Deranged Transcription and Replication in MYC-Induced B and T Cell Leukemias

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 2-2
Author(s):  
Arpan A. Sinha ◽  
Pilar I. Andrade ◽  
Courtney Sansam ◽  
Megan Malone-Perez ◽  
Christopher Sansam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cancer Biology ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Fluorescent Microscopy ◽  
Gene Expression Profiles ◽  
Replication Timing ◽  
Transcription And Replication

MYCis a key oncogene overexpressed by many cancers, however, its oncogenic mechanisms are poorly understood. MYC is also central to acute lymphoblastic leukemia (ALL), the most common and second most lethal pediatric malignancy. Much of MYC's oncogenicity has been attributed to its transcription factor function, but data suggest MYC also deregulates replication in transcription-independent fashion. As a known master regulator of cancer transcriptomes and epigenomes, we hypothesize that MYC dramatically alters both gene expression and replication timing (non-random spatiotemporal process where some part of the genome replicates early, and other late) in both types of ALL - B-ALL and T-ALL. Conceivably, MYC exerts oncogenic effects upon the ALL transcription and replication programs, with some changes shared by B- and T-ALL, and others unique to only one. We aim to address two novel questions not been investigated before. First, in ALL, do the same genetic loci show aberrant RNA transcriptionandDNA replication? Second, how similar are the affected loci in two closely-related, yet distinct, ALL types driven by the same oncogene? The basis of our project is a unique double-transgenicrag2:hMYC,lck:GFPzebrafish pre-clinical model we established, which is the only animal model proven to develop both B-ALL and T-ALL. We previously showed that gene expression profiles (GEP) differentiating zebrafish B- and T-ALL also distinguish human B- and T-ALL, making this an ideal model system to study human ALL. In this model, B-ALL and T-ALL are induced by human MYC(hMYC) regulated by aD.rerio(zebrafish)rag2promoter.Since B and T lymphoblasts both expressrag2, both lineages over-express MYC, causing highly-penetrant B- and T-ALL. Differential activity of aD. rerio lckpromoter causes B cells to fluoresce dimly and T cells to fluoresce brightly, allowing us to identify and purify B-ALL and T-ALL by fluorescent microscopy and fluorescence-based flow cytometry, respectively. This unique model enables comparing B- and T-ALL in one genetic background. We have purified >20 zebrafish ALL (both T-ALL and B-ALL) and isolated their RNA and DNA. We are now analyzing RNA-seq gene expression profiles (GEP) and replication timing (RT) profiles via next generation sequencing (NGS). We will compare both ALL types to identify mRNA signatures that are unique to, or shared by, both types. We seek loci that shift DNA replication from early-to-late, or late-to-early, to define the regions that replicate at the same time in both ALL types, versus loci that vary by ALL type. We will also interrogate these data to determine whether GEP and RT profiles correlate with each other, and with known MYC target genes. In conclusion, GEP and RT have never been analyzed in the same cancer sample, or in related cancers driven by the same oncogene. Exploiting our expertise with thehMYCzebrafish model, we are delineating how MYC alters transcription and replication, to ascertain if these affect the same loci and define which loci are unique to one ALL type or shared by both. MYC hyper-activity is seen ~70% of human cancers - making MYC a crucial oncogene in human cancer biology, so our findings are likely to inform not only mechanisms operative in ALL, but also other MYC-driven cancers. Disclosures No relevant conflicts of interest to declare.

MLL-Rearranged ALL Cells Are Highly Dependent On the Expression of EP300-Interacting Inhibitor of Differentiation 1 (EID1)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1462-1462
Author(s):  
Marieke Hendrika van der Linden ◽  
Pauline Schneider ◽  
Rob Pieters ◽  
Ronald Stam
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Treatment Options ◽  
Gene Expression Profiles ◽  
Pcr Analysis ◽  
Rapid Reduction ◽  
Adequate Treatment ◽  
Inhibitor Of Differentiation 1

Abstract Abstract 1462 MLL-rearranged acute lymphoblastic leukemia (ALL) remains a difficult to treat type of leukemia, for which alternative and more adequate treatment options are still urgently needed. Various genome-wide transcriptome studies, including ours, have shown that MLL-rearranged ALL patients display highly unique gene expression profiles. In search of new and valid therapeutic targets to which more adequate treatments could be developed, we constantly screen our gene expression profiling data for potential candidates. In the present study we set out to investigate EID1 (EP300-interacting inhibitor of differentiation 1), which we found highly and specifically expressed in MLL-rearranged ALL cells. Validating this observation, quantitative RT-PCR analysis confirmed that EID1 expression was significantly higher in MLL-rearranged ALL cells as compared with other ALL subtypes or healthy bone marrow samples. Next, we performed shRNA-mediated knockdown of EID1 in MLL-rearranged ALL cells, which resulted in a dramatic reduction of viable cells. Subsequently, flow cytometry analyses demonstrated that this rapid reduction of cell viability was due to massive induction of apoptosis as well as an abrupt cell cycle arrest. Consequently, it appeared difficult to study the actual function of EID1 over-expression in MLL-rearranged ALL cells. On the other hand, these features make EID1 an attractive target for therapeutic intervention in MLL-rearranged ALL. Hence, further studies on the function of EID1 in MLL-rearranged ALL and exploring the possibilities to inhibit it are warranted. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Altered Transcription and Replication Driving MYC-Induced B and T Cell Leukemias

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3315-3315
Author(s):  
Arpan A. Sinha ◽  
Clay Foster ◽  
Courtney Sansam ◽  
Pilar I. Andrade ◽  
Katie Foster ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cancer Biology ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Clustering Algorithms ◽  
Fluorescent Microscopy ◽  
Cycle Phase ◽  
Unexpected Result ◽  
Rna Seq

Abstract MYC is over-expressed by many cancers, yet its oncogenic mechanisms are incompletely understood. MYC is central to acute lymphoblastic leukemia (ALL) - the most common and second most lethal pediatric malignancy, and ALL afflicts even more adults. Much of MYC's oncogenic function is attributed to its role as a transcription factor, but MYC has been shown to deregulate DNA replication independent of transcription. As master regulator of transcriptomes and epigenomes, we predict that MYC impacts both biologic features in both ALL types, B- and T-ALL. We hypothesize that MYC alters both RNA expression and DNA replication (the ordered spatio-temporal process where genomic domains replicate in either early or late S-phase) in B- and T-ALL, and that these perturbations-some shared, others unique to one ALL type-drive leukemogenesis. Our project utilizes a unique double-transgenic rag2:hMYC, lck:GFP zebrafish ALL model that we established, which is the only animal model that develops both highly penetrant B- and T-ALL. In this model, B- and T-ALL are induced by human MYC (hMYC) that is regulated by a zebrafish (Danio rerio) rag2 promoter. Because B and T lymphoblasts each express rag2, both lineages over-express MYC, inducing B- and T-ALL. The differential activity of the D. rerio lck promoter (regulating GFP) causes B cells to fluoresce dimly and T cells to fluoresce brightly, permitting identification of B- vs. T-ALL by fluorescent microscopy and FACS-purification. Thus, we can compare B- and T-ALL in an isogenic background. We have collected 30 ALL samples (18 T-ALL, 12 B-ALL) and completed two types of analyses on 12 T-ALL and 3 B-ALL. Using RNA-seq, we established gene expression profiles (GEP) for both ALL types; principal component analysis and other clustering algorithms demonstrate B- and T-ALL are distinct. Although we analyze the entire transcriptome, we prioritize genes conserved in humans to focus on translatable targets. To assess DNA replication, we generated Replication Timing (RT) profiles by first FAC-sorting ALL cells based on cell cycle phase (G1, S, G2; defined by DNA content) and then performing whole-genome sequencing to generate RT profiles for the same ALL analyzed by RNA-seq. We identified differentially replicating regions by comparing RT of B-vs. T-ALL, revealing many loci where replication reproducibly shifts from early-to-late, or late-to-early, based on ALL type. Overall, despite their shared genetic driver (MYC) , we found RT differences that distinguish B- vs. T-ALL in ~30% of the genome. Most differences occur in large chromosomal domains, suggesting abnormal chromatin structure in ALL. An additional unexpected result was that many ALL G1 samples had read count differences across large chromosomal regions, indicating the presence of aneuploidies/large CNAs. Several were recurrent and lineage-specific (i.e., exclusive to B- or T-ALL). Together, our data demonstrate differences in RNA transcription, DNA replication, and regions of genomic instability that are lineage-specific, despite a shared MYC oncogene that drives both B- and T-ALL. We will next determine which deranged loci are also perturbed in human ALL, with an overarching goal of finding prognostic biomarkers and therapeutic targets. MYC hyper-activity occurs in ~70% of human malignancies. Thus, MYC is crucial to virtually all cancer biology, making our findings likely to inform not only the mechanisms that drive ALL, but also other cancers where MYC is oncogenic. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification and Validation of Novel Reference Genes in Acute Lymphoblastic Leukemia for Droplet Digital PCR

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 376 ◽  
Author(s):  
Vanessa Villegas-Ruíz ◽  
Karina Olmos-Valdez ◽  
Kattia Alejandra Castro-López ◽  
Victoria Estefanía Saucedo-Tepanecatl ◽  
Josselen Carina Ramírez-Chiquito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Reference Genes ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cellular Processes

Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.

scholarly journals Biosystem Analysis of the Hypoxia Inducible Domain Family Member 2A: Implications in Cancer Biology

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 206 ◽  
Author(s):  
Celia Salazar ◽  
Osvaldo Yañez ◽  
Alvaro A. Elorza ◽  
Natalie Cortes ◽  
Olimpo García-Beltrán ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Cancer Biology ◽  
Chromosomal Abnormalities ◽  
Human Cancer ◽  
Expression Profiles ◽  
Homology Modelling ◽  
B Cell Lymphoma ◽  
Qrt Pcr

The expression of HIGD2A is dependent on oxygen levels, glucose concentration, and cell cycle progression. This gene encodes for protein HIG2A, found in mitochondria and the nucleus, promoting cell survival in hypoxic conditions. The genomic location of HIGD2A is in chromosome 5q35.2, where several chromosomal abnormalities are related to numerous cancers. The analysis of high definition expression profiles of HIGD2A suggests a role for HIG2A in cancer biology. Accordingly, the research objective was to perform a molecular biosystem analysis of HIGD2A aiming to discover HIG2A implications in cancer biology. For this purpose, public databases such as SWISS-MODEL protein structure homology-modelling server, Catalogue of Somatic Mutations in Cancer (COSMIC), Gene Expression Omnibus (GEO), MethHC: a database of DNA methylation and gene expression in human cancer, and microRNA-target interactions database (miRTarBase) were accessed. We also evaluated, by using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), the expression of Higd2a gene in healthy bone marrow-liver-spleen tissues of mice after quercetin (50 mg/kg) treatment. Thus, among the structural features of HIG2A protein that may participate in HIG2A translocation to the nucleus are an importin α-dependent nuclear localization signal (NLS), a motif of DNA binding residues and a probable SUMOylating residue. HIGD2A gene is not implicated in cancer via mutation. In addition, DNA methylation and mRNA expression of HIGD2A gene present significant alterations in several cancers; HIGD2A gene showed significant higher expression in Diffuse Large B-cell Lymphoma (DLBCL). Hypoxic tissues characterize the “bone marrow-liver-spleen” DLBCL type. The relative quantification, by using qRT-PCR, showed that Higd2a expression is higher in bone marrow than in the liver or spleen. In addition, it was observed that quercetin modulated the expression of Higd2a gene in mice. As an assembly factor of mitochondrial respirasomes, HIG2A might be unexpectedly involved in the change of cellular energetics happening in cancer. As a result, it is worth continuing to explore the role of HIGD2A in cancer biology.

Early T-Cell Precursor-ALL in Adult T-ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 911-911 ◽  
Author(s):  
Martin Neumann ◽  
Sandra Heesch ◽  
Stefan Schwartz ◽  
Nicola Gökbuget ◽  
Dieter Hoelzer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Real Time ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Study Group

Abstract Abstract 911 Introduction: Recently, a small subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was described, which is closely associated with the gene expression profile of early T-cell precursors (ETPs). This subtype, termed ETP-ALL, showed a highly unfavorable outcome compared to non-ETP(='typical')-ALL. Based on the results of Coustan-Smith et al. (Lancet Oncology, 2009), the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) and St-Jude Children's hospital modified their treatment in children with ETP-ALL to a more intensive regime including stem cell transplantation. ETP-ALL is characterized by a specific immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers). Here we explored the existence of ETP-ALL in adults and further studied the molecular characteristics of this specific T-ALL subtype. Patients and methods: We examined the gene expression profiles of 86 adult T-ALL patients obtained from the Microarray Innovations in LEukemia (MILE) multicenter study (HG-U133 Plus 2.0, Affymetrix, Haferlach et al., JCO in press). In addition, bone marrow of 296 patients from the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) were analyzed by flow cytometry and expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by real-time-PCR. Results: Using the published list of differentially expressed genes in ETPs (Coustan-Smith et al. 2009) we performed unsupervised clustering analyses of the 86 T-ALL samples. A cluster of 17 samples (19.8%) displayed an ETP-associated gene expression profile and were defined as ETP-ALL. Comparing the gene expression profiles of ETP-ALL and typical T-ALL, 2065 probe sets were differentially expressed in ETP-ALL (FDR 0.05). In addition to genes used for classification, we also identified genes known to be involved in the pathogenesis of T-ALL (e.g. PROM1, BCL2, LMO2, LYL1). In particular, stem cell associated genes such as, BAALC (2.52-fold, p=0.003), IGFBP7 (2.76-fold, p=0.002) or MN1 (3.41-fold, p<0.001) were upregulated in ETP-ALL, whereas HOX11 (45-fold, p=0.004), a marker for thymic T-ALL, was downregulated. An independent cohort of 297 patient samples from the GMALL study group was examined by flow cytometry and real-time PCR. 19 (6.4%) samples revealed the ETP-ALL immunophenotype. As expected, all patient samples were found in the group of early T-ALL, representing 23.5% of all early T-ALLs. There was a significant correlation between a lower leukocyte count at first diagnosis and the classification of ETP-ALL (p=0.001). Gene expression measured by real-time-PCR was performed for genes associated with poor outcome in T-ALL: BAALC (2.11-fold, p<0.001) and IGFBP7 (3.59-fold, p=0.003) were significantly upregulated in the group of ETP-ALL. Similarly, the genes MN1 (4.52-fold, p<0.001) and WT1 (2.76-fold, p=0.036), described as poor prognostic markers in cytogenetically normal AML, were also upregulated in ETP-ALL. Conclusion: In adult T-ALL, a subset of patients shares the gene expression profil and immunophenotype of ETP-ALL, which is in line with recent findings in pediatric patients. The gene expression profile of this subset is significantly correlated to stem cell associated markers predictive for inferior outcome in T-ALL. Interestingly, adverse factors in CN-AML are also aberrantly expressed in ETP-ALL suggesting a myeloid origin of ETPs and indicating a closer relationship between ETP-ALL and AML. The prognostic impact and the determination of the most appropiate set of markers needs to be further investigated. These results support the GMALL strategy to regard early T-ALL patients as high risk with assignment to stem cell transplantation. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

Molecular Determinants of Lymph Node Microenvironment Induced Host Immune Tolerance In CLL: Role for CAV1, PTPN6, and PKCβ In the Process

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1367-1367
Author(s):  
Christine Gilling ◽  
Amit Mittal ◽  
Vincent Nganga ◽  
Vicky Palmer ◽  
Dennis D. Weisenburger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Immune Tolerance ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Receptor ◽  
Differentially Expressed ◽  
Expression Levels ◽  
Aggressive Disease

Abstract Abstract 1367 Previously, we have shown that gene expression profiles (GEP) of CLL cells from lymph nodes (LN), bone marrow (BM), and peripheral blood (PB) are significantly different from each other. Among the major pathways associated with differential gene expression, a “tolerogenic signature” involved in host immune tolerance is significant in regulating CLL progression. The genes associated with the tolerogenic signature are significantly differentially expressed in patient LN-CLL compared to BM-CLL and PB-CLL, suggesting that LN-CLL cells induce this immune tolerance. From 83 differentially expressed genes identified by GEP that are associated with immune dysregulation, we selected eleven genes (CAV1, PTPN6, PKCb, ZWINT, IL2Ra, CBLC, CDC42, ZNF175, ZNF264, IL10, and HLA-G) for validation studies to determine whether these genes are also dysregulated in the Emu-TCL1 mouse model of CLL. The results demonstrate a trend of upregulation of these genes as determined by qRT-PCR in the LN-tumor microenvironment. To further evaluate the kinetics of selected gene expression during tumor progression, we determined the expression levels of Cav1, Ptpn6, and Pkcb at 12, 24, and 36 weeks of CLL development in the Em-TCL1 mouse model. We found that the expression of all three genes increased as a function of age, indicating a correlation of gene expression with disease progression. In addition, as CLL progressed in these mice there was a marked decrease in CD4+ and CD8+ T cells. The murine data were further validated using CLL cells from the same patients with indolent versus aggressive disease indicating a similar trend in expression as CLL progressed (n=4). Furthermore, patient data analyzed by Kaplan Meier analyses of the expression levels of the selected genes indicated a significant association between down-regulation of PTPN6 (p=0.031) and up-regulation of ZWINT (p<0.001) with clinical outcome as determined by a shorter time to treatment (p<0.05). Functional analysis by knockdown of CAV1 and PKCb in primary patient CLL cells determined by MTT assay showed a decrease in proliferation following knockdown of these genes (p<0.005). Protein-interaction modeling revealed regulation of CAV1 and PTPN6 by one another. Additionally, the PTPN6 protein regulates B cell receptor (BCR) signaling and subsequently the BCR regulates PKCb. Therefore, these data from both mice and humans with CLL, argue that an aggressive disease phenotype is paralleled by expression of genes associated with immune suppression. In particular, evidence presented here suggests, dysregulation of CAV1, PTPN6, ZWINT, and PKCb expression promotes CLL progression. Disclosures: No relevant conflicts of interest to declare.

Changes in Gene Expression Profiles in Patients with 5q- Syndrome Caused by Lenalidomide Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5023-5023
Author(s):  
Monika Belickova ◽  
Jaroslav Cermak ◽  
Jitka Vesela ◽  
Eliska Cechova ◽  
Zuzana Zemanova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Differentially Expressed ◽  
Aurora A ◽  
Genes Expression ◽  
Marrow Stroma ◽  
Cell Niche

Abstract Abstract 5023 A direct effects of lenalidomide on gene expression in 5q- patients was studied using HumanRef-8 v2 Expression BeadChips (Illumina). Expression profiles of 6 patients (before treatment and at the time of the first erytroid response) and 6 healthy controls were investigated from CD14+ monocytes of peripheral blood. Differentially expressed genes were identified by Significance Analysis of Microarrays (SAM). Simultaneously, selected genes (TNF, JUN, IL1) were monitored in the course of treatment using Real-Time PCR with Taqman Gene Expression Assays. A comparison of gene expression levels before and during lenalidomide treatment revealed 97 differentially expressed genes (FC >2; p<0.05) related to following biological processes: immune response (16 genes), inflammatory response (11 genes), response to bacteria (8 genes), anti-apoptosis (7 genes), regulation of MAP kinase activity (5 genes), oxygen transport (4 genes), and regulation of cell proliferation (11 genes). An overexpression of a number of cytokines (e.g. TNF, IL8, IL1B, CCL3L, CXCL2, and TNFAIP3) was detected in patients before treatment, after lenalidomide administration expression of the majority of the up-regulated cytokine genes decreased to the control baseline level. Detected overproduction of the cytokines in 5q- syndrome may lead to an increased apoptosis of hematopoietic progenitor cells and together with excessive oxidative stress may contribute to the damage the hematopoietic niche. In the same manner, untreated patients showed suppressed expression of two genes (CXCR4, CRTAP) which play an important role in the stem cell niche. After treatment, we detected increased expression of these genes. Both the observations might explain favorable effects of lenalidomide on the bone marrow stroma defect seen in 5q- syndrome. On the other hand, a substantial increase of the ARPC1B gene (an activator and a substrate of Aurora A) expression was detected after lenalidomide treatment. Since overexpression of Aurora A leads to polyploidy and chromosomal instability, ARPC1B might play a role in the disease progression observed in some patients treated with lenalidomide. To conclude, described changes in genes expression may contribute to identification of the pathways affected by lenalidomide and to the explanation of some effects of this drug that have not been fully understood yet. Supported by grants NS/9634 MZCR, UHKT2005 00023736, MSM0021620808 and COST EUGESMA Disclosures: No relevant conflicts of interest to declare.

Kruppel-Like Factor, Klf9, Promotes Proliferation of Notch-Independent Precursor T-Cell Lymphoblastic Lymphomas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3496-3496
Author(s):  
Peggy P. C. Wong ◽  
Daniele Merico ◽  
Irina Matei ◽  
Vicki Ling ◽  
Shaheena Bashir ◽  
...  
Keyword(s):  
T Cell ◽  
Conformational Changes ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Functional Dependence ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Notch1 Signaling ◽  
The Difference ◽  
Notch1 Mutations

Abstract Abstract 3496 Notch1 signaling is required at multiple stages of normal T-lymphocyte development. Notch1 is a transmembrane receptor that is physiologically activated when Notch ligands induce conformational changes that allow Notch1 cleavage by the intramembranous γ-secretase complex, releasing active intracellular Notch1 (ICN1) fragment from the plasma membrane. Activating NOTCH1 mutations are very frequent in human and mouse T-cell lymphoblastic leukemia/lymphoma (T-LL). Typically, these mutations promote ligand-independent Notch1 cleavage by γ-secretase or increase ICN1 stability by truncating the C-terminal PEST domain. Understandably, much effort has focused on elucidating mechanisms of normal and oncogenic Notch1 signaling. However, some studies suggest that the absence of NOTCH1 mutations portends a worse prognosis for human T-LL. Therefore, we set out to define signals that promote proliferation and survival of T-LL cells lacking activated Notch1. We used Western blotting to detect γ-secretase cleaved ICN1 protein in a cohort of 35 primary T-LLs that developed spontaneously in mice lacking the Ataxia telangiectasia mutated (Atm) tumor suppressor. We identified 3 ICN1 subgroups: 63% expressed PEST-truncated ICN1 (T-ICN1); 17% expressed non-truncated ICN1 (NT-ICN1); and 20% had undetectable ICN1 (UD-ICN1), most lacked Notch1 mRNA. We confirmed the difference in Notch transcriptional activity and functional dependence between the UD-ICN1 and T-ICN1 subgroups and then compared their gene expression profiles to define pathways unique to the UD-ICN1 group. Gene set enrichment analyses revealed that UD-ICN1 T-LLs expressed higher levels of Klf9 and other transcription factors associated with a highly proliferative stage of normal T-cell development. siRNA knock-down studies demonstrated that Klf9 promoted proliferation of UD-ICN1 but not T-ICN1 T-LL cells. Collectively, these data demonstrate that Klf9 can regulate proliferation of Notch-independent T-LLs and suggest that Klf9 may provide a novel therapeutic target for human T-LLs lacking activating NOTCH1 mutations. Disclosures: No relevant conflicts of interest to declare.

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