Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene

The wild-type (wt) p53 tumor suppressor gene is commonly inactivated in human malignancies, either by mutations or by loss of expression. An additional proposed mechanism for inactivation of wt-p53 is amplification of the murine double minute 2 (MDM2) gene and overexpression of the MDM2 protein, which binds to p53 and eliminates its tumor suppressor function. To investigate a potential role for MDM2 in the inactivation of wt-p53 in pediatric acute lymphoblastic leukemia (ALL), we examined the expression of MDM2 and p53, as well as the occurrence of p53 mutations and possible amplification of the MDM2 gene, in 19 pediatric ALL cell lines and one pediatric acute myelogenous leukemia (AML) line. Although we did not find significant amplification of the MDM2 gene in any of the leukemic lines, we detected overexpression of MDM2 in all 10 lines that expressed wt-p53. Of the 10 lines without overexpression of the MDM2 gene, six (including the AML line) did not express p53, and four expressed mutant p53 with single point mutations in exons 7 and 8. To determine whether primary leukemic cells showed a similar correlation, we analyzed the original cryopreserved leukemic bone marrow cells from seven patients from whom cell lines were established. We obtained similar results from both the primary leukemic cells and the corresponding cell lines: overexpression of MDM2 was present in primary cells that expressed wt-p53 but not in cells that lacked expression of wt-p53. These findings suggest an important role for MDM2 in the pathogenesis of pediatric ALL in which leukemic cells express wt-p53.

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The receptor tyrosine kinase p185HER2 is expressed on a subset of B- lymphoid blasts from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia

Blood ◽

10.1182/blood.v86.5.1916.bloodjournal8651916 ◽

1995 ◽

Vol 86 (5) ◽

pp. 1916-1923 ◽

Cited By ~ 23

Author(s):

HJ Buhring ◽

I Sures ◽

B Jallal ◽

FU Weiss ◽

FW Busch ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Tyrosine Kinase ◽

Cell Lines ◽

Receptor Tyrosine Kinase ◽

Lymphoblastic Leukemia ◽

Blast Crisis ◽

Hematopoietic Cell ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

B Lymphoid

The class I receptor tyrosine kinase (RTK) HER2 is an oncoprotein that is frequently involved in the pathogenesis of tumors of epithelial origin. Here we report mRNA expression in peripheral blood and bone marrow cells from healthy donors in hematopoietic cell lines and leukemic blasts from patients with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and chronic myeloid leukemia (CML). However, cell surface expression of HER2 protein (p185HER2) was found exclusively on a subset of leukemic cells of the B-lymphoblastic lineage. p185HER2 expression was found on blasts in 2 of 15 samples from infants, 9 of 19 samples from adult patients with C-ALL (CD19+CD10+), and 1 of 2 samples from patients with pro-B ALL (CD19+CD10-), whereas none of the leukemic cells from patients with AML (0/30), T-ALL (0/7), CLL (0/5) (CD19+CD5+), or CML in chronic and accelerated phase (0/5) or in blast crisis with myeloid differentiation (0/14) were positive for p185HER2. However, cells from 3 of 4 patients with CML in B-lymphoid blast crisis (CD19+CD10+) expressed high levels of p185HER2, which was also found on the surface of the CML-derived B-cell lines BV-173 and Nalm-1. Our study shows p185HER2 expression on malignant cells of hematopoietic origin for the first time. Aberrant expression of this oncogenic receptor tyrosine kinase in hematopoietic cell types may be an oncogenic event contributing to the development of a subset of B- lymphoblastic leukemias.

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The MDM2 Antagonist Nutlin-3 Is a Potent Inducer of Apoptosis in Pediatric Acute Lymphoblastic Leukemia Cells with Wild-Type p53 and Overexpression of MDM2.

Blood ◽

10.1182/blood.v110.11.4277.4277 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4277-4277

Author(s):

Lubing Gu ◽

Ningxi Zhu ◽

Harry W. Findley ◽

Muxiang Zhou

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Cytotoxic Effect ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Wild Type ◽

Potent Inducer ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Mdm2 Antagonist ◽

Mdm2 Expression

In pediatric acute lymphoblastic leukemia (ALL), overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and chemoresistance. A recently-developed small-molecule antagonist of MDM2, nutlin-3, inhibits the MDM2-p53 interaction, resulting in induction of p53 activity and apoptosis. In the present study, we evaluated the cytotoxic effect of nutlin-3 on ALL cells with different p53 status and MDM2 expression, using 18 cell lines and 30 primary leukemia samples. We found that both ALL cell lines and primary ALL samples with wt-p53 are sensitive to nutlin-3. No cytotoxic effect of nutlin-3 was detected in ALL cells with either p53-mutant or null phenotypes. In wt-p53 ALL cells, there was a significant positive correlation between MDM2 expression levels and sensitivity to nutlin-3. Nutlin-3-induced cell death was mediated by p53-induced activation of pro-apoptotic proteins and by p53-induced repression of the anti-apoptotic protein survivin. Because p53 function is inhibited by MDM2 in chemoresistant, MDM2-overexpressing ALL cells, potent killing of these cells by nutlin-3 suggests that this agent may be a novel therapeutic for refractory ALL.

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Expression of Aberrantly Spliced Oncogenic Ikaros Isoforms in Childhood Acute Lymphoblastic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.1999.17.12.3753 ◽

1999 ◽

Vol 17 (12) ◽

pp. 3753-3766 ◽

Cited By ~ 86

Author(s):

Lei Sun ◽

Patricia A. Goodman ◽

Carla M. Wood ◽

Mya-Lisa Crotty ◽

Martha Sensel ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Dna Binding ◽

Subcellular Localization ◽

Cell Lines ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Normal Lymphocyte ◽

Wild Type

PURPOSE: We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children. PATIENTS AND METHODS: We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver–derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations. RESULTS: In each of the ALL cases, we found high-level expression of a non–DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver–derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non–DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition. CONCLUSION: Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non–DNA-binding Ikaros isoforms that are reminiscent of the non–DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.

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A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance of Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-116309 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 653-653 ◽

Cited By ~ 5

Author(s):

Jianping Li ◽

Catalina Troche ◽

Alok Swaroop ◽

Marta Kulis ◽

Jon Oyer ◽

...

Keyword(s):

Cell Cycle ◽

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Mutant Allele ◽

Epithelial Mesenchymal Transition ◽

Lymphoblastic Leukemia ◽

Rna Seq ◽

Wild Type ◽

Mesenchymal Transition ◽

Pediatric All

Abstract Acute lymphoblastic leukemia (ALL) is the most common diagnosed pediatric cancer. Despite improvements in chemotherapy that have increased the 5-year survival rate to close to 90%, 15-20% of these patients may relapse with the majority of such children succumbing to this disease. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biological processes mediated by mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in two B-ALL cell lines (RCH-ACV and SEM) and one T-ALL cell line (RPMI-8402) and inserted the E1099K mutation into three ALL cell lines (697, CEM, MOLT4). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells compared to cells in which the mutation is removed demonstrate enhanced cell growth, colony formation and migration. NSD2 mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system (CNS) in xenografts. The NSD2 mutation is found prominently in children who relapse early from therapy for ALL, and NSD2E1099K cells are particularly resistant to glucocorticoids (GC). Reversion of NSD2E1099K mutation to wild type NSD2 conferred glucocorticoid sensitivity to both B and T cell lines. GC response upon disruption of mutant NSD2 was accompanied by cell cycle arrest and apoptosis. Mice xenografted with NSD2E1099K cells were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2 wild-type cells led to significant tumor reduction and survival extension. RNA-Seq analysis showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2 wild-type cells. Furthermore, in NSD2 mutant cells, there was lower basal expression level of glucocorticoid receptor (GR) and GR levels were not significantly induced by GC. Accordingly, after treatment with GC, there was significantly less DNA-binding activity of the GR in NSD2E1099K cells than that of NSD2 wild-type cells. The key pro-apoptotic regulators Bim and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. In conclusion, these studies demonstrate that the NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by causing GC resistance. Future studies will determine how NSD2 which generally activates genes paradoxically blocks the ability of GC and the GR to induce critical pro-death genes. Disclosures Licht: Celgene: Research Funding.

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Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽

10.1182/blood.v52.4.712.712 ◽

1978 ◽

Vol 52 (4) ◽

pp. 712-718 ◽

Cited By ~ 28

Author(s):

SD Smith ◽

EM Uyeki ◽

JT Lowman

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Lymphoid Cells ◽

Assay System ◽

Leukemic Cells ◽

Specific Cell ◽

Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

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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.

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Triptolide Induces Apoptosis In Acute Lymphoblastic Leukemia Cells by Inhibition of the Oncoprotein MDM2

Blood ◽

10.1182/blood.v116.21.4934.4934 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4934-4934

Author(s):

Mei Huang ◽

Lubing Gu ◽

Muxiang Zhou

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Biological Action ◽

Leukemic Cells ◽

Transcriptional Level ◽

Antitumor Effects ◽

Mdm2 Expression ◽

Pediatric All ◽

Chinese Plant

Abstract Abstract 4934 Triptolide, a nature product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. Recent studies indicate that the antitumor activity of triptolide is associated with its biological action to inhibit expression of many oncoproteins and anti-apoptotic or survival factors that were expressed in the cancer cells. Herein, we demonstrate that triptolide induces apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells that overexpress MDM2 oncoprotein by inhibiting the MDM2 expression. In pediatric ALL, overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and resistance to conventional chemotherapeutic drugs such as doxorubicin. In the present study, we evaluated the role of triptolide in regulating MDM2 and in inducing apoptosis, as compared to doxorubicin, using ALL lines and primary ALL samples. In contrast to doxorubicin, which induced p53 activation and a subsequent upregulation of MDM2, triptolide strongly induced persistent inhibition of MDM2 followed by a steady-state activation of p53, which resulted in potent apoptosis of the MDM2-overexpressing ALL cells tested, even if they were doxorubicin-resistant. We discovered that triptolide's inhibition of MDM2 in ALL cells occurred at the post-transcriptional level through inhibition of mRNA synthesis. Because p53 function is inhibited by MDM2 in chemoresistant/MDM2-overexpressing ALL cells, potent killing of these cells by triptolide suggests that this naturally-derived agent may be a novel therapeutic for refractory ALL. Disclosures: No relevant conflicts of interest to declare.

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Targeting Survivin with YM155 As a Potential Therapy in Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2490.2490 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2490-2490

Author(s):

Abdusebur Jemal ◽

Jeffrey W Tyner ◽

Mathew Thayer ◽

Markus Muschen ◽

Brian J. Druker ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Survivin Expression ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Pediatric All

Abstract Abstract 2490 Background: Pediatric Acute Lymphoblastic Leukemia (ALL) remains the most common pediatric malignancy. Despite advances in treatment and outcomes, there continue to be subsets of patients that are refractory to standard intensive chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. Survivin, a member of the chromosome passenger complex and inhibitor of apoptosis has been shown to be over-expressed in malignant cells and in relapsed ALL. Therefore, survivin may be a potential target for therapy in pediatric ALL. The selective survivin suppressant, YM155 (Astellas) has been shown to inhibit survivin expression and activate cell death in multiple cell lines. Early phase I studies show promise in both tolerability and possible efficacy in B-cell malignancies. Therefore, this drug may have the potential of improving treatment for pediatric B-cell precursor ALL. Design/methods: Pediatric lymphoblastic cell lines, fresh primary lymphoblast cells from newly diagnosed patients with ALL and xenografted patient samples were used in this study. Cells were incubated in the presence of YM155 at doses ranging from 1nM to 10μM. Viability was measured using a standard methane-thiosulfonate viability assay. Activation of apoptosis was identified using the Guava nexin Annexin V binding assay for cell lines. Results: Treatment of ALL cell lines, primary patient samples and xenograft samples show a dose-dependent sensitivity to YM155 by both activation of apoptosis and by cell viability. IC50 doses for the majority of the samples are in the low nanomolar range (Table). Interestingly, there is some variability amongst patient samples suggesting possible variable responses in vivo. Ectopic expression of survivin in cell lines treated with YM155 rescues the effect of the drug. Further, t(9;22) positive ALL samples, including primary patient, xenograft, and dasatinib resistant samples remain significantly sensitive to YM155. For dasatinib sensitive Ph+ALL samples, combination therapy suggest an additive effect by isobologram analysis. Conclusion: Pediatric ALL samples remain sensitive to treatment with YM155 in cell lines, primary patient and xenografted samples. The results of these experiments will be used as a foundation to develop a comprehensive understanding of the mechanisms of survivin dependence in pediatric ALL. Future studies will also be designed to develop YM155 as an additional therapy for pediatric acute lymphoblastic leukemia. Disclosures: Druker: Cylene:; MolecularMD:; Novartis:; Bristol-Myers-Squibb:.

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NKX3.1 is a direct TAL1 target gene that mediates proliferation of TAL1-expressing human T cell acute lymphoblastic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20100745 ◽

2010 ◽

Vol 207 (10) ◽

pp. 2141-2156 ◽

Cited By ~ 40

Author(s):

Sophie Kusy ◽

Bastien Gerby ◽

Nicolas Goardon ◽

Nathalie Gault ◽

Federica Ferri ◽

...

Keyword(s):

T Cell ◽

Tumor Suppressor ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Gene Promoter ◽

Leukemic Cells ◽

Regulatory Sequence ◽

Human T Cell ◽

T Cell Leukemogenesis

TAL1 (also known as SCL) is expressed in >40% of human T cell acute lymphoblastic leukemias (T-ALLs). TAL1 encodes a basic helix-loop-helix transcription factor that can interfere with the transcriptional activity of E2A and HEB during T cell leukemogenesis; however, the oncogenic pathways directly activated by TAL1 are not characterized. In this study, we show that, in human TAL1–expressing T-ALL cell lines, TAL1 directly activates NKX3.1, a tumor suppressor gene required for prostate stem cell maintenance. In human T-ALL cell lines, NKX3.1 gene activation is mediated by a TAL1–LMO–Ldb1 complex that is recruited by GATA-3 bound to an NKX3.1 gene promoter regulatory sequence. TAL1-induced NKX3.1 activation is associated with suppression of HP1-α (heterochromatin protein 1 α) binding and opening of chromatin on the NKX3.1 gene promoter. NKX3.1 is necessary for T-ALL proliferation, can partially restore proliferation in TAL1 knockdown cells, and directly regulates miR-17-92. In primary human TAL1-expressing leukemic cells, the NKX3.1 gene is expressed independently of the Notch pathway, and its inactivation impairs proliferation. Finally, TAL1 or NKX3.1 knockdown abrogates the ability of human T-ALL cells to efficiently induce leukemia development in mice. These results suggest that tumor suppressor or oncogenic activity of NKX3.1 depends on tissue expression.

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Cryptic Truncating Rearrangements of the Erythropoietin Receptor in Ph-like Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.128.128 ◽

2014 ◽

Vol 124 (21) ◽

pp. 128-128

Author(s):

Ilaria Iacobucci ◽

Kathryn G. Roberts ◽

Yongjin Li ◽

Debbie Payne-Turner ◽

Marcus Valentine ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

Ex Vivo ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Erythropoietin Receptor ◽

Leukemic Cells ◽

Wild Type ◽

Fetal Liver Cells

Abstract Introduction: BCR-ABL1-like, or Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), is characterized by a gene expression profile similar to BCR-ABL1-positive ALL, with a broad range of genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We previously reported a rearrangement of EPOR, encoding the erythropoietin receptor, into the immunoglobulin heavy chain locus (IGH). The aims of this study were to define the frequency and genomic architecture of EPOR rearrangements in B-ALL and to examine their role in kinase signaling and lymphoid transformation. Methods: Whole genome and/or transcriptome sequencing was performed on 154 Ph-like ALL cases. Sanger sequencing and fluorescent in situ hybridization were used to confirm and map the EPOR rearrangements. Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of erythropoietin (EPO). EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Epor-/- fetal liver cells were transduced with empty vector, EPOR wild-type or rearranged alleles and used for erythroid colony forming unit (CFU-E) and erythroid burst-forming unit (BFU-E) assays. Luciferase-marked xenografts of human EPOR-rearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases (5.2% of Ph-like ALL) harbored rearrangements of EPOR into either the IGH or immunoglobulin kappa light chain (IGK) loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR. Such rearrangements resulted in overexpression of a C-terminal truncated receptor that retained the phosphorylation site required for STAT5 activation, but lacked multiple intracytoplasmic tyrosine residues whose phosphorylation is required for normal negative regulation of the receptor. Notably, the locations of the truncation sites overlap with those arising from inherited mutations in primary familial congenital polycythemia, in which frameshift and nonsense mutations truncate the receptor. A real-time quantitative PCR assay was established to provide a diagnostic tool and to confirm that primary leukemia cells with these EPOR rearrangements overexpress N-terminal exons but lack expression of C-terminal truncated exon eight. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/- mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Expression of wild-type or truncated EPOR in Epor-/- fetal liver cells promoted erythroid differentiation with formation of CFU-E and BFU-E colonies, indicating that truncated receptors sustain erythroid development. Xenografted EPOR-rearranged leukemic cells exhibited high levels of mutant EPOR on the cell surface, constitutive STAT5 phosphorylation and sensitivity to the JAK2 inhibitor ruxolitinib ex vivo and in vivo. Conclusions: We have identified a subset of Ph-like ALL cases characterized by rearrangements of truncated EPOR into the IGH/IGK chain loci. This represents an entirely new mechanism of EPOR deregulation and unexpectedly implicates EPOR signaling as an important factor influencing B-lymphoid malignancies that are amenable to JAK-STAT5 inhibition. Clinical trials testing ruxolitinib in ALL patients with EPOR rearrangements are warranted. Disclosures No relevant conflicts of interest to declare.

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