Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

A common problem in the study of human malignancy is the elucidation of cancer driver mechanisms associated with recurrent deletion of regions containing multiple genes. Taking B-cell acute lymphoblastic leukaemia (B-ALL) and large deletions of 6q [del(6q)] as a model, we integrated analysis of functional cDNA clone tracking assays with patient genomic and transcriptomic data, to identify the transcription factors FOXO3 and PRDM1 as candidate tumour suppressor genes (TSG). Analysis of cell cycle and transcriptomic changes following overexpression of FOXO3 or PRDM1 indicated that they co-operate to promote cell cycle exit at the pre-B cell stage. FOXO1 abnormalities are absent in B-ALL, but like FOXO3 , FOXO1 expression suppressed growth of TCF3::PBX1 and ETV6::RUNX1 B-ALL in-vitro. While both FOXOs induced PRDM1 and other genes contributing to late pre-B cell development, FOXO1 alone induced the key transcription factor, IRF4 , and chemokine, CXCR4 . CRISPR-Cas9 screening identified FOXO3 as a TSG, while FOXO1 emerged as essential for B-ALL growth. We relate this FOXO3-specific leukaemia-protective role to suppression of glycolysis based on integrated analysis of CRISPR-data and gene sets induced or suppressed by FOXO1 and FOXO3. Pan-FOXO agonist Selinexor induced the glycolysis inhibitor TXNIP and suppressed B-ALL growth at low dose (ID 50 < 50 nM).

Being able to clinically target the interaction of leukaemic cells with their microenvironment has remained a key therapeutic obstacle. Leukaemia cells re-program their microenvironment to provide support and protection from standard chemotherapy, molecularly targeted therapies as well as immunotherapy. To address this challenge, we have developed experimentally accessible human induced pluripotent stem cell engineered (iPSC) niches ex vivo to reveal insights into druggable cancer-niche interactions. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells support ex vivo proliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. Mesenchymal stem cells protected both cycling and non-cycling blasts against dexamethasone treatment while vascular niche-like cells only protected dormant cells. Growth support and protection from dexamethasone induced-apoptosis was dependent on direct cell-cell contact and was mediated by N-cadherin (CDH2). To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft acute lymphoblastic leukaemia mouse model. ADH-1 showed high in vivo efficacy and combination therapy with ADH-1 and dexamethasone was superior compared with dexamethasone alone. These findings provide a proof-of-concept starting point to develop novel and potentially safer anti-cancer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies.

Leukemia cells re-program their microenvironment to augment blast proliferation and enhance treatment resistance. Means of clinically targeting such niche-driven treatment resistance remain ambiguous. We develop human induced pluripotent stem cell (hiPSC)-engineered niches to reveal druggable cancer-niche dependencies. We reveal that mesenchymal (iMSC) and vascular niche-like (iANG) hiPSC-derived cells support ex vivo proliferation of patient-derived leukemia cells, affect dormancy, and mediate treatment resistance. iMSCs protect dormant and cycling blasts against dexamethasone, while iANGs protect only dormant blasts. Leukemia proliferation and protection from dexamethasone-induced apoptosis is dependent on cancer-niche interactions mediated by CDH2. Consequently, we test CDH2 antagonist ADH-1 (previously in Phase I/II trials for solid tumors) in a very aggressive patient-derived xenograft leukemia mouse model. ADH-1 shows high in vivo efficacy; ADH-1/dexamethasone combination is superior to dexamethasone alone, with no ADH-1-conferred additional toxicity. These findings provide a proof-of-concept starting point to develop improved, potentially safer therapeutics targeting niche-mediated cancer dependencies in blood cancers.

Pal, Deepali*, Blair, Helen, Boyd, Sophie, Angel Hanmy Sharon, Nizami, Salem., Isa, Asmida., Beckett, Melanie., Nelson, Ryan, Wilson, Aaron, Singh, Mankaran., Sankar, Shalini., Tirtakusuma, Ricky., Sirintra, Nakjang., Knill, Carly., Fuller, Andrew., McNeill, Hesta., Russell, Lisa., Schwab, Claire., Zhous, Peixun., Sinclair, Paul., Coxhead, Jonathan., Filby, Andrew., Halsey, Christina., Allan, James,M., Harrison, J. Christine., Moorman, Anthony., Heidenreich Olaf. Vormoor, Josef.

In more than 30% of B-cell precursor acute lymphoblastic leukaemia (B-ALL), chromosome 21 sequence is overrepresented through aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). Although frequent, the mechanisms by which these abnormalities promote B-ALL remain obscure. Intriguingly, we found copy number neutral loss of heterozygosity (CN-LOH) of 12q was recurrent in iAMP21-ALL, but never observed in B-ALL without some form of chromosome 21 gain. As a consequence of CN-LOH 12q, mutations or deletions of the adaptor protein, SH2B3 , were converted to homozygosity. In patients without CN-LOH 12q, bi-allelic abnormalities of SH2B3 occurred, but only in iAMP21-ALL, giving an overall incidence of 18% in this sub-type. Review of published data confirmed a tight association between overrepresentation of chromosome 21 and both CN-LOH 12q and SH2B3 abnormalities in B-ALL. Despite relatively small patient numbers, preliminary analysis linked 12q abnormalities to poor outcome in iAMP21-ALL ( p = 0.03). Homology modelling of a leukaemia-associated SH2 domain mutation and in vitro analysis of patient-derived xenograft cells implicated the JAK/STAT pathway as one likely target for SH2B3 tumour suppressor activity in iAMP21-ALL.

Introduction: Acute lymphoblastic leukaemia (ALL) is the most common paediatric cancer, of which, precursor B-cell ALL (B-ALL) accounts for approximately 80% of diagnoses. B-ALL is a heterogeneous disease, with patients characterised and risk stratified according to their cytogenetic profile. TCF3-PBX1 B-ALL was associated with a poor prognosis, but on current therapies outcome has greatly improved. However, approximately 10% of these patients relapse with their disease and at this stage have a dismal prognosis (Moorman et. al. Lancet Oncology 2010). Thus, this subset of patients pose a clinical challenge, and further understanding of disease mechanisms in relapsed TCF3-PBX1 is required to aid the discovery of novel targets for therapy.

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of cases of childhood precursor B-cell acute lymphoblastic leukemia. These abnormalities are too complex to engineer faithfully in animal models and are unrepresented in leukemia cell lines. As a resource for future functional and preclinical studies, we have created xenografts from the leukemic blasts of patients with intrachromosomal amplification of chromosome 21 and characterized them by in-vivo and ex-vivo luminescent imaging, flow immunophenotyping, and histological and ultrastructural analyses of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia-initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumor suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor corepressor, LCOR, in the progression of B-cell acute lymphoblastic leukemia. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-cell acute lymphoblastic leukemia and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions.

Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP, are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors.

Intrachromosomal amplification of chromosome 21 (iAMP21) identifies a high-risk subtype of acute lymphoblastic leukaemia (ALL), requiring intensive treatment to reduce their relapse risk. Improved understanding of the genomic landscape of iAMP21-ALL will ascertain whether these patients may benefit from targeted therapy. We performed whole-exome sequencing of eight iAMP21-ALL samples. The mutation rate was dramatically disparate between cases (average 24.9, range 5–51) and a large number of novel variants were identified, including frequent mutation of the RAS/MEK/ERK pathway. Targeted sequencing of a larger cohort revealed that 60% (25/42) of diagnostic iAMP21-ALL samples harboured 42 distinct RAS pathway mutations. High sequencing coverage demonstrated heterogeneity in the form of multiple RAS pathway mutations within the same sample and diverse variant allele frequencies (VAFs) (2–52%), similar to other subtypes of ALL. Constitutive RAS pathway activation was observed in iAMP21 samples that harboured mutations in the predominant clone (⩾35% VAF). Viable iAMP21 cells from primary xenografts showed reduced viability in response to the MEK1/2 inhibitor, selumetinib, in vitro . As clonal (⩾35% VAF) mutations were detected in 26% (11/42) of iAMP21-ALL, this evidence of response to RAS pathway inhibitors may offer the possibility to introduce targeted therapy to improve therapeutic efficacy in these high-risk patients.

Abstract Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of 2% childhood acute lymphoblastic leukaemia (ALL). iAMP21-ALL patients have precursor B-cell ALL, are older (median age 9 years), generally present with low white cell counts and have an inferior outcome when treated with standard therapy. Stratification to high risk treatment arms has significantly reduced their relapse risk, thus accurate diagnosis is essential. We have identified iAMP21 as a complex structure of one copy of chromosome 21, comprising multiple regions of gain, amplification, inversion and deletion, initiated through breakage-fusion-bridge cycles and chromothripsis. Currently, fluorescence in situ hybridisation (FISH), using probes directed to RUNX1, is the most reliable and convenient detection method, in which four or more copies of RUNX1 on a single abnormal chromosome 21 defines iAMP21. From our examination of several hundred cases of iAMP21-ALL, we have noted that, in the absence of metaphase FISH and/or in cases with an unusual cytogenetic presentation, reliance on FISH alone for accurate detection may be problematic. Among a collection of 210 patients with iAMP21-ALL, we performed SNP6.0 and Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA kit P327 iAMP21-ERG (MRC Holland) on 57 and 45 patients, respectively. Although chromosome 21 structure is highly variable between patients, a characteristic copy number profile emerged from the SNP6.0 data (Figure 1). In common, all cases were amplified across a 5.1Mb region, between 32,813,553 and 37,941,425bp that includes RUNX1 and 46 other known protein-coding genes. Asub-telomeric deletion occurred in 88% patients. We propose that this distinctive chromosome 21 copy number profile is used in addition to FISH for the definitive diagnosis of iAMP21-ALL in problematic cases. Support for this approach is provided by results from 5 patients from the Children's Oncology Group (North America, Australia and New Zealand) and ALL2003 (UK) treatment trials who met the iAMP21 FISH criteria but had atypical karyotypes or an unusual distribution of the additional RUNX1 signals that made confident diagnosis challenging. Clinical and cytogenetic data were collected and SNP6.0 and MLPA were performed to clarify the genomic alterations present in these cases. In patient #1, although five RUNX1 signals were observed per cell, two normal copies of chromosome 21 (each with one RUNX1 signal) were present with only three signals located to the abnormal chromosome 21. In patient #2, the additional signals were distributed between 2 different abnormal copies of chromosome 21. Metaphase FISH of patient #3 indicated that the RUNX1 signals were distributed between one normal copy of chromosome 21 and four small chromosomes, which were identified to originate from chromosome 21 by chromosome painting with a chromosome 21 specific probe (wcp21). In a further 2 cases (patients #4 and #5) the signals were too tightly clustered for the number to be discerned. In these 5 cases the characteristic SNP6.0 profile definitively confirmed the suspected diagnosis of iAMP21-ALL (Figure 2). Our previous SNP6.0 data have shown that the copy number profile of the iAMP21 chromosome remains stable between diagnosis and relapse, as well as in serial xenografts successfully transplanted with iAMP21-ALL cells over several generations of mice. However, while FISH analysis of patient #6 at diagnosis and relapse showed the same signal pattern at both time points, there was a change in the karyotype, involving translocation of part of the iAMP21 chromosome onto chromosome 11 at relapse. These observations indicate that the iAMP21 chromosome may become fragmented and distributed throughout the genome without changing its genomic profile. Collectively these observations indicate that the amplified segments resulting from the formation of iAMP21 chromosomes, with typical copy number profiles, can be distributed throughout the genome, either by translocation or fragmentation. Thus FISH together with chromosome 21 copy number profiling provide more accurate diagnosis of iAMP21-ALL than cytogenetics, which may be misleading. For laboratories with no access to SNP6.0 or other copy number arrays, we have shown that MLPA, with a kit specifically designed to detect chromosome 21 copy number changes, provides a reliable alternative (Figure 3). Disclosures No relevant conflicts of interest to declare.

1 Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK. 2 Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. 3 Northern Institute for Cancer Research, Newcastle University, UK. 4 Functional Genomics and Childhood Leukemia Research Center, Sheba Medical Center, Tel-Hashomer, Ramat Gan, Israel 5 Department of Human Molecular Genetics and Biochemistry, Sackler Medical School, Tel Aviv University, Tel Aviv, Israel 6 Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel. 7 Institute of Cancer and Genomic Sciences, University of Birmingham, UK. 8 Department of Paediatric and Adolescent Haematology and Oncology, Great North Children’s Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. Blood First Edition Paper, prepublished online February 11, 2016; DOI 10.1182/blood-2015-08-665034

Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is accompanied by genomic mutations and rearrangements that commonly affect cytokines, transcription factors or signalling molecules that drive B-cell development or contribute to the pre-B cell receptor (pre-BCR) checkpoint. Deletions of the long arm of chromosome 6 [del(6q)] occur in ~10% of BCP-ALL and are also frequent in mature B and T-cell malignancies. Loss of function of the 6q genes EPHA7 and PRDM1, have been implicated in the genesis of lymphoma and BACH2, as a mediator of pre-BCR negative selection, is functionally a candidate tumour suppressor gene. However loss of these or other 6q genes have not been demonstrated, for example through biallelic inactivation, to contribute to BCP-ALL.

The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

Deletions of the short arm of chromosome 12 (12p) are found in around 6% of acute myeloid leukaemia (AML). Particularly in paediatric AML they often occur as the sole cytogenetic change and impart a poor prognosis (Harrison et al J Clin Oncol 2010). Despite multiple deletion mapping studies, a single gene has not been identified from this region that is responsible for driving leukaemic progression, thus it is clear that a functional approach is required. This study aimed to functionally implicate a significant gene through the use of a competitive selection assay.