Clelia Tiziana Storlazzi

Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP)

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Delta4-7) and by removal of exons 2 through 7 (Delta2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Delta4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Delta2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Pre–B cell receptor–mediated cell cycle arrest in Philadelphia chromosome–positive acute lymphoblastic leukemia requires IKAROS function

B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre–B cell receptor–dependent stages. The Philadelphia chromosome–positive (Ph+) subtype of ALL accounts for 25–30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre–B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph+ ALL cells. Pre–B cell receptor–mediated cell cycle arrest in Ph+ ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre–B cell receptor signaling pathway, even if expression of the pre–B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre–B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph+ ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre–B cell receptor–mediated tumor suppression.

Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure

Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks of genomic amplification in cancer. Different mechanisms have been proposed to explain their genesis. Recently, our group showed that the MYC-containing dmin in leukemia cases arise by excision and amplification (episome model). In the present paper we investigated 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr. Particularly revealing results were provided by the two subclones of the neuroblastoma cell line STA-NB-10, one showing dmin-only and the second hsr-only amplification. Both subclones showed a deletion, at 2p24.3, whose extension matched the amplicon extension. Additionally, the amplicon structure of the dmin and hsr forms was identical. This strongly argues that the episome model, already demonstrated in leukemias, applies to solid tumors as well, and that dmin and hsr are two faces of the same coin. The organization of the duplicated segments varied from very simple (no apparent changes from the normal sequence) to very complex. MYCN was always overexpressed (significantly overexpressed in three cases). The fusion junctions, always mediated by nonhomologous end joining, occasionally juxtaposed truncated genes in the same transcriptional orientation. Fusion transcripts involving NBAS (also known as NAG), FAM49A, BC035112 (also known as NCRNA00276), and SMC6 genes were indeed detected, although their role in the context of the tumor is not clear.

A 76-kb duplicon maps close to the BCR gene on chromosome 22 and the ABL gene on chromosome 9: Possible involvement in the genesis of the Philadelphia chromosome translocation

A patient with a typical form of chronic myeloid leukemia was found to carry a large deletion on the derivative chromosome 9q+ and an unusual BCR-ABL transcript characterized by the insertion, between BCR exon 14 and ABL exon 2, of 126 bp derived from a region located on chromosome 9, 1.4 Mb 5′ to ABL. This sequence was contained in the bacterial artificial chromosome RP11-65J3, which in fluorescence in situ hybridization experiments on normal metaphases was found to detect, in addition to the predicted clear signal at 9q34, a faint but distinct signal at 22q11.2, where the BCR gene is located, suggesting the presence of a large region of homology between the two chromosomal regions. Indeed, blast analysis of the RP11-65J3 sequence against the entire human genome revealed the presence of a stretch of homology, about 76 kb long, located approximately 150 kb 3′ to the BCR gene, and containing the 126-bp insertion sequence. Evolutionary studies using fluorescence in situ hybridization identified the region as a duplicon, which transposed from the region orthologous to human 9q34 to chromosome 22 after the divergence of orangutan from the human-chimpanzee-gorilla common ancestor about 14 million years ago. Recent sequence analyses have disclosed an unpredicted extensive segmental duplication of our genome, and the impact of duplicons in triggering genomic disorders is becoming more and more apparent. The discovery of a large duplicon relatively close to the ABL and BCR genes and the finding that the 126-bp insertion is very close to the duplicon at 9q34 open the question of the possible involvement of the duplicon in the formation of the Philadelphia chromosome translocation.

Identification of RUNX1/AML1 as a classical tumor suppressor gene

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

Breakpoint characterization of der(9) deletions in chronic myeloid leukemia patients

Deletions adjacent to the 9/22 translocation breakpoint on the derivative chromosome 9 have recently been described in a substantial number of chronic myeloid leukemia (CML) cases, but their extension has not been characterized in detail. Using FISH with an appropriate set of BAC/PAC probes, we have characterized the deletion in 10 CML cases, identified by screening 71 patients at diagnosis. Five patients showed a complex chromosome rearrangement and 3 of them were deleted. The size of the deletion was variable, ranging from few hundreds kb to 8 Mb. A minimally deleted region on both chromosomes 9 and 22 was identified and was found to contain the ASS gene on chromosome 9 and IGLL1 on chromosome 22.

A Novel Fusion Gene, SS18L1/SSX1, in Synovial Sarcoma

Synovial sarcoma is an aggressive soft tissue tumor that is characterized cytogenetically by the t(X;18)(p11;q11) translocation, resulting in fusion between the SS18 gene on chromosome 18 and one of the SSX genes on the X chromosome. The three fusion genes that have been detected thus far, SS18/SSX1, SS18/SSX2, and SS18/SSX4, account for more than 95% of the synovial sarcomas. Because SS18/SSX fusions do not seem to occur in other tumor types, and because synovial sarcomas may sometimes be difficult to distinguish from other spindle cell tumors, molecular genetic analysis has become established as an important diagnostic tool. Upon cytogenetic analysis of a soft‐tissue tumor that showed classic synovial sarcoma morphology, we detected two supernumerary marker chromosomes but no rearrangement of chromosomes X or 18. By fluorescence in situ hybridization, the marker chromosomes were shown to contain material from chromosomes X and 20, including the SSX gene cluster on the X chromosome and the SS18L1 gene, which shows strong homology with the SS18 gene, on chromosome 20. Further RT‐PCR analysis and sequencing of the amplified products revealed a novel SS18L1/SSX1 fusion transcript in which nucleotide 1216 (exon 10) of SS18L1 was fused in‐frame with nucleotide 422 (exon 6) of SSX1. Thus, the existence of genetic heterogeneity has to be taken into account when RT‐PCR is used for the diagnosis of synovial sarcoma.

Identification of a novel amplicon at distal 17q containing the BIRC5/SURVIVIN gene in malignant peripheral nerve sheath tumours

Previous studies have suggested that amplification of genes, notably the TOP2A gene, on chromosome arm 17q may be important for the development of malignant peripheral nerve sheath tumour (MPNST). In order to study the frequency, distribution, and chromosomal organization of rearrangements at 17q, interphase and metaphase fluorescence in situ hybridization (FISH) were used to evaluate copy number changes at 17q in 28 MPNSTs. Increased copy numbers were seen for the ERBB2 and TOP2A genes in eight and nine cases, respectively, supporting a potential role for these two genes in MPNST tumourigenesis. Net gain of distal 17q material was observed in 16 of the 28 MPNSTs, with high‐level gain in three cases, and was associated with poor outcome. Among the 26 patients for whom follow‐up data were available, gain of distal 17q was present in 11 of 12 tumours that had metastasized, compared with 4 of 14 of those that had not metastasized. Detailed FISH mapping analysis of metaphase spreads identified a 2 Mb commonly gained/amplified region at 17q25. Among the genes mapping to this region, BIRC5, which encodes the baculoviral IAP repeat‐containing protein 5/survivin protein, is a strong candidate target gene for amplification, as it has been previously shown to be overexpressed in neurofibromatosis type 1‐associated MPNST. Three other genes that co‐amplified with BIRC5 represent other potential candidate genes: PTDSR involved in apoptosis; SEPT9 overexpressed in human malignant brain tumours; and SOCS3 involved in cell survival and differentiation of neurons. Copyright

Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates Hippo signalling and supports glioblastoma growth

Human glioblastoma is the most frequent and aggressive form of brain tumour in the adult population. Proteolytic turnover of tumour suppressors by the ubiquitin–proteasome system is a mechanism that tumour cells can adopt to sustain their growth and invasiveness. However, the identity of ubiquitin–proteasome targets and regulators in glioblastoma are still unknown. Here we report that the RING ligase praja2 ubiquitylates and degrades Mob, a core component of NDR/LATS kinase and a positive regulator of the tumour-suppressor Hippo cascade. Degradation of Mob through the ubiquitin–proteasome system attenuates the Hippo cascade and sustains glioblastoma growth in vivo. Accordingly, accumulation of praja2 during the transition from low- to high-grade glioma is associated with significant downregulation of the Hippo pathway. These findings identify praja2 as a novel upstream regulator of the Hippo cascade, linking the ubiquitin proteasome system to deregulated glioblastoma growth.

IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia.

Background Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. Principal Findings Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. Conclusions Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.