Disruption to the FOXO-PRDM1 axis resulting from deletions of chromosome 6 in acute lymphoblastic leukaemia.

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 (ID50 < 50 nM).

SH2B3 inactivation through CN-LOH 12q is uniquely associated with B-cell precursor ALL with iAMP21 or other chromosome 21 gain.

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.

Dynamic clonal progression in xenografts of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

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 co-repressor, 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.

Analysis of a breakpoint cluster reveals insight into the mechanism of intrachromosomal amplification in a lymphoid malignancy.

A distinct sub-group of B-cell precursor acute lymphoblastic leukemia, defined by intrachromosomal amplification of chromosome 21 (iAMP21), is restricted to older children and has been associated with a poor outcome. Accurate diagnosis is important for appropriate risk stratification for treatment. It could be improved by understanding the initiating mechanism. iAMP21 is characterized by amplification of a 5.1-24 Mb region of chromosome 21, which includes the RUNX1 gene. It is thought to arise through a breakage-fusion-bridge (BFB) mechanism. Breakpoints initiating BFB cycles were determined from recent array data from 18 patients. Three occurred within the PDE9A gene. Other patients with breakpoints in PDE9A were identified by fluorescence in situ hybridization and molecular copy number counting. Sequencing defined a 1.7 Kb breakpoint cluster region, positioned 400 bp distal to an extensive region enriched for CA repeats with the potential to form Z-DNA. None of the rearranged sequences showed the inverted repeat structure characteristic of BFB; instead PDE9A was fused to intergenic regions of chromosome 21 or to genes on other chromosomes. These observations indicated that previously unrecognized complex events, involving microhomology-mediated end joining, preceded or accompanied initiation of the BFB cycle. A chi-like heptomer, CCTCAGC, contained four of the breakpoints, two within PDE9A and two within partner Alu-repeat sequences. This heptomer was closely homologous to a breakpoint hotspot within the TCF3 gene, suggesting involvement of a common novel recombinogenic mechanism that might also contribute to the recombinogenic potential of Alu repeats. These findings provide insight into potential mechanisms involved in the formation of iAMP21.

The facultative heterochromatin of the inactive X chromosome has a distinctive condensed ultrastructure.

The mammalian inactive X chromosome (Xi) is a model for facultative heterochromatin. Increased DNA compaction for the Xi, and for facultative heterochromatin in general, has long been assumed based on recognition of a distinct Barr body using nucleic-acid staining. This conclusion has been challenged by a report revealing equal volumes occupied by the inactive and active X chromosomes. Here, we use light and electron microscopy to demonstrate in mouse and human fibroblasts a unique Xi ultrastructure, distinct from euchromatin and constitutive heterochromatin, containing tightly packed, heterochromatic fibers/domains with diameters in some cases approaching that of prophase chromatids. Significant space between these packed structures is observed even within condensed regions of the Xi. Serial-section analysis also reveals extensive contacts of the Xi with the nuclear envelope and/or nucleolus, with nuclear envelope association being observed in all cells. Implications of our results for models of Xi gene silencing and chromosome territory organization are discussed.

A fluorescence in situ hybridization map of 6q deletions in acute lymphocytic leukemia: identification and analysis of a candidate tumor suppressor gene.

With the objective of identifying candidate tumor suppressor genes, we used fluorescence in situ hybridization to map leukemia-related deletions of the long arm of chromosome 6 (6q). Twenty of 24 deletions overlapped to define a 4.8-Mb region of minimal deletion between markers D6S1510 and D6S1692 within chromosome 6 band q16. Using reverse transcription-PCR, we found evidence of expression in hematopoietic cells for 3 of 15 genes in the region (GRIK2, C6orf111, and CCNC). Comparison between our own and published deletion data singled out GRIK2 as the gene most frequently affected by deletions of 6q in acute lymphocytic leukemia (ALL). Sequence analysis of GRIK2 in 14 ALL cases carrying heterozygous 6q deletions revealed a constitutional and paternally inherited C to G substitution in exon 6 encoding for an amino acid change in one patient. The substitution was absent among 232 normal alleles tested, leaving open the possibility that heterozygous carriers of such mutations may be susceptible to ALL. Although low in all normal hematopoietic tissues, quantitative reverse transcription-PCR showed higher baseline GRIK2 expression in thymus and T cells than other lineages. Among T-cell ALL patients, 6q deletion was associated with a statistically significant reduction in GRIK2 expression (P = 0.0001). By contrast, elevated GRIK2 expression was measured in the myelomonocytic line THP-1 and in one patient with common ALL. Finally, we detected significant levels of GRIK2 expression in prostate, kidney, trachea, and lung, raising the possibility that this gene may be protective against multiple tumor types.