs in haematological malignancies: molecular, clinical and therapeutic implications. Leukemia 2008;22(6):1095-105.28. Morlando M, Ballarino M, Fatica A. Long Non-Coding RNAs: New Players in Hematopoiesis and Leukemia. Front Med (Lausanne) 2015;2:23. 29. Ghazavi F, De Moerloose B, Van Loocke W, et al. Unique long non-coding RNA expression signature in ETV6/RUNX1-driven B-cell precursor acute lymphoblastic leukemia. Oncotarget 2016;7(45):73769-80.HEMATOTHESIS

Novel molecular genetic insights in paediatric B-cell precursor acute lymphoblastic leukaemia

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Novel molecular genetic insights in paediatric B-cell precursor acute lymphoblastic leukaemia

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