LMO2 Oncogene Research Articles

Ldb1 is required for Lmo2 oncogene–induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia

AbstractProlonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

Abstract 2537: Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia

Abstract LIM domain only protein 2 (LMO2) is an oncogene that is overexpressed in a subset of B-cell acute lymphoblastic leukemia (B-ALL). The mechanisms that regulate LMO2 expression in B-ALL are still unknown. Here, we show that transcription of LMO2 in B-ALL is regulated by Ikaros, a tumor suppressor protein, which is encoded by the IKZF1 gene, and whose deletion is associated with development of high-risk B-ALL. The use of global chromatin immunoprecipitation coupled with the next-generation sequencing (ChIP-seq) studies in primary human B-ALL cells and in cell lines revealed a strong Ikaros occupancy at the promoter of the LMO2 gene. Ikaros binding to the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP). We tested the role of Ikaros in regulating LMO2 transcription in B-ALL using gain-of-function and loss-of-function experiments. A luciferase reporter assay with the LMO2 promoter showed that Ikaros directly represses LMO2 transcription. Overexpression of Ikaros in B-ALL via retroviral transduction was associated with strongly reduced expression of LMO2. Furthermore, Ikaros knock-down with shRNA, resulted in increased transcription of LMO2 in B-ALL cells. These results suggest that Ikaros represses transcription of LMO2 in B-ALL. Since Ikaros' function in B-ALL is negatively regulated by pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition affects Ikaros-mediated repression of LMO2. Both molecular inhibition with shRNA, and pharmacological inhibition of CK2 with a specific CK2 inhibitor, CX-4945, resulted in reduced expression of LMO2. Inhibition of CK2 was also associated with increased Ikaros occupancy at the LMO2 promoter. In high-risk B-ALL that have deletion of a single copy of the IKZF1 gene, Ikaros does not bind to the LMO2 promoter. Treatment of primary B-ALL cells that have IKZF1 haploinsufficiency restores Ikaros binding to the LMO2 promoter and results in reduced LMO2 expression. In conclusion, our data demonstrate that Ikaros and CK2 regulate transcription and overall expression of the LMO2 oncogene in B-ALL. Our results identify a novel mechanism of therapeutic action of CK2 inhibitors in high-risk B-ALL—repression of LMO2 expression via restoration of Ikaros' tumor suppressor function. Citation Format: Yali Ding, Jonathon L. Payne, Shriya Kane, Elanora Dovat, Mario Soliman, Chunhua Song, Sinisa Dovat. Regulation of LMO2 oncogene expression in high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2537.

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia

AbstractSomatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5′-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy–induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Small genomic insertions form enhancers that misregulate oncogenes

The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers.

Activation of the LMO2 Oncogene in T-ALL through a Somatically Acquired Neomorphic Promoter

LMO2is a crucial regulator of normal hematopoiesis but is progressively silenced from the early T-cell progenitor stage of thymic development. Aberrant LMO2 expression leads to a block in differentiation, increased self-renewal and aggressive T-cell acute lymphoblastic leukemia (T-ALL). Although ≈50% of T-ALL patients overexpress LMO2, this can be attributed to a cytogenetic lesion in only ≈10% of patients, leaving a significant portion mechanistically unaccounted for. We recently discovered somatic mutations that create an oncogenic super-enhancer driving TAL1 expression (Mansour et al., 2014, Science). Therefore, we investigated whether comparable mutations may be causing dysregulated LMO2 expression.Aberrant H3K27ac marks indicative of active chromatin were identified prior to and encompassing the non-coding exon 2 of the LMO2 gene by ChIP-seq in DU.528 and PF-382 T-ALL cell lines, both of which exhibit upregulated LMO2 expression but lack chromosomal lesions at this locus. Sequencing across this peak revealed a heterozygous 20bp duplication in PF-382 cells and a heterozygous 1bp deletion in DU.528 cells, located close to a region recently described as an intermediate promoter. Both indels generated a de novo MYB consensus motif. MYB ChIP-seq showed that ≥96% of reads aligned to the mutant rather than the wild-type allele, suggesting that MYB was preferentially recruited to the mutant allele. Heterozygous mutations were also detected in diagnostic samples from 3% (5/159) of pediatric and 6% (10/164) of adult T-ALL patients. Absence of the mutations in 7 available patient-matched remission samples confirmed that they were somatic. The mutations were densely distributed around highly conserved native ETS1, MYB and GATA motifs; 5 patients had an additional MYB site, 3 both a MYB and an ETS1 site, 1 an ETS1 site alone, and 4 had new RUNX1 binding sites. This suggests that this region may have a regulatory role that is exploited by the mutations to constitutively activate LMO2.All 6 mutant-positive patients with available RNA showed LMO2 overexpression as determined by qRT-PCR, consistent with the hypothesis that these mutations activate gene expression. Furthermore, monoallelic LMO2 expression could be demonstrated in DU.528 cells and 3 of 4 informative T-ALL samples using a heterozygous germline SNP. Expression of the mutations in luciferase reporter assays also indicated that they all markedly activated luciferase activity to between 2x and 57x compared to the wild-type sequence.To assess causality between the mutations and LMO2 dysregulation, we used CRISPR/Cas9 genome-editing with a guide RNA designed to target the mutant MYB site in PF-382 cells. Reduction of LMO2 expression to ≤10% of PF-382 mutant activity was observed in a clone where the mutant allele had been reverted to wild-type and in another clone with a single T>C substitution disrupting the mutant MYB binding site. Interestingly, 2 clones that increased the distance between the native and the mutant MYB sites also resulted in a reduction in LMO2 expression to 19% and 25% of PF-382 activity, suggesting that there are functionally limiting spatial constraints on the mutant MYB site in relation to other neighboring transcription factor binding sites. This was further validated by the lack of reduction in LMO2 expression in a clone where the sequence between the two MYB sites was altered but the spacing distance was unchanged.In conclusion, we have identified and functionally validated a novel recurrent mutation hotspot occurring in a non-coding site whereby introduction of additional binding sites for a number of different transcription factors drives monoallelic LMO2 overexpression from a neomorphic promoter in a substantial proportion of both adult and pediatric T-ALL patients. This mechanism of oncogene activation may be relevant to a wide variety of human cancers. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

Abstract B21: Genetic characterization and therapeutic targeting of MYC translocated pediatric T-cell acute lymphoblastic leukemia

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive class of hematologic tumors caused by malignant transformation and abnormal proliferation of T-cell progenitors. T-ALL represents 10-15% of pediatric ALL and, despite the improved survival rate during the last decades, 20% of cases still experience relapsed and therapy failure. MYC serves as a pivotal oncogene in the pathogenesis of T-ALL that can be activated through a number of direct and indirect mechanisms. For example, a rare subgroup of primary T-ALL (~1%) is characterized by T cell receptor (TCR) driven translocations that cause massive activation of the MYC oncogene and present an unfavorable prognosis, rapid disease progression and poor response to conventional therapy. Here, we performed detailed molecular genetic characterization of an extensive series of primary T-ALL that present TCR driven MYC translocations and evaluated new therapeutic strategies for this poor prognostic subtype of human leukemia. Molecular genetic characterization of a cohort of 28 pediatric T-ALL patients carrying the t(8;14)(q24;q11) translocation was performed by copy number profiling, gene expression and mutational screening. Xenograft experiments with primary patient material were used to evaluate the therapeutic relevance of JQ1 in the context of MYC translocation positive T-ALL. Copy number analysis of MYC translocated T-ALL cases revealed loss of the previously reported T-ALL tumor suppressor genes PTEN (18%), CDKN2A/B (68%) and LEF1 (7%). Moreover, about 30% of patients harbored genomic deletions that cause aberrant activation of the SIL-TAL1 or LMO2 oncogenes. Interestingly, sequencing analysis revealed no aberrations in NOTCH1 or FBXW7 and a high number (30%) of loss-of-function mutations targeting PTEN. Therefore, TCRAD-MYC translocation positive T-ALL seems to represent a NOTCH1 independent subtype of leukemia that is frequently addicted to activated PI3K/AKT signaling. In line with this notion, the t(8;14)(q24;q11) positive T-ALL cell lines KE-37 and MOLT16 lack NOTCH1/FBXW7 mutations but both present loss of PTEN. Indeed, these tumor lines show aberrant pAKT activation but lack enhanced NOTCH1 activity at the protein level. In accordance with the frequent identification of TAL1 and/or LMO2 rearrangements, genome-wide transcription profiling analysis revealed that TCRAD-MYC positive T-ALL showed a uniform gene expression signature reminiscent of late cortical thymocyte T-ALL that express CD4, CD8 and membrane CD3. Importantly, gene set enrichment analysis revealed a negative enrichment of NOTCH1 target genes in MYC translocated late cortical T-ALLs, including NOTCH3, HES4, DLL4, PTCRA and DTX1. From a therapeutic perspective, in vitro drug sensitivity screening showed that MYC rearranged T-ALL cell lines were amongst the most sensitive to JQ1 treatment (IC50 values: MOLT16, 199nM; KE37, 497nM). MYC mRNA and protein downregulation were confirmed after JQ1 treatment for both the lines. Moreover, in vivo drug treatment experiments using xenografts generated from primary t(8;14)(q24;q11) positive T-ALL patients cells showed the in vivo activity of the JQ1 against this aggressive subtype of human T-ALL, as evaluated by percentage of leukemic blasts in blood or bone marrow and spleen size. Given the high frequency of genetic abnormalities targeting the PI3K/AKT pathway in these NOTCH1 independent human T-ALL, we are currently evaluating the synergistic activity of JQ1/MK-2206 combination therapy using the same in vivo model systems. In this study, we identified TCRAD-MYC rearranged T-cell leukemia as a novel subclass of NOTCH1-independent late cortical T-ALL that frequently depend on aberrant PI3K/AKT signaling. Moreover, we showed that JQ1 might serve as a promising new therapeutic strategy for the treatment of this high-risk subtype of pediatric leukemia. Citation Format: Gloria Milani, Kaat Durinck, Filip Matthijssens, Sofie Peirs, Tim Pieters, Lindy Reunes, Beatrice Lintermans, Niels Vandamme, Tim Lammens, Yunlei Li, Claire Schwab, Susana Raimondi, Barbara De Moerloose, Yves Benoit, Geert Berx, Christine Harrison, Giuseppe Basso, Helene Cavé, Rosemary Sutton, Vahid Asnafi, Charles Mullighan, Jules Meijerink, Mignon Loh, Pieter Van Vlierberghe. Genetic characterization and therapeutic targeting of MYC translocated pediatric T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B21.

LMO2 Regulates DNA Replication in Hematopoietic Cells

Oncogenic transcription factors are major drivers in acute leukemias. These oncogenes are believed to subvert normal cell identity via the establishment of gene expression programs that dictate cell differentiation and growth. The LMO2 oncogene, which is commonly activated in T-cell acute lymphoblastic leukemia (T-ALL), has a well-established function in transcription regulation. We and others previously demonstrated that LMO1 or LMO2 collaborate with the SCL transcription factor to activate a self-renewal program that converts non self-renewing progenitors into pre-leukemic stem cells. Here we demonstrate a non-transcriptional role of LMO2 in controlling cell fate by directly promoting DNA replication, a hitherto unrecognized mechanism that might also account for its oncogenic properties.To address the question whether LMO2 controls other functions via protein-protein interactions, we performed a proteome-wide screen for LMO2 interaction partners in Kit+ Lin- cells. In addition to known LMO2-interacting proteins such as LDB1 and to proteins associated with transcription, we unexpectedly identified new interactions with three essential DNA replication enzymes, namely minichromosome 6 (MCM6), DNA polymerase delta (POLD1) and DNA primase (PRIM1). First, we show that in Kit+ hematopoietic cells (TF-1), all components of the pre-replication complex co-immunoprecipitate with LMO2 but not with SCL, suggesting a novel SCL-independent function. Second, LMO2 is recruited to DNA replication origins in these cells together with MCM5. Third, tethering LMO2 to synthetic DNA sequences is sufficient to transform these into origins of replication. Indeed, we show by DNA capture that LMO2 fused to the DNA binding domain of GAL4 is sufficient to recruit DNA replication proteins to GAL4 binding sites on DNA. In vivo, this recruitment is sufficient to drive DNA replication in a manner which is dependent on the integrity of the GAL4 binding sites. These results provide unambiguous evidence for a role of LMO2 in directly controlling DNA replication.Cell cycle and cell differentiation are tightly coordinated during normal hematopoiesis, both during erythroid differentiation and during thymocyte development. We next addressed the functional importance of LMO2 in these two lineages. Erythroid cell differentiation proceeds through different stages from the CD71+Ter119- to the CD71-Ter119+. These stages are also distinguishable by morphological criteria. We observe that LMO2 protein levels directly correlate with the proportion of cells in S phase, i.e. both LMO2 levels and the proportions of cycling cells decrease with terminal erythroid differentiation. Strikingly, lowering LMO2 levels in fetal liver erythroid progenitors via shRNAs decreases the proportion of cells in S phase and arrests Epo-dependent cell growth. Despite a drastic decrease in the numbers of erythroid precursors, these cells differentiate readily to the CD71-Ter119+ stage. Therefore, LMO2 levels dictate cell fate in the erythroid lineage, by favoring DNA replication at the expense of terminal maturation.Conversely, ectopic expression in thymocytes induces DNA replication and drives cells into cell cycle, causing differentiation blockade. Our results define a novel role for the oncogenic transcription factor LMO2 in directly promoting DNA synthesis. To our knowledge, this is the first evidence for a non-transcriptional function of the LMO2 oncogene that drives cell cycle at the expense of differentiation, favouring progenitor cell expansion in the thymus, and causing T-ALL when ectopically expressed in the T lineage. We propose that the non-transcriptional control of DNA replication uncovered here for LMO2 may be a more common function of oncogenic transcription factors than previously appreciated. DisclosuresNo relevant conflicts of interest to declare.

Identification of Candidate Oncogenes and Chromosomal Breakpoint Sequencing By Targeted Locus Amplification in T-Cell Acute Lymphoblastic Leukemia

Background: T-cell acute lymphoblastic leukemia is characterized by clonal and mutual exclusive chromosomal rearrangements that recurrently activate TAL1, LMO2, TLX1, NKX2-1, TLX3, HOXA or MEF2C oncogenes. Most of these translocations or chromosomal rearrangements occur as erroneous D-J or V-DJ rearrangement attempts of T-cell receptor beta (TCRB) or TCR alpha/delta (TCRAD) genes, mostly positioning oncogenes under the transcriptional control of TCR enhancer elements. Alternatively, oncogenes can also be activated as consequence of BCL11B chromosomal rearrangements. Although many oncogenes are known in T-ALL, the driving oncogenic lesion in particular T-ALL cases remains unknown.Aims: In this study, we aimed to clone reciprocal breakpoint sequences to elucidate cellular mechanisms that lead to recurrent BCL11B -TLX3 chromosomal translocations. Moreover, we want to identify oncogene candidates in various T-ALL patient samples with BCL11B-, TCRB- or TCRAD-translocations for which the candidate oncogene so far has not been identified.Methods: We used Targeted Locus Amplification procedure, a recently developed method that relies on the crosslinking of DNA in live cells, DNA digestion and re-ligation to allow formation of circular DNA ligation fragments and inverted polymerase chain reaction amplification from specific view-point loci. Amplified DNA fragments are sequenced by next generation sequencing, allowing sequence identification in a region covering 2MB around selected regions of interest, including TLX3, TLX1, TAL1, LMO2, BCL11B, TCRAD (TRAJ61), TCRB (TCRBC2)Results: TLA was successfully performed on 10 T-ALL patients having FISH validated TAL1 translocations (2 patients), LMO2 translocations (3 patients), TLX3 translocations (3 patients), TLX1 translocations (2 patients) or an inversion targeting NKX2.1 (1 patient). Analysis of both TAL1 translocated cases revealed a TAL1-TCRAD genomic fusion due to a classical t(1;14)(q32;q11) in 1 patient, but surprisingly reveal a TAL1-TCF7 genomic fusion due to a t(1;5)(q32;q31.1) chromosomal translocation in the second patient. For the LMO2 translocated cases, two patients showed classical LMO2-TCRAD (t(11;14)(p13;q11)) or LMO2-TCRB (t(7;11)(q35;p13) translocations, whereas the third patient presented with an unusual LMO2-BCL11B genomic fusion due to a t(11;14)(p13;q32). Two out of 3 TLX3-translocated patients had classical t(5;14)(q35;q32) translocations, whereas the TLX3 gene in the third patient was rearranged to the calcyphosine-like gene (CAPSL, which flanks the IL7Ra gene) on chromosomal 5p13.2 due to a t(5;5)( p13.2;q35) or an inv(5). One patient had an inversion on chromosome 14, i.e. inv(14)(q11;q13), that brings the NKX2.1 oncogene under the transcriptional control of the TCRAD enhancer. Finally, one TLX1-rearranged patient had a classical TLX1-TCRAD translocation, whereas the other presented with a chromosomal inversion involving the chromosomal band 10q24 (which included TLX1), but also revealed a novel translocation involving the centromere protein P gene (CENPP) on chromosome 9q22.31 with the TCRAD locus.Summary/Conclusions: Targeted Locus Amplification identification of chromosomal rearrangements and genomic breakpoint sequences reveals novel complex translocations in 3 out of 10 T-ALL cases analyzed thus far, indicating higher complexity of chromosomal translocations of known T-ALL oncogenes as thus far anticipated. It further proved a useful tool to identify novel translocation partners from various loci such as the TCR or BCL11B genes that are recurrently involved in these chromosomal rearrangements in T-ALL. Cloning of molecular translocation breakpoints of diagnostic T-ALL patient samples may further provide excellent minimal residual disease markers for disease monitoring during the course of treatment. DisclosuresSplinter:Cergentis BV: Employment. van Min:Cergentis BV: Employment.

445. A Delectable Lentiviral Vector System for Testing Oncogene Addiction in Hematological Malignancies

Mouse models of hematological malignancies can be established by overexpressing oncogenes in hematopoietic cells followed by transplantation. It is often unclear whether the oncogene is only necessary for initiating the malignancies or if it is also required for maintenance, known as oncogene addiction. An addicted oncogene could then serve as target for therapy. We have developed a dual lentiviral vector system to distinguish these possibilities. We chose the oncogene LMO2 as our model because it is involved in a number of leukemias caused by gamma-retroviral vector insertion in gene therapy of SCID-X1 and Wiskott-Aldrich syndrome. Our previous studies showed that overexpression of LMO2 using a gamma-retroviral vector in early thymic progenitors (ETP) caused a differentiation block at the DN2 stage of development and subsequent transplant of these cells gave rise to leukemia with 100% penetrance in mice. We inserted the LoxP sequence into the 3’ U3 region of a self-inactivating CL20 lentiviral backbone and subcloned a MSCV-LMO2-i-GFP expression cassette into the vector (Fig 1A) to make a CL20-tLMO2-i-GFP vector. To deliver Cre recombinase in an inducible fashion, we constructed a second lentiviral vector in which a MSCV-CreERT2-i-mCherry expression cassette is inserted. Vectors were made by transiently transfecting 293T cells and the unconcentrated titer of these vectors were 2E6 and 3E6 TU/ml, respectively. We first tested these vectors in NIH3T3 cells. NIH3T3 cells transduced with these two vectors were subsequently sorted for GFP+mCherry+ double positive cells. We then treated the cells with various concentrations of 4-hydroxytamoxifen (4-OHT) to induce nuclear entry of Cre recombinase in order to delete the MSCV-LMO2-i-GFP expression cassette. Seven days later, 79.3% of the cells lost GFP expression in the group that was treated with 100nM 4-OHT (Fig 1B), indicating efficient cassette deletion. The loss of LMO2 protein is also confirmed by Western blot (Fig 1C). We then transduced ETP cells with the CL20-tLMO2-i-GFP vector and cultured the cells on an OP9-DL1 stroma expressing the Delta-like ligand DL1 to induce differentiation. At day 14, 90.5% of the transduced cells were blocked at the DN2 stage, demonstrating that this deletable lentiviral vector was able to express sufficient levels of LMO2 to induce the DN2 block (Fig 1D). These cells have been transplanted into sublethally irradiated Rag2-/-IL2rg-/- mice to establish the leukemia. One leukemia has been established with a GFP+ tumor, enlarged spleen of 0.534g and an erythroid phenotype as analyzed via flow cytometry. Tumor cells have been transduced with CreERT2 vector for secondary transplant and treatment with tamoxifen to establish whether LMO2 is required for tumor maintainence. In summary, we have established a dual lentiviral vector system that can be used conveniently to study oncogene addiction in hematological malignancies. View Large Image | Download PowerPoint Slide

Genetic barcode sequencing for screening altered population dynamics of hematopoietic stem cells transduced with lentivirus

Insertional mutagenesis has been associated with malignant cell transformation in gene therapy protocols, leading to discussions about vector security. Therefore, clonal analysis is important for the assessment of vector safety and its impact on patient health. Here, we report a unique approach to assess dynamic changes in clonality of lentivirus transduced cells upon Sanger sequence analysis of a specially designed genetic barcode. In our approach, changes in the electropherogram peaks are measured and compared between successive time points, revealing alteration in the cell population. After in vitro validation, barcoded lentiviral libraries carrying IL2RG or LMO2 transgenes, or empty vector were used to transduce mouse hematopoietic (ckit+) stem cells, which were subsequently transplanted in recipient mice. We found that neither the empty nor IL2RG encoding vector had an effect on cell dynamics. In sharp contrast, the LMO2 oncogene was associated with altered cell dynamics even though hematologic counts remained unchanged, suggesting that the barcode could reveal changes in cell populations not observed by the frontline clinical assay. We describe a simple and sensitive method for the analysis of clonality, which could be easily used by any laboratory for the assessment of cellular behavior upon lentiviral transduction.

Abstract 3598: Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele.

Abstract Mouse thymic lymphoma (TL) has been used for study of radiation carcinogenesis because of its high incidence. The lymphoma or leukemia is a mouse model of human T-cell acute lymphoblastic leukemias (T-ALL), which is a malignant clonal expansion of thymocytes that accounts for about 15% of ALL cases. Ionizing radiation can damage DNA, and the cells with damaged DNA are assumed to be the cells of origin in tumors. However, which cell in the thymus or the bone marrow leads to TL remains obscure. Bcl11b gene encodes zinc-finger transcription proteins, mutations or deletion of which has been found in 10-16% of T-ALL. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes within atrophic thymus. Resembling clonal expansion of thymocytes was reported in the T-ALL model overexpressing LMO2 oncogene. Interestingly, LMO2-induced clonally expanding thymocytes persist long-term and comprise cells with the stem cell-like self-renewal property that function for the bone marrow stem cells before TL development. In this study, we developed Bcl11bflox/+;Lck-Cre and Bcl11bflox/+;CD4-Cre mice, in which loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage without expression of CD4 and CD8 cell surface markers and at the CD4+CD8+ double-positive (DP) stage, respectively. Those mice were subjected to γ-irradiation of 3Gy at 8 weeks of age and their thymocytes were characterized of clonal expansion, differentiation and cell number. The clonal expansion was observed in only the former mice, suggesting the origin of the premaligant thymocytes not in the DP cells, the cell type seen in a majority of TL. The clonally expanded thymocytes possessed common rearrangement sites at the TCRβ locus but they underwent rearrangement at various different sites at the TCRα locus, and their majority showed mature phenotypes, a higher expression of TCRβ and the CD8 expression. This suggests the presence of a subpopulation of immature thymocytes that is capable to rearrange DNA at the TCRα locus in DP cells to continuously produce further differentiated thymocytes. We also found the decrease in thymocyte number in irradiated Bcl11bflox/+;Lck-Cre mice. This probably reflects the reduced proliferation of bone marrow-derived progenitors, and this reduction may weaken the competition in niche with premaligant thymocytes and help for them to survive and proliferate. Citation Format: Ryo Kominami, Rieka Go, Satoshi Hirose, Yukio Mishima. Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3598. doi:10.1158/1538-7445.AM2013-3598

Modelling acute leukemias in mice: clonal evolution and the emergence of leukemic stem cells.

Modelling acute leukemias in mice: clonal evolution and the emergence of leukemic stem cells.

C-Myc Inhibition Targets the Leukemia-Initiating Cells in a Tal1/Lmo2 mouse Model of T-ALL

Abstract 1880Thymic expression of the Tal1 and Lmo2 oncogenes in mice results in rapid development of T-ALL, and similar to T-ALL patients, over half the leukemic mice develop spontaneous mutations in Notch1. We have previously shown that Notch1 inhibition reduces and in some cases eliminates leukemia-initiating cells (L-ICs), however the use of anti-Notch1 therapies in the clinic has been limited due to on-target effects of Notch inhibition in the intestine. Due to the toxicities associated with Notch inhibition in vivo, we hypothesized that it may be more effective to inhibit c-Myc, a critical Notch1-regulated gene in T-ALL. We demonstrate that silencing of c-Myc in primary mouse T-ALL cells significantly prolongs survival by reducing L-IC frequency and self-renewal. A novel bromodomain inhibitor, JQ1, has been developed and shown to inhibit c-Myc in multiple hematopoietic malignancies, but its effects on LSCs or L-ICs remain unclear. Our preliminary studies reveal that JQ1 treatment prolongs survival in our mouse T-ALL model, at least in part, by reducing c-Myc mRNA and protein levels. Whether JQ1 has anti-L-IC activity in mouse T-ALL models and relapsed pediatric T-ALL patient samples will be discussed. Disclosures:No relevant conflicts of interest to declare.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Notch1 inhibition targets the leukemia-initiating cells in a Tal1/Lmo2 mouse model of T-ALL

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy largely caused by aberrant activation of the TAL1/SCL, LMO1/2, and NOTCH1 oncogenes. Approximately 30% of T-ALL patients relapse, and evidence is emerging that relapse may result from a failure to eliminate leukemia-initiating cells (LICs). Thymic expression of the Tal1 and Lmo2 oncogenes in mice results in rapid development of T-ALL; and similar to T-ALL patients, more than half the leukemic mice develop spontaneous mutations in Notch1. Using this mouse model, we demonstrate that mouse T-ALLs are immunophenotypically and functionally heterogeneous with approximately 1 of 10 000 leukemic cells capable of initiating disease on transplantation. Our preleukemic studies reveal expansion of Notch-active double-negative thymic progenitors, and we find the leukemic DN3 population enriched in disease potential. To examine the role of Notch1 in LIC function, we measured LIC activity in leukemic mice treated with vehicle or with a γ-secretase inhibitor. In 4 of 5 leukemias examined, Notch inhibition significantly reduced or eliminated LICs and extended survival. Remarkably, in 2 mice, γ-secretase inhibitor treatment reduced LIC frequency below the limits of detection of this assay, and all transplanted mice failed to develop disease. These data support the continued development of Notch1 therapeutics as antileukemia agents.

Targeting the Leukemia Initiating Cells in a Mouse T-ALL Model.

Abstract 3474Poster Board III-411T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that is largely caused by aberrant activation of the TAL1/SCL, LMO1/2 and NOTCH1 oncogenic pathways. Although most patients respond to cytotoxic therapy, 20-30% relapse and currently we cannot distinguish children likely to relapse from those who will respond to therapy. Evidence is emerging that some malignancies are driven by a rare initiating population that is capable of extensive self-renewal and is resistant to conventional chemotherapy. Although the leukemia-initiating cells (L-ICs) have been well documented in AML and CML, it is unclear whether T-ALL is driven by a rare L-IC and whether relapse reflects an inability to eliminate L-ICs. To determine whether T-ALL is driven by a rare L-IC, we utilized a murine model of T-ALL in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development via E47/HEB inhibition and 75% of tumors develop spontaneous gain of function mutations in Notch1. We have shown that treatment with γ-secretase inhibitors (GSIs) to inhibit Notch1 activity induces apoptosis of mouse T-ALL cells in vitro and when administered in vivo extends the survival of leukemic mice. We demonstrate that clonal mouse T-ALL tumors are phenotypically heterogeneous, containing immature CD4- and CD8-negative, double negative (DN) 3 and 4 thymic progenitors and differentiated double positive (DP) and/or single positive (SP) leukemic blasts. Importantly, the DN3 or DN4 progenitors are maintained upon serial transplantation of the tumor into syngeneic recipient mice. Injection of serial dilutions of murine T-ALL cells reveal that the tumors are also functionally heterogeneous; with 1/5000-1/50,000 tumor cells exhibiting leukemia initiating activity. We found the CD44-, CD25+ DN3 progenitors enriched in disease potential, whereas mice injected with DP leukemic blasts failed to develop leukemia. Consistently, our preleukemic studies reveal a 3.5-fold increase in the percentage of Notch1 active, DN3/4 thymic progenitors, raising the possibility that Notch1 drives L-IC growth. Collectively, these studies suggest that activation of the Notch1-c-Myc pathway may confer self-renewal capabilities on committed thymic progenitors. The effects of Notch inhibitors on mouse L-IC survival and activity will be discussed. Disclosures:No relevant conflicts of interest to declare.

EBV MicroRNA Expression in Virus Driven B-Cell Differentiation and Lymphomagenesis.

Abstract 93Lymphomagenesis is a complex process, in part reflecting the nature of the transforming event, as well as the developmental stage of the cell. In the B-cell differentiation represents a continuum that is initiated when a naïve B-cell encounters antigen, undergoes a germinal centre (GC) reaction and ends with terminal differentiation into either a memory or plasma B-cell. Interruption of this process by a transforming event may result in a clonal proliferation where differentiation of the cell is blocked at this stage. The majority of B-cell lymphomas are derived from GC or post-GC B-cells. As physiologically relevant human models that emulate the various stages of B-cell differentiation are lacking we rationalized that in-vitro utilization of the B-cell lymphotrophic Epstein-Barr virus (EBV) would provide insights into this process. In one scenario, EBV infects naïve B-cells and drives a differentiation process paralleling the GC reaction through a well-characterized series of latency gene expression programs. EBV is also implicated in a range of GC and post-GC derived B-cell lymphomas (including Burkitt's, Hodgkin's, PTLD and DLBCL). Using high efficiency EBV infection of isolated naïve B-cells from EBV seronegative subjects, we have demonstrated that EBV infection provides a highly relevant in-vitro model that accurately reflects three distinct phases in the GC differentiation process. Alterations in the expression of a broad range of genes associated with the differentiation of the naïve B-cell were observed within 24 hours of infection and within four days of infection a process exhibiting many similarities to the GC reaction had taken place. These included BCL6, the levels of which were rapidly down-regulated within 24 hours indicating activation of the naïve B-cell. Levels of the memory cell marker CD27 steadily increased over 24 to 96 hours, while BLIMP1 expression increased, peaking at 48 hours. An increase in AID expression over 8 to 48 hours was consistent with somatic hypermutation and isotype switching. Finally a dramatic elevation in expression of the GC associated oncogene LMO2 was observed after two days followed by an equally dramatic downregulation after two weeks. Within two weeks of infection (phase 1), B-cells progressed through a GC-like phase followed by a one week transition state (phase 2) after which continued culture resulted in further differentiation to cells with the phenotypic hallmarks of post-GC cells (phase 3). MicroRNAs (miRNAs) are small non-coding RNAs, which act as negative regulators of gene expression. miRNA expression reflects the developmental lineage and differentiation state of several human cancers and over-expression is implicated in lymphomagenesis. They are also associated with the development of the GC reaction. EBV expresses at least 39 unique miRNAs from the BART and BHRF1 clusters within the viral genome. These EBV miRNAs are differentially expressed in tumour cell lines, suggesting roles during EBV-driven B-cell differentiation and lymphomagenesis. The relationship between EBV miRNAs and the kinetics of EBV driven B-cell differentiation has not been characterized. In our model we find distinct miRNA expression kinetics, coincidental with gene expression changes during B-cell differentiation, suggesting that these regulatory molecules may be involved in the GC process. Although a small number of EBV miRNAs were expressed at low levels early in the GC-like phase 1, the majority were up-regulated during the transition phase 2, exhibiting a subsequent partial down-regulation in the post-GC-like phase 3. The three phases were coincident with differential BART and BHRF1 promoter usage and alternate splicing. Strikingly, application of the infection model to primary patient samples and lymphoma cell-lines revealed that lymphomas clustered within distinct phases, reflecting the full continuum of the B-cell differentiation process. Interestingly, the majority of PTLD samples clustered within the transition phase, whereas Burkitt's and Hodgkin's lymphoma sample segregated with the GC stage. Application of our gene expression and miRNA data to cell-lines and a range of GC and post-GC EBV-positive lymphomas of various histological types indicate that our B-cell differentiation model can be used to accurately classify B-cell lymphomas in a physiologically relevant manner according to the stage of arrested B-cell differentiation. Disclosures:No relevant conflicts of interest to declare.

JAK2 phosphorylates histone H3Y41 and excludes HP1α from chromatin

Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. Here we show that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1alpha (HP1alpha), but not HP1beta, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1alpha at the same site. Tauhese results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia.

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2locus

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 104 cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.