Single-Cell Phospho-Profiling in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia Reveals Signaling Differences in Cytogenetic and Prognostic Subgroups.

Abstract Abstract 2653 Poster Board II-629 Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in combination with surface staining in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level. We anayzed 22 xenograft samples derived from NOD/SCID-mice transplanted with primary pediatric B- cell precursor acute lymphoblastic leukemia (BCP- ALL) cells. The cells were isolated from the spleens of leukemia bearing mice and stimulated ex vivo in vitro with different stimulants and cytokines. Activation of various phosphoepitopes was analyzed by flow cytometry and compared to the basal state of unstimulated samples. TEL/AML1 fusion and MLL-rearrangements are the most common cytogenetic aberrations in childhood BCP- ALL and are associated with a good or very poor prognosis, respectively. Although there were no differences detectable in basal phosphorylation between the different cytogenetic subgroups, TEL/AML1- positive samples (n= 5) displayed a significantly lower phosphorylation of extracellular regulated kinase (ERK1/2) after stimulation with PMA (Phorbol-12-myristat-13-acetate, activator of protein kinase C) or interleukin 7 (IL-7), while they showed a significantly higher activation of p38 after stimulation with PMA, compared to samples without translocation (n= 13). Additionally, the fusion gene negative samples showed a downregulation of STAT1-phosphorylation after stimulation with interleukin 10 (IL-10) whereas the TEL/AML1-positive samples showed no change. Interestingly, the MLL- positive samples (n= 3) also did not show a difference in STAT1-phosphorylation after IL-10, but showed significantly stronger STAT1 activation in response to interferon alpha (IFN-a) compared to samples without fusion genes. Moreover, the MLL- positive samples also displayed a weaker reaction in ERK-phosphorylation after IL-7 compared to the leukemia samples without cytogenetic aberrations. Differences in other prognostic subgroups analysed include a weaker phosphorylation of p38 and JNK after anisomycin in samples where the patient initially presented with hyperleucocytosis (> 100.000 WBC/μl) (n= 3), an indicator of poor prognosis. A decrease in STAT3- activation after IL-10 was observed in samples where the patients displayed bone marrow remission on day 15 of therapy (n= 8), compared to no change in the samples of patients with > 5% residual blasts (n= 8), indicative of therapy resistance, at this timepoint. Similar to the results for the cytogenetic subgroups, there were no differences detectable at basal phosphorylation levels between the prognostic subgroups. Taken together, these data show that basal phosphorylation states of specific signaling molecules do not discriminate between the different prognostic subgroups of BCP- ALL analyzed. Cytogenetic and other prognostic subgroups however display specific profiles of signaling networks after stimulation. This strategy will prove helpful to identify mechanisms by which different subgroups with distinct clinical outcomes interpret environmental signals and hereby define pathways important for continued survival, proliferation and resistance eventually leading to novel biomarkers and targeted therapies. Disclosures: No relevant conflicts of interest to declare.

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Novel MEIS1-FOXO1 Fusion Gene in a Case of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-116354 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5283-5283

Author(s):

Chuang Jiang ◽

Jiabi Qian ◽

Wenge Hao ◽

Wei LIU ◽

Shuhong Shen ◽

...

Keyword(s):

Cell Cycle ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Residual Disease ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Functional Study ◽

Cell Precursor ◽

Foxo1 Gene

Abstract Background: Thanks to the total therapy and systemic basic-translation research, the overall survival rate in children with acute lymphoblastic leukemia (ALL) has dramatically improved to almost 90% over these past few decades. FOXO1 gene belongs to the forkhead family of transcription factors, which play roles in myogenic growth and differentiation. Translocation of FOXO1 with PAX3 has been reported in pediatric alveolar rhabdomyosarcoma. In B-cell precursor ALL, two cases with FOXO1 fusions have been identified already, while its function on ALL remains unknown. Here, we report a novel MEIS1-FOXO1 fusion gene in a case with B-ALL. Methods: Flowcytometery, karyotype, RT-PCR and fluorescence in were employed, MEIS1-FOXO1 was identified as novel fusion gene in a case of pediatric BCP-ALL. Using IL-3 dependent BaF3 cells as study model to test the leukemia transformation potential of MEIS1-FOXO1. Results: A novel MEIS1-FOXO1 fusion was identified in one cease of pediatric B-ALL. Panel next generation sequencing (NGS) showed that the leukemia clone had concurrent NRASG12D, TP53R273H, WHSC1E1099K, ABCC1R1166X, PHGR1H37P, HOXA3P219L and DSTP4606L somatic mutation. This patient was enrolled in CCCG-ALL2015 clinical trial (ChiCTR-IPR-14005706) and achieved completed remission and low minimal residual disease (MRD) level (MRD<0.01%) at day 19 from induction therapy. Functional study showed that MEIS1-FOXO1 fusion gene can potentiate BaF3 cells growth independent of IL3 supplement, as compared to those without MEIS1-FOXO1 fusion transduction. In the meanwhile, we have found that MEIS1-FOXO1 fusion gene can drive cells into S-phase with concurrent decreased G0/G1 phase, which might be its oncogenic role in leukemogenesis. Using qPCR methods, we have found that MEIS1-FOXO1 fusion gene altered the cell cycle related genes expression. Conclusions: Integrating the FOXO1-fusion reports, our data have added more evidence to underline the role of FOXO1 deregulation in the pathogenesis of acute lymphoblastic leukemia. Novel fusion of MEIS1-FOXO1 can potentiate B-ALL via cell cycle entry. Detailed mechanisms involved into the MEIS1-FOXO1 should be further investigated. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Single-Cell Developmental Classification of B-Cell Precursor Acute Lymphoblastic Leukemia at Diagnosis Reveals Predictors of Relapse

Experimental Hematology ◽

10.1016/j.exphem.2018.06.027 ◽

2018 ◽

Vol 64 ◽

pp. S33-S34 ◽

Cited By ~ 1

Author(s):

Kara Davis ◽

Zinaida Good ◽

Jolanda Sarno ◽

Astraea Jager ◽

Nikolay Samusik ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Single Cell ◽

Lymphoblastic Leukemia ◽

Cell Precursor

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NUTM1 is a recurrent fusion gene partner in B-cell precursor acute lymphoblastic leukemia associated with increased expression of genes on chromosome band 10p12.31-12.2

Haematologica ◽

10.3324/haematol.2018.206961 ◽

2019 ◽

Vol 104 (10) ◽

pp. e455-e459 ◽

Cited By ~ 4

Author(s):

Femke M. Hormann ◽

Alex Q. Hoogkamer ◽

H. Berna Beverloo ◽

Aurélie Boeree ◽

Ilse Dingjan ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Chromosome Band ◽

Cell Precursor ◽

Expression Of Genes

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PAX5-AUTS2: A recurrent fusion gene in childhood B-cell precursor acute lymphoblastic leukemia

Leukemia Research ◽

10.1016/j.leukres.2012.04.015 ◽

2012 ◽

Vol 36 (8) ◽

pp. e178-e181 ◽

Cited By ~ 17

Author(s):

Dagmar Denk ◽

Karin Nebral ◽

Jutta Bradtke ◽

Gertrud Pass ◽

Anja Möricke ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Cell Precursor

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ERG Intragenic Deletion Characterizes a Distinct Oncogenic Subtype of B-Cell Precursor Acute Lymphoblastic Leukemia with a Favourable Outcome Despite Frequent IKZF1 Deletions

Blood ◽

10.1182/blood.v120.21.121.121 ◽

2012 ◽

Vol 120 (21) ◽

pp. 121-121

Author(s):

Emmanuelle Clappier ◽

André Baruchel ◽

Jérôme Rapion ◽

Aurélie Caye ◽

Ahlème Khemiri ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Favourable Outcome ◽

Fish Analysis ◽

Cell Precursor ◽

Pcr Assay ◽

Genetic Lesion ◽

Intragenic Deletion

Abstract Abstract 121 The genetic landscape of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children above 10 years and adolescents remains poorly defined. Specifically, more than half of these patients have none of the cytogenetic abnormalities that define oncogenic subtypes and underlie risk stratification. To uncover new genetic abnormalities in these unassigned cases, we studied 85 BCP-ALL from patients aged 10 to 17 diagnosed at St-Louis hospital (Paris, France), for which the main classifying genetic lesions were assessed (i.e. high hyperdiploidy, t(12;21)/ETV6-RUNX1, t(1;19)/TCF3-PBX1, t(9;22)/BCR-ABL1, iAMP21, MLL translocations, low hypodiploidy, and near haploidy). Fifty of these BCP-ALL presented no classifying genetic lesions. Paired leukemic and remission samples could be analysed by high density array-CGH (Agilent 1M arrays) in 17 of these unassigned cases. We focused on acquired, focal, and recurrent copy-number abnormalities. A mono-allelic intragenic deletion of the ETS-related Gene (ERG) was found in 3 cases. ERG belongs to the ETS family of transcription factors and is implicated in chromosomal translocations associated with several cancer types including acute myeloid leukemia. The possibility of a cryptic unbalanced translocation was ruled out in the 3 cases by FISH analysis. The deletions encompassed exons 3 to 7, or 3 to 9, and the breakpoints were tightly clustered. Based on the breakpoint sequences we designed a PCR assay that allowed us to screen ERG intragenic deletions in the whole cohort. ERG deletion was identified in 9 additional cases, none of them having any of the known classifying genetic lesions, bringing up to 25% (12 out of 50) the frequency of ERG deletion in unassigned BCP-ALL of children older than 10. These results suggested that ERG deletion characterized a novel oncogenic subtype of BCP-ALL. Of note, these results were consistent with independent data of Harvey et al. (2010) that reported ERG deletions in a distinct gene-expression cluster. To confirm and extend these findings in the whole population of paediatric BCP-ALL, we used our breakpoint-specific PCR assay to screen ERG deletions in an independent cohort of 822 unselected patients aged 1 to 17, enrolled in the EORTC 58951 trial. ERG deletion was identified in 31/822 (3.7%) patients. Again, none of them had another known classifying genetic lesion, confirming that ERG deletion characterizes a distinct oncogenic subtype. Patients with ERG deletion were significantly older compared to other patients (median 7.0 vs 4.0, p=0.002), but they had similar white blood counts at diagnosis. They had a favourable outcome, with a 8-year event free survival (EFS) of 82.4% and overall survival (OS) of 96.0%, which is similar to EFS of 83.4% and OS of 91.6% obtained for patients having no very high risk initial features (i.e. no t(9;22)/BCR-ABL1, MLL rearrangement or haploidy/low hypodiploidy). IKZF1 deletion is a cooperative genetic lesion that has been recently shown to be associated with a poor outcome in BCP-ALL. Remarkably, the incidence of IKZF1 deletions in patients with ERG deletion was significantly higher than in other BCR-ABL1-negative patients, especially when considering the IKZF1 intragenic deletion Δ4-7 (10/31, 32.3% vs 34/744, 4.6%, P<0.001), and this regardless of age. Surprisingly, IKZF1 deletion had no impact on the prognosis of ERG deleted patients. Indeed, patients combining ERG and IKZF1 Δ4-7 deletions had a better outcome than other BCR-ABL1-negative patients with IKZF1 deletions (8-year EFS 83.3% vs 53.0%, hazard ratio (HR) 0.19, 95% CI 0.02–1.41; p=0.069). Altogether, we have identified a novel oncogenic subtype of BCP-ALL characterized by ERG deletion. This subtype is frequently associated with IKZF1 deletions, suggesting a preferred oncogenic cooperation. Importantly, despite having older age and frequent IKZF1 deletions, which are factors usually predictive of a poor prognosis, patients with ERG deletion have a favourable outcome. Therefore, this genetic abnormality may be systematically assessed as part of the diagnostic work-up of BCP-ALL and taken into account when considering treatment stratification. Disclosures: No relevant conflicts of interest to declare.

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Epigenetic Status Of CEBPA Promoter and Expression Of CEBPα Correlates With Molecular Genetic Subtype and CD2 Aberrant Expression In B Cell Precursor ALL

Blood ◽

10.1182/blood.v122.21.2573.2573 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2573-2573

Author(s):

Lucie Slamova ◽

Julia Starkova ◽

Karel Fiser ◽

Eva Fronkova ◽

Leona Rezkova Reznickova ◽

...

Keyword(s):

B Cell ◽

Early Phase ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Methylation Status ◽

Fisher Exact Test ◽

Cell Precursor ◽

Aberrant Expression ◽

Mll Gene

Abstract CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A). In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p<0.0001). Interestingly, in the only hypomethylated ETV6-RUNX1pos case we found aberrant CD2 expression on blasts, which is exceptional in ETV6-RUNX1pos ALL. We next asked whether methylation of CEBPA promoter correlates with CEBPα expression. It is generally accepted that promoter hypomethylation is often associated with increased expression of the relevant gene. Our data prove that in general, this holds true also for BCP-ALL. However, in two genetically defined subsets we observed either high expression despite hypermethylation (MLL-rearranged ALL) or low expression despite hypomethylation (mBCR-ABLpos ALL) (Figure 1B). In BCP-others hypomethylation of CEBPA promoter was significantly associated with upregulation of myeloid antigens (CD14 and/or CD33) and downregulation of B cell marker CD19 on blasts during the first weeks of the treatment (Fisher test, p=0.0009). In summary Methylation status of CEBPA promoter correlates with genetic subtypes of BCP-ALL. The notion that hypomethylation leads to overexpression was confirmed in majority of BCP-ALLs, while in mBCR-ABLpos and MLL gene rearranged BCP-ALL it did not follow this pattern. Hypomethylation of CEBPA promoter in BCP- others correlates with CD2 expression on blasts and increased CEBPα gene expression. During the early phase of the treatment in other BCP-ALLs with hypomethylated CEBPA promoter increase of myeloid and decrease of B lymphoid markers on blasts was observed. Supported by: GACR P301/10/1877, GACR P304/12/2214, GAUK 914613, UNCE 204012, NT13462, NT12397- 4, project for conceptual development of research organization (Ministry of Health, CZ) 00064203, the FACS Aria instrument was supported by EU-Prague project CZ.2.16/3.1.00/24022 Disclosures: No relevant conflicts of interest to declare.

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Modeling the evolution of ETV6-RUNX1–induced B-cell precursor acute lymphoblastic leukemia in mice

Blood ◽

10.1182/blood-2011-02-338848 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1041-1051 ◽

Cited By ~ 48

Author(s):

Louise van der Weyden ◽

George Giotopoulos ◽

Alistair G. Rust ◽

Louise S. Matheson ◽

Frederik W. van Delft ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Mouse Model ◽

B Cell ◽

Insertional Mutagenesis ◽

Fusion Gene ◽

Single Nucleotide Polymorphism Array ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Sleeping Beauty ◽

Cell Precursor

Abstract The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.

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JAK2 Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.583.583 ◽

2016 ◽

Vol 128 (22) ◽

pp. 583-583

Author(s):

Elisabeth M.P. Steeghs ◽

Isabel S. Jerchel ◽

Willemieke de Goffau-Nobel ◽

Alex Q. Hoogkamer ◽

Judith M. Boer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Induced Resistance ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Leukemic Cell ◽

Leukemic Cells ◽

Cell Precursor

Abstract Background In high risk pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, gain of function mutations and translocations affecting JAK2 have been described. These mutations and translocations result in aberrant kinase signaling and may therefore serve as an ideal target for precision medicines. Aim Evaluate the frequency and prognosis of JAK2 lesions among different subtypes of childhood BCP-ALL, and study the efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib. Methods This study comprised 77 BCR-ABL1-like cases and 76 B-other cases which were screened for JAK2 translocations using RT-PCR. Furthermore a representative pediatric cohort of 461 newly diagnosed BCP-ALL cases was screened for JAK2 mutations using targeted next-generation sequencing. Clinical analyses were performed in 341 BCP-ALL patients. Patient-derived-xenograft (PDX) cells were isolated from NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which were injected with primary leukemic cells. Purity of PDX cells was enriched to over 90% and presence or absence of JAK2 lesions was validated. PDX and primary leukemic cells were exposed to a dilution series of momelotinib or ruxolitinib for four days. Where indicated, cells were pre-incubated with 25 ng/ml TSLP for 1 hour. In mono-culture assays, cytotoxicity was quantified using MTT and in co-culture assays flow cytometry was used. Leukemic cells were discriminated from mesenchymal stromal cells (MSCs) using CD19 and viability was assessed by Annexin V and Propidium Iodide. Western blotting was used to study protein expression levels. Results JAK2 translocations were detected in 6.5% of BCR-ABL1-like cases (3 PAX5-JAK2 cases, 1 TERF2-JAK2 case and 1 BCR-JAK2 case), but not in B-other cases. JAK2 mutations were identified in 3.5% of all BCP-ALL cases, which included JAK2 mutations in BCR-ABL1-like (7.6%), B-other (11.9%), and high hyperdiploid cases (1.6%), but not in MLL rearranged, BCR-ABL1-positive, ETV6-RUNX1-positive or TCF3-PBX1-positive cases. Cumulative incidence of relapse in patients harboring JAK2 lesions was as poor as in JAK2 wildtype BCR-ABL1-like and B-other patients. Efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib was examined in JAK2 lesion positive (primary and PDX) leukemic cells. Inhibitors were cytotoxic in both translocated and mutated cells, although efficacy in JAK2 mutated cells highly depended on CRLF2 activation by TSLP. CRLF2 activation resulted in downstream STAT5 activation and sensitization towards ruxolitinib compared to unstimulated cells (p < 0.05). Cells harboring JAK2 translocations signaled independently of CRLF2. Although momelotinib and ruxolitinib exposure blocked downstream STAT1/5 phosphorylation, both inhibitors also induced accumulation of phosphorylated JAK2Y1007. Consequently, release of the inhibitors resulted in a profound re-activation of JAK2 signaling, observed by upregulation of downstream STAT1/5 signaling. Furthermore, we observed microenvironment-induced resistance. Culturing leukemic cells in the presence of primary bone marrow MSCs induced resistance to ruxolitinib, compared to leukemic cells in single cultures (p < 0.05). A similar trend was observed for momelotinib. In addition, patients harboring JAK2 mutations displayed a heterogeneous leukemic cell population. Mouse xenograft models revealed different outgrowth patterns of leukemic cells, in which the JAK2 mutated clone persisted, decreased or even disappeared, resulting in outgrowth of JAK2 wildtype leukemic cells. Moreover, JAK2 mutations were not mutually exclusive for other pathway mutations (e.g. KRAS). Conclusion JAK2 translocations and mutations were detected in poor prognostic BCP-ALL cases. In ex vivo assays, the JAK1/2 inhibitors momelotinib and ruxolitinib were cytotoxic in JAK2 aberrant cells. Despite these promising findings, we identified certain limitations of these inhibitors. Inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon their release. Furthermore, our data suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and by microenvironment-induced resistance. Taken together, our data yield important directives for the clinical use of JAK inhibitors in pediatric BCP-ALL. Disclosures No relevant conflicts of interest to declare.

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