8 research outputs found
Molecular And Therapeutic Implications Of Notch1 Signaling In Pediatric T-Cell Acute Lymphoblastic Leukemia
T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric ALL cases and is associated with early relapse and inferior outcome. The poorer prognosis of T-ALL compared to B-precursor ALL may in part reflect the lack of unique features on which to base therapy. NOTCH1 mutations are of particular interest since these were reported in 37-71% of T-ALLs. The prognostic value of NOTCH1 mutations remains controversial as both favorable and unfavorable associations were reported, whereas in other studies, there were no associations between NOTCH1 mutations and treatment outcome. We explored the impact of mutations in NOTCH1, FBW7 and PTEN on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia. We identified a high frequency of mutations in NOTCH1 (16 patients), FBW7 (5 patients) and PTEN (26 patients) in a well defined cohort of 47 pediatric T-ALL specimens. NOTCH1 mutations showed a 1.3-3.3-fold increase in activation over wild-type NOTCH1 in reporter assays; mutant FBW7 resulted in further augmentation of NOTCH1 activity. NOTCH1 and FBW7 mutations were accompanied by increases in median transcripts for NOTCH1 target genes (HES1, DELTEX1 and cMYC). However, none of these mutations were associated with treatment outcome. Increased HES1, DELTEX1 and cMYC transcript levels were associated with significant increases in the transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltransferase, Bcl-2 and dihydrofolate reductase. Our results suggest (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL and (2) that, depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.
Relapse is the most common caused of off-therapy events and is responsible for the majority of ALL treatment failures. Relapse can arise from the (i) the induction of resistance via acquisition of new genetic alterations after diagnosis, (ii) the selection and expansion of an already present resistant-subpopulation at the time of diagnosis, or very rarely as (iii) a secondary, de novo ALL. To determine the contribution of genetic alteration to the development of relapse in T-ALL, we assessed the frequency of mutations in NOTCH1 alone or in combination with mutations in FBW7 and PTEN at the time of diagnosis and relapse in 11 paired clinical T-ALL specimens. We observed that the 7 patients harboring mutations in NOTCH1 and/or PTEN at some stage in their disease had a longer remission period (13 months vs. 5.5 months), and were typically diagnosed at an early age (120 months vs. 132 months). In these 7 patients, nearly 70% of relapse appeared to be associated with the emergence of a new leukemic clone, an assumption made by the presence of a new mutation or loss of a mutation at relapse. Using real-time PCR techniques with specific hybridization probes, we were able to determine that the leukemic clone for one patient was present at the time of diagnosis, but at a very low expression level. This suggests that the clone responsible for relapse was resistant to the initial chemotherapy treatment. For another patient, the relapse clone could not be detected at diagnosis, suggesting that it was induced following chemotherapy. This study strongly warrants future studies with a larger patient cohort to systematically identify specific hallmarks of relapse.
NOTCH1 is a potentially attractive therapeutic target for T-ALL since constitutively activating effects of mutant NOTCH1 can be abolished with -secretase inhibitors (GSIs). Because of possible effects of GSIs on other cellular targets in addition to NOTCH1, we explored shRNA knockdown of NOTCH1 to identify novel NOTCH1-regulated genes that may serve as prognostic indicators or therapeutic targets in T-ALL. NOTCH1 expression was knockeddown in Jurkat T-ALL cells using lentivirus expressing shRNAs for NOTCH1 or a non-targeted control (J.ntc) sequence. NOTCH1 knockdown was verified using western blots to measure activated NOTCH1 (ICN1) protein levels, and real-time RT-PCR to measure transcript levels of known NOTCH1 targets (e.g., HES1). Two clonal sublines (J.N1KD 2-4 and J.N1KD 2-7) were identified with significantly decreased expression of NOTCH1 compared to J.ntc. The J.N1KD 2-4 and J.N1KD 2-7 sublines showed minimal changes in cell growth, cell cycle progression and apoptosis. To characterize genotypic changes accompanying NOTCH1 knockdown, we performed microarray analysis with Agilent Whole Genome oligonucleotide microarrays and microRNA (miR) HumanV2 arrays. The microarray identified Rictor, a key component to in the mTOR2 complex, as a novel downstream target of NOTCH1 signaling. Upon NOTCH1 inhibition, an increase in the expression of Rictor was observed, both at the transcript and protein levels. Initial computational analysis of the Rictor promoter suggests that NOTCH1 may regulate its expression directly (via RBPJ) or indirectly (via HES1). The miR array identified 20 miRs in J.N1KD 2-4 and J.N1KD 2-7 cells with altered expression compared to J.ntc greater than 1.5-fold (p〈0.05) and ranging from 3-to10-fold. miRs hsa-Let-7e, hsa-miR-125a-5p and hsa-miR-99b, reportedly derived from a polycistronic transcript, were decreased 10-fold accompanying NOTCH1 knockdown. Using miR qPCR, we confirmed decreased levels of hsa-miR-125a-5p and hsa-miR-99b in the J.N1KD 2-4 and J.N1KD 2-7 sublines. In conclusion, we have developed novel T-ALL cell line models to study the impact of decreased NOTCH1 levels and activity independent of GSI treatment. Our results implicate NOTCH1 in regulating levels of Rictor and hsa-miR-125a-5p, and suggest that caution may be warranted in targeting NOTCH1 with GSIs in the therapy of T-ALL, reflecting the potential promotion of cell survival via the upregulation of Rictor. The downstream effect of regulating hsa-miR-125a-5p has yet to be determined
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Transcriptome Sequence Analysis of Pediatric Acute Megakaryoblastic Leukemia Identifies An Inv(16)(p13.3;q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein As a Recurrent Lesion in 39% of Non-Infant Cases: A Report From the St. Jude Children's Research Hospital – Washington University Pediatric Cancer Genome Project
Abstract
Abstract 757
Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2. Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains. GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin. Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3). Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case. Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9). Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL. In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%).
To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation.
Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. *TAG and ALG contributed equally to this work.
Disclosures:
Biondi: BMS, Novartis, Micromed: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding. Doehner:Hoffmann La Roche: Honoraria
The genomic landscape of core-binding factor acute myeloid leukemias
Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes
An Inv(16)(p13.3q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein Defines an Aggressive Subtype of Pediatric Acute Megakaryoblastic Leukemia
To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.
► CBFA2T3-GLIS2 is a recurrent fusion gene in pediatric AMKL ► CBFA2T3-GLIS2 AMKL has a distinct expression profile and an inferior outcome ► CBFA2T3-GLIS2 induces BMP signaling and enhanced self-renewal of progenitor cell
The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.
Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL