ALBERT

Description: In genome-wide analyses of DNA copy number abnormalities (CNAs) and loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) microarrays, we previously reported a high frequency of genetic alterations of regulators of B lymphoid development and cell cycle in B-progenitor acute lymphoblastic leukemia (B-ALL), and a near-obligate deletion of the transcription factor Ikaros (IKZF1) in BCR-ABL1 ALL (Nature2007;446:758 and Nature2008;453:110). To assess the prognostic significance of genetic alterations and their effects on the leukemic cell transcriptome, we have now extended these studies to a cohort of 221 NCI high risk pediatric ALL cases treated on the Children’s Oncology Group (COG) P9906 trial. The majority of these cases (N=170) lacked known cytogenetic abnormalities, and none were BCR-ABL1 positive. CNA and LOH data were generated using Affymetrix 500K SNP arrays, and gene expression data was available for 207 cases. Genomic resequencing was performed for PAX5 and IKZF1. These analyses revealed mutations in genes regulating B lymphoid development in 57.5% of cases, including PAX5 CNAs (31.7%), PAX5 sequence mutations (8.1%), IKZF1 deletions (24.9%) and IKZF1 sequence mutations (2.6%). In addition, recurring CNAs were detected in a number of other genes known to play roles in transformation and drug response including CDKN2A/B (45.7%), RB1 (11.3%), BTG1 (10.4%), IL3RA (6.8%), KRAS (6.3%), NRAS (2%), NR3C1 (2%), NR3C2 (3%), and ERG (6.3%). To examine associations between genetic alterations and outcome in a genome-wide fashion, we used a semi-supervised principal components approach and identified a predictor of outcome driven by deletion/mutation of IKZF1, EBF, and BTLA (hazard ratio 3.74, P=8.1×10−5). IKZF1 lesions were most strongly associated with poor outcome (cumulative incidence of relapse 68.8% in IKZF1-mutated ALL vs. 29.1% for IKZF1 wild type cases, P=0.002). This copy number predictor also predicted outcome in an independent cohort of 258 B-progenitor ALL cases treated at St Jude Children’s Research Hospital (HR 3.76, P=0.00023). Importantly, the St Jude cohort had a much higher proportion of cases with conventional recurring cytogenetic abnormalities including hyperdiploidy and translocations than the COG P9906 cohort (60.4% versus 23%, respectively), including BCR-ABL1 (N=21; 8.1%). Notably, the association of IKZF1 mutation and poor outcome was also observed in the cases lacking BCR-ABL1. The presence of an IKZF1 mutation was also associated with a high-level of minimal residual disease following induction therapy in both cohorts. We next defined a gene expression profile of high risk (IKZF1-mutated) ALL in each cohort. Using gene set enrichment analysis (GSEA), we found the high risk gene expression signature to be positively enriched for hematopoietic stem cell genes and genes associated with minimal residual disease, and negatively enriched for genes regulating cell cycle progression and genes upregulated during B cell maturation. Moreover, GSEA also demonstrated significant similarity of the expression signatures of high risk COG and St Jude ALL, a striking finding given the marked differences in sample composition of the two groups. As IKZF1 mutation is a near obligate lesion in BCR-ABL1 lymphoid leukemia (Nature2008;453:110) and in this study is a strong independent predictor of poor outcome in BCR-ABL1 negative ALL, we next examined similarity of the gene expression signatures of high risk (IKZF1-mutated) BCR-ABL1 negative ALL and BCR-ABL1 positive ALL. Using GSEA and direct comparison of differentially expressed genes, there was highly significant similarity between the two signatures. These findings indicate that IKZF1 mutation is a key determinant of the gene expression signature of both BCR-ABL1 positive and negative ALL, and is thus likely to be central to the pathogenesis and poor outcome of both leukemia subtypes. Thus, IKZF1 deletion is a new prognostic factor in ALL, and is associated with a gene expression profile indicative of impaired maturation of the leukemic blasts, an observation compatible with the known role of IKZF1 in regulating early lymphoid development. These findings have important implications for risk stratification of ALL.

Description: Using genome-wide profiling of DNA copy number abnormalities using high-resolution single nucleotide polymorphism arrays, we recently identified a high frequency of genomic aberrations involving the PAX5 gene in pediatric B-progenitor ALL. PAX5 is a critical transcriptional regulator of B lymphocyte commitment and differentiation. Mutations, including partial tandem duplication, complete and focal deletions, point mutations in the DNA-binding or transactivation domain, and three translocations that encode PAX5 fusion proteins were observed in 31.7% of B-ALL. The PAX5 deletions were mono-allelic and resulted in either loss of the entire gene, or the deletion of only a subset of the exons leading to the production of PAX5 proteins that lacked the DNA-binding paired domain (exons 2–4) and/or the transcriptional activation domain (exons 7–10). In murine systems, the complete absence of PAX5 results in the arrest of B-cell development at the pro-B-cell stage prior to immunoglobulin heavy chain rearrangement, whereas haploinsufficiency leads to a partial block in B-cell development. Importantly, in the primary leukemia samples, the mono-allelic loss of PAX5 was associated with reduced expression of PAX5 by flow cytometry and quantitative RT-PCR, suggesting that haploinsufficiency contributes to the block in differentiation characteristic of B-progenitor ALL. To determine if the other identified PAX5 mutations result in hypomorphic alleles, we analyzed the DNA-binding and transcriptional activity of the encoded proteins. DNA-binding activity was assessed by electrophoretic mobility gel-shift assays using a labeled oligonucleotide probes from the promoters of the PAX5 target genes CD19 and CD79A (mb-1), and transcriptional activity was assessed by a luciferase-based reporter assays using the PAX5-dependent reporter plasmid, luc-CD19. Analysis was performed on the paired-domain mutants P80R and P34Q, the focal deletions Δe2-5, Δe2-6, Δe2-7, Δe2-8, and Δe6-8, and the PAX5-ETV6 and PAX5-FOXP1 translocation-encoded fusion proteins. As expected, DNA-binding was abrogated in deletion mutants that lacked the paired domain (Δe2-5, Δe2-6, Δe2-7, Δe2-8). In contrast, the PAX5 Δe6-8, which retains the paired DNA binding domain but lacks a significant portion of the transcriptional regulatory domain, had normal DNA binding activity. Importantly, the paired domain point mutants impaired DNA-binding in a promoter specific manner, with P80R having a marked reduction in binding to both the CD19 and mb-1 promoters, whereas P34Q showed reduced binding only to the mb-1 promoter. Surprisingly, the PAX5-ETV6 and the PAX5-FOXP1 translocations had markedly reduced DNA-binding activity despite retention of the PAX5 paired domain. As expected each of the mutants with impaired or absent DNA-binding activity were found to have markedly reduced transcriptional activity when compared to wild type PAX5. Similarly, those mutants with altered or deleted transcriptional activation domains had reduced transcriptional activity, as did the two PAX5 translocation-encoded fusion proteins (PAX5-ETV6 and PAX5-FOXP1). Moreover, transfection of increasing amounts of PAX5-ETV6 or PAX5-FOXP1 together with a fixed amount of wild type PAX5 revealed that the fusion proteins competitively inhibit the transcriptional activation of wild type PAX5. Taken together, these data indicate that the identified PAX5 mutations impair DNA-binding and/or transcriptional activity. This loss of normal PAX5 function in turn would contribute to the observed arrest in B-cell development seen in ALL.

Description: Genes regulating B lymphoid development are somatically mutated in over 40% of B-progenitor acute lymphoblastic leukemia (ALL) cases, with the most common targets being the transcription factors PAX5, IKZF1 (encoding Ikaros), and EBF1. Notably, BCR-ABL1 ALL is characterized by a high frequency of mutations of IKZF1 (85%), PAX5 (55%), and CDKN2A/B (encoding INK4/ARF, 55%), suggesting that these lesions cooperate with BCR-ABL1 in lymphoid leukemogenesis. To examine cooperativity between Pax5 haploinsufficiency and BCR-ABL1, we transplanted Pax5+/+ and Pax5+/− bone marrow cells transduced with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 transduced Pax5+/− marrow developed B progenitor cell ALL with significantly higher penetrance and decreased latency when compared to animals transplanted with BCR-ABL1 transduced Pax5+/+ marrow (median survival 36 vs. 60 days, P=0.0003). The latency of tumor onset was further decreased in the presence of Arf haploinsufficiency (Pax5+/+Arf+/+ 60 days, Pax5+/−Arf+/+ 36 days, Pax5+/−Arf+/− 21 days, P

Description: Abstract 678 Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified a near-obligate deletion of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. To test this hypothesis, we have examined the effect of Ikaros haploinsufficiency in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from either Ikzf1 wild type mice or mice heterozygous for an Ikzf1 null allele was transduced with MSCV bicistronic retrovirus expressing GFP and p185 BCR-ABL1 and transplanted into lethally irradiated C57BL/6 recipients. Recipients of MSCV-GFP-p185 BCR-ABL1-transduced Ikzf1+/− marrow developed leukemia with markedly increased penetrance and reduced latency in comparison to recipients of wild-type marrow (46 vs. 122 d, P

Description: Using high-resolution SNP arrays and genomic resequencing, we recently reported deletions, translocations, and mutations involving regulators of B cell development in 40% of pediatric B-progenitor ALL (Nature2007;446:758). The most frequently involved genes were PAX5, EBF1, IKZF1 (Ikaros), IKZF3 (Aiolos) and LEF1. Ikaros is a transcription factor required for normal lymphoid development and acts as a tumor suppressor in mice. Focal deletions of IKZF1 were observed in 7 B-progenitor ALL cases, suggesting that the previously reported expression of dominant-negative, non-DNA binding Ikaros isoforms may be caused by genomic IKZF1 abnormalities. We have now extended the analysis to 283 pediatric ALL cases, including B-progenitor ALL with high hyperdiploidy (N=39), hypodiploidy (N=10), rearrangement of MLL (N=23), TCF3-PBX1 (N=17), ETV6-RUNX1 (N=48) and BCR-ABL1 (N=21), as well as cases with low hyperdiploid, normal or miscellaneous cytogenetics (N=75), and T-lineage ALL (N=50). We also examined 22 adult BCR-ABL1 positive ALL cases, 37 acute leukemia cell lines and 49 samples from 23 chronic myeloid leukemia (CML) cases at various stages of disease, including 15 with matched blast crisis samples (12 myeloid, 3 lymphoid). All samples were examined with 500,000 feature Affymetrix SNP arrays (250k Sty and Nsp). The 50k Hind and Xba arrays were also used in 252 ALL cases. Sixty-two (20.3%) ALL cases harboured IKZF1 deletions, including 36 of 43 (83.7%) BCR-ABL1 positive B-ALL cases (76.2% of 21 childhood cases, and 90.9% of 22 adult cases). The deletions were limited to IKZF1 in 25 BCR-ABL1 ALL cases, and in 19 cases deleted an internal subset of IKZF1 exons, most commonly 3–6 (Δ3–6). Remarkably, chronic phase CML samples lacked evidence of IKZF1 deletion, whereas four of 15 matched CML blast crisis samples (66% of lymphoid and 17% of myeloid) had acquired an IKZF1 deletion. The IKZF1 Δ3–6 deletion was also detected in the BCR-ABL1 B-progenitor cell lines BV173, OP1 and SUP-B15, the Δ1–6 deletion in the MYC-IGH/BCL2-IGH B-progenitor cell line 380, and Δ1–7 in the BCR-ABL1 B-progenitor cell line TOM-1. RT-PCR analysis for IKZF1 transcripts demonstrated complete concordance between the extent of IKZF1 deletion and the expression of aberrant Ikaros transcripts lacking internal exons. Importantly, on quantitative RT-PCR analysis and western blotting, expression of the dominant-negative Ik6 transcript and protein, which lacks exons 3–6, was exclusively observed in those cases with IKZF1 Δ3–6, demonstrating that the Ik6 transcript is the result of a specific genetic lesions and not alternative splicing of wild-type IKZF1. Lastly, sequence analysis of the IKZF1 Δ3–6 breakpoints indicated that the deletions arise from aberrant activity of RAG-mediated V(D)J recombination. Taken together, these data demonstrate that deletion of IKZF1, resulting in either haploinsufficiency or the expression of a dominant negative form of the transcription factor, is a central event in the pathogenesis of both pediatric and adult BCR-ABL1 B-progenitor ALL.

Description: Abstract 10 Over 60% of pediatric B progenitor acute lymphoblastic leukemia (ALL) cases contain somatic mutations in genes regulating B cell development, with PAX5 being the most common target of mutation (〉32% of cases). The transcription factor PAX5 is required for commitment and maintenance of the B lymphoid lineage. A variety of PAX5 mutations has been identified including, mono-allelic deletions, sequence mutations, internal deletions, frame-shift mutations and translocations. We have previously shown that these PAX5 mutations result in reduced transcriptional activity either as a result of haploinsufficiency or the generation of altered PAX5 isoforms with reduced DNA-binding and/or transcriptional activity. However, the direct effect of the mutations on normal B cell development remains unknown. To address this question, we assessed the ability of a series of PAX5 mutations to rescue normal B cell development in Pax5-/- bone marrow (BM) cells using a murine in vitro culture system. Whole BM or transduced cells were grown in IL-7 producing stromal-supported cultures for two weeks and then assessed for their extent of B cell differentiation using flow cytometry. Under these in vitro conditions, both Pax5+/+ and Pax5+/− BM cells differentiated to a Hardy fraction D pre-B cell stage of differentiation (CD43−/B220+/CD19+/BP1+), with only a slight decrease in the level of expression of BP1 detected in the Pax5+/− cells. By contrast, Pax5-/- cells failed to undergo significant differentiation under these in vitro growth conditions and were arrested at an early pro-B stage of development (CD43+/−/B220+/CD19−/BP1−). To assess the biological activity of the identified PAX5 mutants, we then transduced lineage-depleted BM cells from Pax5+/+, Pax5+/− and Pax5-/- mice with MSCV-based retroviral vectors expressing either wild type (WT) or mutant PAX5 followed by in vitro culture. Three classes of PAX5 mutations were assessed: DNA binding domain mutations (P80R, P34Q, and V26G), an internal deletion mutation (Δe6-8), and translocation-induced PAX5 chimeric genes (PAX5-ETV6, PAX5-FOXP1 and PAX5-ZNF521). As expected, expression of WTPAX5 resulted in full rescue of Pax5-/- cells and induced no significant effects on the ability of Pax5+/+ and +/− cells to differentiate. By contrast, PAX5 DNA-binding domain mutants resulted in only partial rescue of Pax5-/- cells, with P80R inducing B220+/CD19−/BP1−, P34Q producing B220+/CD19+ cells with weak BP1 expression, and V26G yielding CD19+/BP1+ cells with minimally reduced levels of BP1. Similarly, expression of Δe6-8 resulted in partial rescue with the expansion of B220+/CD19+/−/BP1− cells. In stark contrast, expression of the translocation encoded PAX5 fusion proteins failed to induce any evidence of rescue. Moreover, these fusion proteins induced only minimal perturbations in the ability of Pax5+/+ and +/− cells to differentiate, suggesting that these fusion proteins were weak competitive inhibitors of normal Pax5 transcriptional activity under intra-cellular conditions. To further characterize the effects of these PAX5 mutations on B cell differentiation, we next analyzed the gene expression patterns of the resultant cell populations using the Mouse Genome 430 2.0 Arrays (Affymetrix) and compared the profiles to those obtained from purified Hardy fractions from normal murine BM. The expression signatures of the Pax5-/- cells were identical to those for normal Hardy fraction A and shifted to the signature of Hardy fraction C following rescue with WTPAX5. Transduction of Pax5-/- cells with either V26G or P34Q resulted in a near complete rescue with expression signatures similar to those obtained for Hardy fractions B/C. By contrast, transduction with P80R or Δe6-8 yielded a more incomplete rescue with expression profiles that were between Hardy fractions A and B. Interestingly, a number of genes within the B cell receptor signaling pathway were altered in cells rescued by P80R and Δe6-8, including the down regulation of CD19, Btk and Blnk. In summary, our data demonstrate that leukemia-associated PAX5 mutations have a graded effect on the transcriptional network that controls normal B cell development and differentiation. Defining the differential target gene specificity of the various PAX5 mutants should provide valuable insights into the molecular mechanisms through which these genetic lesions contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: In a previous gene expression profiling study of acute lymphoblastic leukemia (ALL), we identified a novel subtype of B-progenitor ALL (4.9% of 284 cases) with a unique gene expression profile, aberrant expression of CD2 and the absence of recurring cytogenetic abnormalities (Cancer Cell2002;1:133). Efforts to identify rearrangement or mutation of many of the top-ranked genes in the novel expression signature failed to identify a causative lesion. To further investigate the genetic basis of this subtype, we performed integrated genomic analysis of 277 ALL cases. Affymetrix 250k Sty and Nsp SNP microarrays were used in all cases, and Affymetrix U133A gene expression profiles were obtained on 183 of the cases. Unsupervised clustering of gene expression data identified 16 cases of the novel subtype, including all of the 14 previously defined cases. Remarkably, focal mono-allelic deletions of the ETS family member ERG (v-ets erythroblastosis virus E26 oncogene homolog) were detected by genome-wide copy number analysis in 11/16 (69%) of the novel cases, but not in any other ALL subtype. Extensive analysis failed to reveal evidence of translocations involving the altered ERG allele, indicating that these are intragenic deletions limited to ERG. The ERG deletions involved a subset of exons (ERG isoform 1 exons 3–7 or 3–9) resulting in the expression of internally deleted ERG transcripts with altered reading frames predicted to produce a prematurely truncated N-terminal protein fragment. However, using an alternative translational start site 5′ to exon 10, the transcripts also encode a ∼28kDa C-terminal ERG fragment that contains the entire C-terminal ETS DNA-binding and transactivation domains, but lacks all N-terminal domains. Importantly, western blot analysis of primary leukemic blasts revealed expression of only the 28kDa C-terminal ERG protein, along with full length ERG expressed from the retained wild type allele. Remarkably, the C-terminal ERG protein was also detected in 4 of 5 novel ALL cases that lacked detectable ERG deletions, but not in any other ALL subtype. In luciferase reporter assays, this aberrant ERG protein acts as a competitive inhibitor of wild type ERG. Analysis of a second cohort of 35 B-progenitor ALL cases lacking recurring cytogenetic abnormalities identified two cases with ERG deletions and a third expressing the aberrant ERG protein, all of which had the novel gene expression profile. Sequencing of ERG in 252 ALL cases identified only one case with an ERG mutation that resulted in a frameshift in the ETS domain. This case did not share the novel signature nor express the aberrant C-terminal ERG protein. Finally, in an analysis of 37 acute leukemia cell lines, the B-progenitor ALL line NALM-6 was found to harbor a focal, internally truncating ERG deletion, expressed the aberrant ERG protein, and shared the novel gene expression profile, thus identifying it as a model of this novel ALL subtype. These data establish focal ERG deletions as the genetic lesion underlying a novel subtype of ALL, and have expanded the genetic mechanisms that lead to the dysregulation of ERG from chromosomal translocations that result in enhanced transcriptional activity, to deletions that generate dominant negative forms of the transcription factor.

Description: Relapsed acute lymphoblastic leukemia (ALL) is the fourth most common pediatric malignancy and carries a dismal prognosis. To gain insights into the genetic alterations responsible for relapse, we performed genome-wide analysis of matched diagnosis and relapse samples from 61 pediatric ALL patients, including 47 B-progenitor and 14 T-ALL cases. All samples were flow sorted as required to ensure at least 80% tumor cell purity prior to DNA extraction. DNA copy number abnormality (CNA) and loss-of-heterozygosity (LOH) data was generated using Affymetrix SNP 6.0 arrays (1.87 million markers) for 47 diagnosis-relapse pairs, and 500k arrays for the remainder. Remission marrow samples were also examined for 48 cases. Analysis of diagnostic samples identified 10.8 CNAs per B-ALL case and 7.4 per T-ALL case. The most frequent target of CNAs at diagnosis were deletions of genes involved in B-lymphoid development (49% of B-ALL cases, involving PAX5 and IKZF1 in 12 cases each), CDKN2A/B in 36% of B-ALL and 71% of T-ALL, and ETV6 (11 B-ALL cases). At relapse, we observed a striking degree of change in the number, extent, and nature of CNAs. In B-ALL the average number of CNAs increased from 10.8/case at diagnosis to 14.0/case at relapse (P=0.0005), with the majority of this change due to an increase in deletions. By contrast, no difference in the number of CNAs was observed between diagnosis and relapse in T-ALL. Most relapse samples (54 of 61) harbored some of the CNAs present at diagnosis, and had identical or similar antigen receptor locus deletions, indicating a common clonal origin. Nevertheless, 92% of relapse cases exhibited significant changes in CNAs, including the acquisition of new lesions (34%), loss or alteration of lesions present at diagnosis (12%), or both acquisition of new lesions and loss of diagnosis lesions (46%). In the cases where a clear clonal relationship existed, almost half of the relapse clones were derived from a pre-leukemic cell and not from the clone predominating at diagnosis. The most frequent targets of relapse-acquired CNAs were CDKN2A/B, ETV6, and regulators of B-lymphoid development. 18 cases developed new CDKN2A/B deletions, with 70% showing bi-allelic loss; 11 developed new ETV6 lesions; and 15 developed new or more extensive CNAs involving B-lymphoid regulators. In contrast to diagnosis where PAX5 is most frequently involved, at relapse Ikaros and related gene family members were most common (IKZF1, 8 cases; IKZF2, 2 cases; IKZF3, 1 case). Other CNAs previously identified at diagnosis were also detected as new lesions at relapse, including deletions of ADD3, ATM, BTG1, FHIT, KRAS, NF1, PTCH, TBL1XR1, TOX and WT1, suggesting that these lesions contribute to treatment resistance. To further define the biological pathways most frequently altered by relapse–acquired CNAs, the genes within altered regions were categorized into 148 different biological pathways and each cases was then assessed for overrepresentation of these pathways. This analysis identified cell cycle and B-cell transcriptional regulatory pathways as the most significantly targeted pathways at relapse. To determine whether CNAs identified at relapse were present at low levels at diagnosis or were acquired during therapy, we developed qualitative genomic PCR assays for deletions involving ADD3, C20orf94, DMD, ETV6, IKZF2 and IKZF3, and tested corresponding diagnosis and relapse samples. This analysis detected evidence of the relapse clone at diagnosis in 7 of 10 cases tested, indicating that in the majority of cases, CNAs that emerge in the predominant clone at relapse are present at low levels at diagnosis and are selected for during treatment. These results provide critical insights into the spectrum of genetic lesions that underlie ALL relapse. Although our data are limited to a single class of mutations (CNAs), they demonstrate that no single genetic lesion or alteration of a single pathway is responsible for relapse. Instead, a diversity of mutations appear to contribute to relapse with the most common alterations targeting key regulators of tumor suppression, cell cycle control, and lymphoid/B cell development. Notably, few lesions involved genes with roles in drug import, metabolism, export and/or response, (an exception being the glucocorticoid receptor gene NR3C1) suggesting that that the mechanism of relapse is more complex than simple “drug resistance”.