Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Abstract 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<0.0001). All leukemias were of B cell lineage and were transplantable to secondary recipients. In addition, Southern blot analysis revealed the Pax5+/−Arf+/+ leukemias to be monoclonal, where as the Pax5+/−Arf+/− leukemias were oligoclonal. Importantly, the Pax5+/− leukemias exhibited a more immature B cell immunophenotype than Pax5 wild type leukemias. Moreover, a proportion of the Pax5+/− leukemias (19%) exhibited a very immature early pro B cell immunophenotype (Cd19−, Bp1−), suggesting the possibility of acquired lesions in other key regulators of normal B cell differentiation. To explore this possibility and to identify the total complement of genetic lesions required to generate overt leukemia, we performed genome-wide copy number analysis on 30 murine leukemias (15 Pax5+/+, 15 Pax5+/−) using a custom CGH microarray (Agilent) that interrogated 477,000 autosomal loci, including 18,000 probes covering 20 genes encoding B lymphoid transcription factors and genes targeted by recurring copy number abnormalities (CNAs) in human BCR-ABL1 ALL (Bcl11a, Cdkn2a, Ebf1, Ikzf1, Ikzf2, Ikzf3, Il7r, Lef1, Mdm2, Mef2c, Myb, Pax5, Pten, Rb1, Sfpi1, Sox4, Stat5a, Tcf3, Tcf4, and Trp53). This analysis identified focal recurring CNAs in multiple genes including Cdkn2a/b, Ebf1, Ikzf1, Ikzf2, Ikzf3, and Pax5, each of which is a target of mutation in human B-ALL. Overall, there were on average 3.5 CNAs in Pax5+/+ leukemias versus 0.7 CNAs in Pax5+/− leukemias. Genomic resequencing was also performed on Pax5 and revealed three missense mutations in the DNA binding paired domain (R38H, P80R and G85R), one of which (P80R) is the most common PAX5 point mutation in human B-ALL. All three point mutations are predicted to impair DNA binding of Pax5. Interestingly, the majority of the pro-B cell leukemias that arose in the Pax5+/−Arf+/+ animals were found to harbor mutations (CNAs or point mutation) of the retained Pax5 allele, consistent with the immature immunophenotype. To further explore the relationship between our murine model and human BCR-ABL1 ALL, we performed gene expression profiling of Pax5+/+ and Pax5+/− leukemias and compared their signatures to those of human BCR-ABL1 ALL and stage-specific murine B lymphoid developmental signatures using gene set enrichment analysis (GSEA). This analysis identified significant similarity between murine and human BCR-ABL1 leukemias, thus providing further evidence that this model closely recapitulates human BCR-ABL1 ALL. Notably, Pax5+/− leukemias, or Pax5+/+ leukemias that acquired additional mutations of B-lymphoid regulators exhibited a less mature gene expression profile than leukemias lacking B-lymphoid regulatory mutations. These data indicate that loss of Pax5 contributes to leukemogenesis, that additional genomic alterations in genes regulating B lymphoid development and cell cycle regulators/tumor suppressors (Arf) are frequent events in BCR-ABL1 acute lymphoblastic leukemia, and that these lesions result in impaired B-lymphoid maturation in B-ALL. The genetic complexity of BCR-ABL1 ALL is likely to have important therapeutic implications for this poor prognosis subtype of leukemia.

Download Full-text

Haploinsufficiency of Ebf1 Collaborates with BCR-ABL1 to Decrease the Latency of Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v112.11.3363.3363 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3363-3363

Author(s):

Salil Goorha ◽

Noel T. Lenny ◽

Christopher B Miller ◽

S. Scott Perry ◽

Xiaoping Su ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

B Cell Development ◽

B Lineage ◽

B Lymphoid

Abstract In previously published genome-wide copy number analysis of leukemic samples from 242 pediatric acute lymphoblastic leukemia (ALL) patients, we reported that mutations in genes regulating B lymphoid development are the most common lesion in B-progenitor ALL, and these include PAX5, IKZF1, and EBF1. Mono-allelic deletion of EBF1 was observed in 8/200 B-progenitor leukemia samples, including a BCR-ABL1 ALL. EBF1 encodes a transcription factor that is required for the development of B cells, and with E2A regulates the expression of B-lineage specific genes. Mice null for Ebf1 arrest B cell development at the pro-B cell stage, whereas Ebf1+/− mice have a 50% reduction in the number of immature and mature B cells but a normal number of pro-B cells. Importantly, neither haploinsufficiency nor the complete loss of Ebf1 results in the development of leukemia in mice. To examine the role of genetic alterations targeting B-lymphoid differentiation in the pathogenesis in BCR-ABL1 ALL, we transduced Ebf1+/+ and Ebf1+/− bone marrow cells with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus and transplanted the resultant cells into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 Ebf1+/− cells developed B lineage ALLs at a shorter latency than observed with BCR-ABL1 Ebf1+/+ cells (median overall survival of 17 days in Ebf1+/− vs 42 days in Ebf1+/+, P<0.0001). All leukemias had a B220+Cd19+Bp1+ pre-B cell immunophenotype; however, the leukemias that developed from the Ebf1+/− cells aberrantly expressed high levels of the stem cell marker Sca1 (mean fluorescence level for Sca1 of 69.6 in Ebf1+/− (n=22) vs 16.8 in Ebf1+/+ (n=14), p<0.0001). To begin to understand how a decrease in the copy number of Ebf1 may contribute to leukemogenesis, we examined early B cell development in bone marrow (BM) cells from two week-old C57BL6 Ebf1+/− and Ebf1+/+ mice. Our analysis confirmed previous reports indicating a 2-fold reduction of B220+CD43− B cells in Ebf1+/− compared to Ebf1+/+ mice. Interestingly, however, we also detected an approximately 6-fold increase in a transitional population of B220loIL-7R+cKitlo Pre-pro B cells that also expressed Sca1 (2194 mean number of Ebf1+/− cells per 100,000 BM cells (n=10) vs 372 mean number of Ebf1+/+ cells per 100,000 BM cells (n=8), p<0.0001), an observation that raises the possibility that Ebf1 haploinsufficiency expands the pool of cells that are susceptible to transformation by BCR-ABL expression. It will be important to examine whether the accelerated tumorigenesis resulting from Ebf1 haploinsufficiency is a consequence of a subtle shift in differentiation, or some alternative mechanism of oncogenic cooperativity. Studies are underway to directly assess the role of B220loIL-7R+cKitlo Sca1+ cells in BCR-ABL1 driven ALL.

Download Full-text

Genomic Analysis of the Clonal Origins of Relapsed Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v112.11.429.429 ◽

2008 ◽

Vol 112 (11) ◽

pp. 429-429

Author(s):

Charles G Mullighan ◽

Letha A Phillips ◽

Xiaoping Su ◽

Jing Ma ◽

Christopher B Miller ◽

...

Keyword(s):

Cell Cycle ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Biological Pathways ◽

Lymphoid Development ◽

Glucocorticoid Receptor Gene ◽

Low Levels ◽

B Lymphoid

Abstract 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”.

Download Full-text

IKZF1 and 22q11.22 Deletions and PDGFRA Gains Are Associated with Poor Outcome in Down Syndrome Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.289.289 ◽

2012 ◽

Vol 120 (21) ◽

pp. 289-289 ◽

Cited By ~ 2

Author(s):

Karen R Rabin ◽

Clinton C Mason ◽

Sivashankarappa Gurusiddappa ◽

Hon-Chiu Eastwood Leung ◽

Debra J. Morrison ◽

...

Keyword(s):

Gene Expression ◽

Down Syndrome ◽

Acute Lymphoblastic Leukemia ◽

Expression Profiling ◽

Copy Number ◽

Poor Prognosis ◽

Lymphoblastic Leukemia ◽

Wild Type ◽

Improve Risk Stratification ◽

The Poor

Abstract Abstract 289 Children with Down syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL), and consistently demonstrate poorer outcomes due to higher rates of both relapse and treatment-related mortality compared to other children with ALL. The biology of ALL in DS is unique, with lower frequency of the classic cytogenetic lesions generally observed in childhood ALL, and increased frequency of JAK2 mutations and CRLF2 rearrangements, which are not clearly associated with adverse prognosis in DS-ALL. In order to improve risk stratification and identify potential novel therapeutic targets in this vulnerable population, we analyzed 90 DS-ALL cases for prognostically significant copy number abnormalities (CNAs) in a collaborative cohort from the Children's Oncology Group (n=30), St. Jude Children's Research Hospital (n=22), AIEOP (n=16), Texas Children's Cancer Center (n=10), UKALL 2003 (n=6) and an archival UK sample (n=1), and Utah's Primary Children's Medical Center (n=5). Copy number profiling was performed using 500K, 6.0, CytoScan HD, and OncoScan FFPE Express arrays (Affymetrix), and Human CNV370-Duo arrays (Illumina). Gene expression profiling was performed using U133 Plus2.0 arrays (Affymetrix). Copy number was analyzed with Nexus Copy Number (BioDiscovery, Inc.) and gene expression was analyzed with Partek Genomics Suite (Partek, Inc.) and Gene Set Enrichment Analysis (Broad Institute). Deletions of a focal region on 22q11.22 (present in 28.9% of cases, similar to the incidence previously reported in a non-DS cohort [Mangum et al, ASH 2011:741]), and deletions of IKZF1 (present in 20.0% of cases), were significantly associated with poor event-free survival (EFS) (5-year EFS was 88.6 ± 6.3% in wild-type cases [n=31], 68.1 ± 13.3% in cases with deletion of 22q11.22 only [n=15], and 60.0 ± 21.9% in those with deletion of IKZF1 only [n=5]; combined deletion [n=6] was associated with an even poorer EFS (33.3 ± 19.3%, p<0.0001, Figure 1A). Patients were observed to have focal deletions on 22q11.22 spanning up to 235 kb; the most common recurring shared region is just under 10 kb in length, and does not contain known coding genes. Increased number of copies of a 14 kb region in the platelet-derived growth factor receptor alpha gene (PDGFRA), located at 4q12, occurred in 35.5% of cases performed on microarrays containing evaluable probes in this region, and were significantly associated with poor outcome (4-year EFS 87.5 ± 8.3% in wild-type cases [n=20] and 33.9 ± 17.6% in PDGFRA gain cases [n=11], p = 0.004, Figure 1B). Five cases contained 3 copies of PDGFRA, and six cases had at least 4 copies, with no correlation between outcome and number of copies gained. PDGFRA rearrangements and point mutations have been reported in other malignancies, but copy number gain is a novel mechanism of alteration. Cases with a focal 22q11.22 deletion were associated with an increased frequency of CRLF2 rearrangement (75% versus 47%, Fisher's exact p=0.028). No other differences in age, initial WBC, or CRLF2 rearrangement in cases with versus without the focal 22q11.22 deletion, IKZF1 deletion, or PDGFRA gain were present, nor were there significant differences in incidence of focal PDGFRA gains in cases with versus without IKZF1 or 22q11.22 deletions. Gene expression profiling in 27 evaluable cases demonstrated upregulation of genes with kinase activity in 12 cases with either focal 22q11.22 or IKZF1 deletions. This study represents one of the largest collaborative cohorts of DS-ALL for genomic profiling, confirms the poor prognosis associated with IKZF1 deletion (Buitenkamp et al., Leukemia 2012), and identifies two additional genomic loci strongly associated with poor prognosis. If validated in additional DS-ALL cohorts, these findings suggest key lesions that contribute to the poor outcomes observed in this population. These specific genomic changes may improve risk stratification of treatment in children with DS and ALL, and may lead to enhanced sensitivity to tyrosine kinase inhibitors in the estimated 60% of cases bearing one or more of these three lesions. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Genomic Determinants of Response to Blinatumomab in Relapsed/Refractory (R/R) B-Cell Precursor Acute Lymphoblastic Leukemia in Adults

Blood ◽

10.1182/blood-2018-99-113604 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1552-1552 ◽

Cited By ~ 2

Author(s):

Yaqi Zhao ◽

Ibrahim Aldoss ◽

Chunxu Qu ◽

Guido Marcucci ◽

Anthony S. Stein ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

B Cell ◽

High Frequency ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Cell Precursor ◽

B Lymphoid

Abstract Introduction: Blinatumomab, a CD19/CD3 bi-specific T-cell engager monoclonal antibody that re-directs CD3-positive T cells towards CD19-positive B cells, has shown promise in the treatment of R/R B-cell precursor acute lymphoblastic leukemia (B-ALL), with superior survival rates compared to salvage chemotherapy. However, many patients do not respond or subsequently relapse, and the mechanisms underlying resistance are unclear. The goals of this study were to characterize the genomic features associated with response to blinatumomab. Methods: We studied 29 patients (pts; median age 28, range 18-70) with R/R B-ALL who were treated with up to 5 cycles of blinatumomab, and predominantly of Hispanic ancestry (66%). Overall, 17 pts (59%) achieved remission with blinatumomab whilst 12 showed no response. Among the 17 responders, 7 (41%) subsequently relapsed or progressed during treatment. We analyzed leukemic blasts obtained before and after blinatumomab treatment whenever available: pre-blinatumomab R/R (n=28), post-blinatumomab refractory (n=5), post-blinatumomab relapsed (n=3) (Figure 1). Leukemia and matched remission samples were studied using transcriptome sequencing (n=34), whole genome sequencing (n=28), whole exome sequencing (n=19) and Infinium Omni2.5Exome-8 (SNP array, n=19). Results: Seventeen of 29 pts (59%) were Ph-like ALL. Twelve of 17 Ph-like ALL pts had high CRLF2 expression, among these we identified P2RY8-CRLF2 (n=4) and IGH-CRLF2 (n=8). Within the remaining 5 Ph-like ALL cases, two pts harbored NUP214-ABL1, two IGH-EPOR and one TERF-JAK2. Fifteen of the 17 (88%) Ph-like ALL cases were of Hispanic ancestry. The prevalence of other known subtypes was relatively low: BCR-ABL1 7%, hypodiploid 7%, KMT2A 3%, TCF3-PBX1 3% and B-other 21%. We observed a high response rate of 83.3% (10/12 cases) in Ph-like_CRLF2 pts, whilst the frequency of response was 60% (3/5, including two IGH-EPOR) for Ph-like_non-CRLF2 pts, and 33% (4/12) for the other subtypes (Ph-like ALL vs. others, P=0.029) (Figure 1). Unsupervised hierarchical clustering of pre-blinatumomab samples identified 3 clusters based on response to blinatumomab: cluster 1 contained non-responders, clusters 2 and 3 were largely made up of responders. By gene expression profiling using CIBERSORT we found reduced infiltration of cytotoxic CD8+ T-cells in cluster 1 compared to clusters 2 and 3 (6.1% vs. 14.9%, P=0.014), which was inversely correlated with the presence of CD4+ T cells (17.9% vs. 11.5%). GSEA showed enrichment for the IFNγ response, JAK-STAT signaling, chemokine and cytokine signaling in responders. In non-responders, differential gene expression analysis identified up-regulation of the H3K4 demethylase KDM5B, an oncogene associated with progression and chemoresistance of glioma and neuroblastoma. We observed a high frequency of alterations affecting B-lymphoid development (IKZF1, PAX5 and EBF1) in the pre-blinatumomab samples (20 of 22, 91%), which were maintained during progression or relapse. The frequency of B-lymphoid alterations did not differ significantly between responders and non-responders (13 of 14, 93% vs. 7 of 8, 88%). Alterations affecting the cell cycle (CDKN2A/B, TP53, RB1) were observed at a high frequency in pre-blinatumomab samples (15 of 22, 68%), with CDKN2A/B deletions enriched in responders compared to non-responders (11 of 14, 79% vs. 2 of 8, 25%; P=0.026). We also observed a high prevalence of alterations affecting epigenetic modifiers (ARID1B, CREBBP, KDM6A, KMT2D, TRRAP, SMARCA4) in pre-blinatumomab samples (17 of 22, 77%), with no difference between responders and non-responders (10 of 14, 71% vs. 7 of 8, 88%; P=0.61). Of the post-blinatumomab R/R samples available for study (n=8), CD19 expression was negative (n=1), dim (n=2) or positive (n=5). In contrast to previous reports of CD19 escape in CAR T-cell treated patients, there was no evidence of aberrantly spliced CD19 mRNA species, CD19 mutation or deletion in the three negative/dim cases. Conclusion: We show that a heightened immune response through the infiltration of cytotoxic T-cells and activation of IFNγ and JAK-STAT signaling in leukemic cells is an important determinant of response to blinatumomab. Importantly, blinatumomab is a valid therapeutic approach for patients harboring high-risk CRLF2 and EPOR-rearrangements. CD19 escape is not associated with genetic alterations at the CD19 locus. Figure. Figure. Disclosures Stein: Amgen Inc.: Speakers Bureau; Celgene: Speakers Bureau. Mullighan:Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

Download Full-text

A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽

10.1182/blood-2006-05-025221 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3417-3423 ◽

Cited By ~ 55

Author(s):

Marina Bousquet ◽

Cyril Broccardo ◽

Cathy Quelen ◽

Fabienne Meggetto ◽

Emilienne Kuhlein ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Quantitative Polymerase Chain Reaction ◽

Dominant Negative ◽

Leukemic Cells ◽

Dominant Negative Effect ◽

Wild Type ◽

Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

Download Full-text

Gene Expression Profiling Data in Lymphoma and Leukemia: Review of the Literature and Extrapolation of Pertinent Clinical Applications

Archives of Pathology & Laboratory Medicine ◽

10.5858/2006-130-483-gepdil ◽

2006 ◽

Vol 130 (4) ◽

pp. 483-520 ◽

Cited By ~ 2

Author(s):

Cherie H. Dunphy

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Gene Expression Profiling ◽

Hodgkin Lymphoma ◽

Expression Profiling ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Clinical Applications

Abstract Context.—Gene expression (GE) analyses using microarrays have become an important part of biomedical and clinical research in hematolymphoid malignancies. However, the methods are time-consuming and costly for routine clinical practice. Objectives.—To review the literature regarding GE data that may provide important information regarding pathogenesis and that may be extrapolated for use in diagnosing and prognosticating lymphomas and leukemias; to present GE findings in Hodgkin and non-Hodgkin lymphomas, acute leukemias, and chronic myeloid leukemia in detail; and to summarize the practical clinical applications in tables that are referenced throughout the text. Data Source.—PubMed was searched for pertinent literature from 1993 to 2005. Conclusions.—Gene expression profiling of lymphomas and leukemias aids in the diagnosis and prognostication of these diseases. The extrapolation of these findings to more timely, efficient, and cost-effective methods, such as flow cytometry and immunohistochemistry, results in better diagnostic tools to manage the diseases. Flow cytometric and immunohistochemical applications of the information gained from GE profiling assist in the management of chronic lymphocytic leukemia, other low-grade B-cell non-Hodgkin lymphomas and leukemias, diffuse large B-cell lymphoma, nodular lymphocyte–predominant Hodgkin lymphoma, and classic Hodgkin lymphoma. For practical clinical use, GE profiling of precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, and acute myeloid leukemia has supported most of the information that has been obtained by cytogenetic and molecular studies (except for the identification of FLT3 mutations for molecular analysis), but extrapolation of the analyses leaves much to be gained based on the GE profiling data.

Download Full-text

Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

Blood ◽

10.1182/blood-2011-03-341412 ◽

2011 ◽

Vol 118 (11) ◽

pp. 3080-3087 ◽

Cited By ~ 186

Author(s):

Jinghui Zhang ◽

Charles G. Mullighan ◽

Richard C. Harvey ◽

Gang Wu ◽

Xiang Chen ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

B Cell ◽

High Frequency ◽

Lymphoblastic Leukemia ◽

Cell Development ◽

B Cell Development ◽

Ras Signaling ◽

Somatic Alterations

Abstract We sequenced 120 candidate genes in 187 high-risk childhood B-precursor acute lymphoblastic leukemias, the largest pediatric cancer genome sequencing effort reported to date. Integrated analysis of 179 validated somatic sequence mutations with genome-wide copy number alterations and gene expression profiles revealed a high frequency of recurrent somatic alterations in key signaling pathways, including B-cell development/differentiation (68% of cases), the TP53/RB tumor suppressor pathway (54%), Ras signaling (50%), and Janus kinases (11%). Recurrent mutations were also found in ETV6 (6 cases), TBL1XR1 (3), CREBBP (3), MUC4 (2), ASMTL (2), and ADARB2 (2). The frequency of mutations within the 4 major pathways varied markedly across genetic subtypes. Among 23 leukemias expressing a BCR-ABL1-like gene expression profile, 96% had somatic alterations in B-cell development/differentiation, 57% in JAK, and 52% in both pathways, whereas only 9% had Ras pathway mutations. In contrast, 21 cases defined by a distinct gene expression profile coupled with focal ERG deletion rarely had B-cell development/differentiation or JAK kinase alterations but had a high frequency (62%) of Ras signaling pathway mutations. These data extend the range of genes that are recurrently mutated in high-risk childhood B-precursor acute lymphoblastic leukemia and highlight important new therapeutic targets for selected patient subsets.

Download Full-text

Clinical application of whole transcriptome sequencing for the classification of patients with acute lymphoblastic leukemia

BMC Cancer ◽

10.1186/s12885-021-08635-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wencke Walter ◽

Rabia Shahswar ◽

Anna Stengel ◽

Manja Meggendorfer ◽

Wolfgang Kern ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Copy Number Variations ◽

Current Standard ◽

Genetic Classification ◽

Genetic Characteristics ◽

Standard Methods

Abstract Background Considering the clinical and genetic characteristics, acute lymphoblastic leukemia (ALL) is a rather heterogeneous hematological neoplasm for which current standard diagnostics require various analyses encompassing morphology, immunophenotyping, cytogenetics, and molecular analysis of gene fusions and mutations. Hence, it would be desirable to rely on a technique and an analytical workflow that allows the simultaneous analysis and identification of all the genetic alterations in a single approach. Moreover, based on the results with standard methods, a significant amount of patients have no established abnormalities and hence, cannot further be stratified. Methods We performed WTS and WGS in 279 acute lymphoblastic leukemia (ALL) patients (B-cell: n = 211; T-cell: n = 68) to assess the accuracy of WTS, to detect relevant genetic markers, and to classify ALL patients. Results DNA and RNA-based genotyping was used to ensure correct WTS-WGS pairing. Gene expression analysis reliably assigned samples to the B Cell Precursor (BCP)-ALL or the T-ALL group. Subclassification of BCP-ALL samples was done progressively, assessing first the presence of chromosomal rearrangements by the means of fusion detection. Compared to the standard methods, 97% of the recurrent risk-stratifying fusions could be identified by WTS, assigning 76 samples to their respective entities. Additionally, read-through fusions (indicative of CDKN2A and RB1 gene deletions) were recurrently detected in the cohort along with 57 putative novel fusions, with yet untouched diagnostic potentials. Next, copy number variations were inferred from WTS data to identify relevant ploidy groups, classifying an additional of 31 samples. Lastly, gene expression profiling detected a BCR-ABL1-like signature in 27% of the remaining samples. Conclusion As a single assay, WTS allowed a precise genetic classification for the majority of BCP-ALL patients, and is superior to conventional methods in the cases which lack entity defining genetic abnormalities.

Download Full-text

Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Blood ◽

10.1182/blood-2018-99-113056 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 549-549

Author(s):

Jason H. Rogers ◽

Rohit Gupta ◽

Jaime M. Reyes ◽

Lorenzo Brunetti ◽

Michael C. Gundry ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Sanger Sequencing ◽

Lymphoblastic Leukemia ◽

Clonal Cell ◽

Ribonucleoprotein Complexes ◽

Clonal Cell Lines ◽

Cell Acute Lymphoblastic Leukemia

Abstract Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models. We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D). We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/-clonal cell lines are distinct from their isogenic IKZF1-/-counterparts. Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/-cells and IKZF1IK6/+cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Engineered Bcor Mutations Lead to Acute Lymphoblastic Leukemia of Progenitor B-1 Lymphocyte Origin in a Sensitized Background

Blood ◽

10.1182/blood-2018-99-115388 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1331-1331

Author(s):

Mianmian Yin ◽

Yang Jo Chung ◽

R. Coleman Lindsley ◽

Yeulin Zhu ◽

Robert L. Walker ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Regulatory Elements ◽

Leukemic Transformation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Indel Mutation

Abstract Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway. To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes. A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. Disclosures Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

Download Full-text