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Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs), leading to the development of leukemia stem cells. Previously, using a zebrafish model of AE and a whole-organism chemical suppressor screen, we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here, we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition, treatment with nimesulide, a COX-2 selective inhibitor, dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary, our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation, growth, and self-renewal, and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments.

Acute myeloid leukemia (AML) is a clonal disease characterized by heterogeneous involvement of hematopoietic stem cell/progenitor cell populations. Using FLT3 internal tandem duplication (FLT3/ITD) as a molecular marker, we tested the hypothesis that clinical outcome in AML correlates with disease involvement of CD34(+)/CD33(-) precursors. Diagnostic specimens from 24 children with FLT3/ITD-positive AML were sorted by fluorescence-activated cell sorting (FACS), and resultant CD34(+)/CD33(-) and CD34(+)/CD33(+) progenitors were analyzed directly and after colony-forming cell (CFC) assay for the presence of FLT3/ITD. FLT3/ITD was present in all CD34(+)/CD33(+) patient samples. In contrast, FLT3/ITD was detected in CD34(+)/CD33(-) progenitors in only 19 of 24 samples. A bipotent progenitor was affected in a subset of patients, as evidenced by the presence of FLT3/ITD in both granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies. Those patients in whom CD34(+)/CD33(-) precursors harbored the FLT3/ITD had worse clinical outcome; actuarial event-free survival (EFS) at 4 years from study entry for those patients with and without FLT3/ITD detection in CD34(+)/CD33(-) progenitors was 11% +/- 14% versus 100% +/- 0%, respectively (P = .002). This study suggests that FLT3/ITD involvement in CD34(+)/CD33(-) precursors is heterogeneous and that detection of the mutation in the less-mature progenitor population may be associated with disease resistance.

Activating mutations of receptor tyrosine kinases (RTKs) and their downstream affectors are common in acute myeloid leukemia (AML). We performed mutational analysis of FLT3, c-kit, c-fms, vascular endothelial growth factor (VEGF) receptors (Flt-1, KDR [kinase domain receptor]), and ras genes in a group of 91 pediatric patients with AML treated on Children's Cancer Group clinical trial CCG-2891. Forty-six percent of patients had activating mutations of FLT3 (24.5%), c-kit (3%), or ras (21%) genes. Mutation-positive patients had a higher median diagnostic white blood cell (WBC) count (71.5 vs 19.6 × 109/L; P = .005) and lower complete remission rate (55% versus 76%; P = .046) than mutation-negative patients. The Kaplan-Meier estimate of overall survival (OS) for patients with and without an activating mutation was 34% versus 57%, respectively (P = .035). However, within this group, patients with FLT3/ALM (activation loop mutation) had good outcomes (OS, 86%). Exclusion of the FLT3/ALM from analysis decreased the OS for the remaining mutation-positive patients to 26% (P = .003). Ten of the 23 mutation-positive and 11 of the 34 mutation-negative patients received an allogeneic bone marrow transplant (BMT) in first complete remission (CR). In the mutation-positive group, the disease-free survival (DFS) for the allogeneic BMT recipients was 72% versus 23% for the 13 patients who received chemotherapy or autologous BMT (P = .01). DFS for the mutation-free patients with and without allogeneic BM transplantation was 55% and 40%, respectively (P = .38). Activating mutations in the RTK/ras signaling pathway are common in pediatric AML, and their presence may identify a population at higher risk of poor outcome who may benefit from allogeneic BM transplantation.

A genome-wide screening for loss of heterozygosity (LOH), a marker for possible involvement of tumor suppressor genes, was conducted in 53 children with de novo acute myelogenous leukemia (AML). A total of 177 highly polymorphic microsatellite repeat markers were used in locus-specific polymerase chain reactions. This comprehensive allelotyping employed flow-sorted cells from diagnostic samples and whole-genome amplification of DNA from small, highly purified samples. Nineteen regions of allelic loss in 17 patients (32%) were detected on chromosome arms 1q, 3q, 5q, 7q (n = 2), 9q (n = 4), 11p (n = 2), 12p (n = 3), 13q (n = 2), 16q, 19q, and Y. The study revealed a degree of allelic loss underestimated by routine cytogenetic analysis, which failed to detect 9 of these LOH events. There was no evidence of LOH by intragenic markers for p53, Nf1, or CBFA2/AML1. Most lymphocytes lacked the deletions, which were detected only in the leukemic myeloid blast population. Analysis of patients' clinical and biologic characteristics indicated that the presence of LOH was associated with a white blood cell count of 20 x 10(9)/L or higher but was not correlated with a shorter overall survival. The relatively low rate of LOH observed in this study compared with findings in solid tumors and in pediatric acute lymphoblastic leukemia and adult AML suggests that tumor suppressor genes are either infrequently involved in the development of pediatric de novo AML or are inactivated by such means as methylation and point mutations. Additional study is needed to determine whether these regions of LOH harbor tumor suppressor genes and whether specific regions of LOH correlate with clinical characteristics. (Blood. 2001;98:1188-1194)

The Flt3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication of the Flt3 gene (Flt3/ITD) has been reported in acute myelogenous leukemia (AML) and may be associated with poor prognosis. We analyzed diagnostic bone marrow specimens from 91 pediatric patients with AML treated on Children's Cancer Group (CCG)-2891 for the presence of the Flt3/ITD and correlated its presence with clinical outcome. Fifteen of 91 samples (16.5%) were positive for the Flt3/ITD. Flt3/ITD-positive patients had a median diagnostic white count of 73 800 compared with 28 400 for the Flt3/ITD-negative patients (P = .05). The size of the duplication ranged from 21 to 174 base pairs (bp). Nucleotide sequencing of the abnormal polymerase chain reaction products demonstrated that all duplications involved exon 11 of theFlt3 gene and also preserved the reading frame. Lineage restriction analysis revealed that Flt3/ITD was not present in the lymphocytes, suggesting a lack of stem cell involvement for this mutation. None of the Flt3/ITD-positive patients had unfavorable cytogenetic markers, and there was no predominance of a particular FAB class. The remission induction rate was 40% in Flt3/ITD-positive patients compared with 74% in Flt3/ITD-negative ones (P = .005). The Kaplan-Meier estimates of event-free survival at 8 years for patients with and without Flt3/ITD were 7% and 44%, respectively (P = .002). Multivariate analysis demonstrated that presence of the Flt3/ITD was the single most significant, independent prognostic factor for poor outcome (P = .009) in pediatric AML.

Associated with approximately 8-13% cases of acute myeloid leukemia (AML), t (8;21) results in the formation of the fusion oncoprotein AML1-ETO (AE), which appears to promote hematopoietic precursor expansion through aberrant regulation of canonical AML1 targets. Multiple lines of evidence indicate AE is insufficient for leukemogenesis and requires cooperative secondary mutations. Interstitial deletions of chromosome 9 are one of the more frequent associated cytogenetic abnormalities seen with t(8;21). We previously identified that knockdown of TLE1 and TLE4, members of the Groucho/TLE family of co-repressors residing in a commonly deleted region of chromosome 9 in t (8;21) del (9q) AML cooperates with AE to drive myeloblastic proliferation and cause a leukemic phenotype. In this study, we show how TLE4 loss in the context of AML1-ETO expression results in cell proliferation, decreased apoptosis, blocked differentiation, and increased resistance to chemotherapy and that these effects are mediated through an upregulation of cyclooxygenase (COX)-dependent inflammation. TLE4 knockdown via shRNA (shTLE4) in Kasumi-1 cells (derived from a patient with t (8;21) AML) resulted in increased cell cycling and decreased cell death that is concomitant with significantly lower levels of p16Ink4 and p27Kip1 expression, both major regulators of the G1/S cell cycle checkpoint. RNAseq with GSEA analysis identified enrichment of pathways related to IFN signaling and inflammation within upregulated genes in shTLE4 cells compared to control. Filtering for genes with log2 fold change greater than 0.6, we identified three genes related to inflammation (PTGER4, CEBPb, FOS). As demonstration of the relevance of these findings, we showed these expression levels were also higher in primary human del (9q) and t (8;21) del (9q) leukemia samples compared to normal CD34+ cells; revealing a potential leukemogenic role for inflammatory pathways in del (9q) and t (8;21) del (9q) AML. Using a sub-lethal dose of indomethacin (50uM INDM), we found modulating the COX-dependent inflammatory axis decreased growth of shTLE4 Kasumi-1 cells, decreased cell cycling, and increased dead populations. Concomitantly, INDM treatment was able to reverse shTLE4-induced increases in PTGER4, CEBPb, FOS, and IL1b expression to levels similar to those in control cells. Given the ability to induce myeloblastic proliferation with TLE knockdown and AE expression in zebrafish, we investigated whether shTLE4 causes changes in key myeloid differentiation regulators in Kasumi-1 cells. qPCR analysis demonstrated significantly repressed ELANE, MPO, and PU.1 expression in shTLE4-treated cells, which is relieved with the addition of INDM. TLE4 knockdown in HL60 cells (derived from t (15;17) PML/RARA leukemia) increased levels of FOS, IL1b, CEBPb, and PTGER4. At the same time, shTLE4 significantly reduced CD11b+ and CD11b+ CD14+ populations while decreasing expression of PU.1 and MYB - demonstrating a similar inflammatory gene signature associated with a myeloid differentiation block found in the Kasumi-1 model. We next wanted to examine whether inhibiting COX-dependent inflammation can be synergistic with existing therapies. INDM significantly increased the cell death and apoptosis seen in both control cells as well as cells with TLE4 knockdown treated with AraC. The loss of TLE4 conferred resistance to combination treatment with indomethacin and AraC as evidenced by increased cell proliferation and decreased cell death. However, we observed that cells with TLE4 knockdown were significantly more sensitive to the combination of sunitinib and INDM, pointing to a potentially more therapeutic and less toxic treatment regimen for this otherwise resistant cell population. In this study, we have demonstrated that loss of TLE4 appears to contribute to leukemogenesis through differentiation arrest and increased proliferation mediated through upregulation of COX-dependent inflammatory pathways. Our findings suggest that COX inhibition may serve as an effective adjuvant therapy for AML. Disclosures No relevant conflicts of interest to declare.

Deletions on chromosome 9q are seen in a subset of acute myeloid leukemia (AML) cases and are specifically associated with t(8;21) AML. We previously defined the commonly deleted region in del(9q) AML and characterized the genes in this interval. To determine the critical lost gene(s) that might cooperate with the AML1-ETO fusion gene produced by t(8;21), we developed a set of shRNAs directed against each gene in this region. Within this library, shRNAs to TLE1 and TLE4 were the only shRNAs capable of rescuing AML1-ETO expressing U937T-A/E cells from AML1-ETO–induced cell-cycle arrest and apoptosis. Knockdown of TLE1 or TLE4 levels increased the rate of cell division of the AML1-ETO–expressing Kasumi-1 cell line, whereas forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells. Our data are consistent with a model in which haploinsufficiency of these TLEs overcomes the negative survival and antiproliferative effects of AML1-ETO on myeloid progenitors, allowing preleukemic stem cells to expand into AML. This study is the first to implicate the TLEs as potential tumor suppressor genes in myeloid leukemia.

Abstract 1202 Transducin-like/Enhancer of Split 4 (TLE4), a homolog of the master developmental regulator in Drosophila, Groucho, is a co repressor previously described by our group as the putative tumor suppressor lost in a subset of t(8:21) AML patients also harboring 9q deletion. TLE4 has been found to be of fundamental importance in the repressive function of multiple highly conserved transcriptional complexes, including Hes1, Tcf/Lef, CSL, Runx1-3, and Pax5, all of which have been shown to be intimately connected to normal steady state hematopoiesis and deregulated in malignancy. Owing to its deletion in leukemia and its ability to confer functionality to a variety of signaling networks, our lab sought to investigate its role in hematopoiesis by generating a murine knockout model for Tle4. While normal at birth, within 2–3 weeks of age knockout (KO) animals become runted and eventually moribund by the time of weaning. Gross examination of the spleen and thymus in 3 week old animals reveals significant atrophy of these organs with severe abnormalities in normal tissue architecture. Tle4−/− mice lack clear demarcations between medullary and cortical regions of the thymus, in addition to having a significant increase in apoptosis seen via TUNEL staining. FACS analysis of the thymus shows CD4/CD8 double positive cells are heavily reduced in KOs, with an increase in both single positive mature populations. Additionally, there is a partial, but not complete, block in T cell progenitor development through TCR rearrangement (DN2-DN3 stages). Structural defects are seen in the spleen as well, with a failure in the formation of follicular zones. B cells are significantly reduced with an increase in CD11b+ cells in the spleen, in addition to a shift in the percentages of B1, B2, and marginal zone B cells in some animals. Histological examination of the long bones reveals dramatic abnormalities, both in hematopoietic and bone lineages. Tle4−/− mice exhibit a reduction in calcified bone, a dysplastic and reduced hypertrophic zone, and reabsorption of the trabecular region. These mice also have a gradual clearing of the marrow itself, with 2 week old animals first showing evidence of cytopenia that then progresses to full marrow failure by 3–3.5 weeks of age. The bone marrow exhibits a marked leukocytopenia, with a clear B lymphocytopenia as in the spleen. Furthermore, within the bone marrow, there is an increase in the number of CD4 and decrease in the number of CD8 T cells. ProB staging of developing B cells also trends towards a partial arrest in the transitions from the A through C fractions. When analyzed for progenitor populations, KO mice show severe reductions in the number of common lymphoid progenitors, while myeloid progenitors show no particular shifts in population frequency. Finally, there is a significant decrease in HSCs, defined as lin-Sca1+Ckit++ (LSK). In order to assess whether the hematological phenotype observed in these mice are of a cell intrinsic or extrinsic nature, transplants were performed using embryonic day 13.5 fetal liver and 2 week old bone marrow. Analysis of recipient animals 16 weeks after transplant revealed leukocytopenia with a specific reduction in B cells in the blood, however the T cell and LSK defects seen in donor animals was not observed. Furthermore, when LSK CD34+ Flt3+ or – cells from KO or WT 2 week old animals were plated on OP9 cells over-expressing Deltex1 or GFP, there were no defects in T, B, or CD11b+ cell production. The unique spectrum of phenotypic consequences concomitant to Tle4 deletion argues for a complex yet fundamental role in the maintenance of the hematopoietic niche. As illustrated by the ability to recapitulate some yet not all of the KO hematological aberrations in transplants, it appears that Tle4's involvement in lineage commitment is heterotypic in nature, an unsurprising finding considering its direct involvement in multiple highly conserved signaling pathways. Considering its principal role in those pathways, continued efforts to investigate the role the TLE family of corepressors play in the bone marrow niche may prove vital in reworking our paradigms for both normal and malignant hematopoiesis. Figure 1: Tle4 deletion causes alterations in LSK and common lymphoid progenitor frequencies. (Controls n=7, KO n=8; *p Figure 2: Representative Flow plots illustrating progenitor and LSK defects in Tle4−/− mice. Figure 2:. Representative Flow plots illustrating progenitor and LSK defects in Tle4−/− mice. Disclosures: No relevant conflicts of interest to declare.

Abstract 1197 TLE1 belongs to the Groucho/TLE family of co-repressors that act as master regulators during development affecting segmentation, neurogenesis, myogenesis, and multiple cell fate decisions. TLE1 modulate several major signaling pathways including Wnt and Notch, and specifically interacts with multiple transcription factors involved in hematopoiesis such TCF/LEF, HES1, RUNX1/AML. TLE1 has also been implicated in Crohn's disease via its interaction with NOD2, a regulator of NFkB. Our laboratory identified TLE1 as a likely AML tumor suppressor gene, commonly deleted in subgroups of AML, and others have shown its role as a tumor suppressor gene in myeloid and other hematopoietic malignancies. To better understand the role of TLE1 in hematopoiesis and leukemogenesis we created a line of Tle1 null mice. Tle1 null mice are born normally, but become progressively growth retarded by 3 days of life, with only 50% survival by 4 weeks as compared to heterozygous and wild type littermates. Abnormalities are observed in several organs systems including the hematopoietic system. We characterized the hematopoietic system in Tle1 knock out mice between two and 12 weeks of age. The bone marrow cellularity in the Tle1 knock out mice is comparable to the wild type mice at all time points examined. However, frequency of granulocyte macrophage progenitors in bone marrow mononuclear cells is significantly higher in the Tle1 knockout bone marrow compared to heterozygous and wild type mice. The proportion and number of myeloid cells as evidenced by Gr1, Mac1 expression are significantly higher in the bone marrow, spleen and blood of these knockout mice. There were significantly lower B-cells (B220+cells) in the Tle1 knockout mice compared to heterozygous and wild type. In colony forming assays there was a trend towards higher number of CFU-GM (7.66 vs 5), p=0.07) and CFU-M (27.16 vs 12.5, p=0.05) colonies from Tle1 null bone marrow as compared to wild type bone marrow. The spleens from four week and 17 months old Tle1 knockout mice had higher frequency of Gr1-negative, Mac1-positive and F4/80 positive macrophages. We also observed a significantly higher production of the inflammatory cytokines IL6 and TNFafrom peritoneal macrophages harvested from Tle1 null mice as compared to those from wild type mice in response to TLR ligand stimulation. To investigate the potential mechanism of this inhibitory effect of TLE1 on inflammation we demonstrated that TLE1 expression is able to block the nuclear translocation of NFkB in THP1 cells in response to LPS-K12 (p In summary this work demonstrates that the lack of Tle1 expression biases hematopoiesis towards myeloid differentiation, a finding of potential relevance given the inactivation of TLE1 seen in subsets of myeloid malignancies. We further show that inactivation of Tle1 leads to an increase in macrophages primed to release increased inflammatory cytokines. This is notable given the recent observation that TLE1 may modulate the effects of NOD2 in the pathogenesis of Crohn's disease. These Tle1 null mice will allow the investigation of the potential role of TLE1 as a modulator of a variety of other inflammatory diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 2473 The Groucho/TLE family of corepressors has been described as master regulatory genes during development, affecting multiple cell fate decisions. These proteins bind to a variety of transcription factors and recruit inhibitory proteins to repress transcription. We previously identified TLE1 as a novel tumor suppressor gene that is deleted or methylated in subgroups of acute myeloid leukemia (AML) and other hematological malignancies. We find the loss of Tle1 alone is insufficient to induce leukemia in mice and apparently requires cooperation with additional oncogenes. Our studies, and those from other groups, have shown that over-expression of TLE1 in leukemia cells slows cell cycle progression, colony formation and tumor growth in xenografts, while silencing results in increased cell proliferation. The pathways by which TLE1 affects oncogenesis is unclear, but this gene family is capable of interacting with effectors of Myc, Wnt, Notch, TGFB signaling–prominent pathways dysregulated in malignancies. Myc is important for hematopoietic stem cell proliferation, survival and differentiation and is over-expressed in most AML samples. The TLE homologue Groucho binds and represses Drosophila Myc expression of target genes, thus we postulated that TLE1 could be an important regulator of Myc activity in leukemia. Using hematopoietic progenitor cells from Tle1 knockout and wild-type fetal livers we found that the loss of Tle1 dramatically increased proliferation and serial replating efficiency. Expression of N-Myc by itself in wild type fetal liver cells triggered significant cell death and apoptosis. However, when N-Myc expression was combined with the additional loss of Tle1, not only was N-Myc induced apoptosis inhibited, but a dramatic cell proliferation, well in excess of that seen with Tle1 loss by itself, was seen. Furthermore, mice transplanted with N-Myc transduced hematopoietic cells from Tle1 knockout mice fetal liver developed a more aggressive leukemia, compared to N-Myc transfected wild type mice fetal liver hematopoietic cells, with increased proliferation of leukemic cells as demonstrated by in vitro colony assays and higher secondary transplantability. We extended these studies to a zebrafish model of Rag2-Myc mediated T-ALL. Using these zebrafish we demonstrated over-expression of the TLE homologue, Groucho, completely blocked the initiation and progression of Myc induced leukemia development. Expression of a truncated version of Groucho reduced the initiation of T-ALL and prolonged the survival of fish developing leukemia. These studies demonstrate TLE1 can inhibit the oncogenicity of Myc, and suggests modulation of expression of this gene family may be of importance for a variety of malignancies. Disclosures: No relevant conflicts of interest to declare.

The Groucho (Gro)/TLE family of transcriptional co-repressor proteins have been called master regulatory genes based on their interaction with a variety of transcription factors and their critical role in development. The TLEs have also been shown to play major roles in brain and lymphocyte differentiation. We became interested in this gene family after finding two family members, TLE1 and TLE4, localized to the commonly deleted region on chromosome 9q in acute myeloid leukemia (AML). This deletion is tightly associated with t(8;21), and we recently showed loss of TLE1 and TLE4 cooperated with AML1-ETO to affect myeloid cell proliferation and survival, implicating TLE1 and 4 as potential tumor suppressor genes in AML. Based on their known ability to inhibit the function of several signaling pathways and transcription factors including Wnt/b-catenin, NF-kB, AML1, and Pu.1 known to be important in leukemogenesis and hematopoiesis, we undertook a series of experiments to determine whether the TLEs could affect myeloid cell differentiation and proliferation. We showed that expression of either TLE1 or TLE4 was able to induce differentiation in the HL-60 myeloid cell line as demonstrated by morphological changes, increased expression of the myeloid differentiation makers CD11b, CD14 and CD15, downregulated myeloperoxidase activity, as well as increased nitroblue tetrazolium (NBT) staining. Furthermore, when HL-60 cells were induced to differentiate by exposure to all-trans-retinoic acid (ATRA) we observed a 150-fold transient increase in TLE1 message after three days. Knockdown of TLE1 with specific shRNAs partially blocked ATRA-induced differentiation as monitored by CD11b expression, suggesting this burst of TLE1 expression is critical for ATRA induced myeloid cell differentiation. To determine the role of the TLEs in primary human cells, human cord blood cells were sorted, and TLE mRNA levels were determined in progenitor cells (CD34+/CD33−/CD16−/CD15−/CD14−), early myeloid precursors (CD34−/CD33+/CD16−/CD15−CD14−) and mature granulocytes (CD34−/CD33+/CD16+/CD15−CD14−). Lowest levels of TLE1 and TLE4 were found in progenitor cells with a peak of expression in early myeloid precursors. To help determine the significance of these low TLE levels in hematopoietic stem/progenitor cells, we designed shRNAs to simultaneously knockdown both TLE1 and TLE4. Knockdown of TLE1/4 in cord blood cells resulted in a 6.3-fold expansion of CD34+CD38− cells after 4 days’ coculture with M2-10B4 in the presence of hSCF, hFlt3-ligand, and hTPO, as compared with only 2.6-fold expansion of cells transfected with control shRNA. Similarly, using mouse bone marrow cells cultured in vitro in the presence of mIL-3, mIL-6 and mSCF, we found a 2.1-fold increase in mouse bone marrow Lin−/Sca-1+/c-kit+ (LSK) cells transfected with TLE1/4 shRNA as compared to cells transfected with scrambled control shRNA. Our results indicate that modulation of TLE levels is capable of influencing both myeloid differentiation, as well as expansion of the stem cell and/or progenitor population.

Deletion of the long arm of chromosome 9, del(9q), is one of the most common cytogenetic abnormalities seen with t(8;21) AML. This close association suggests that that the loss of a critical gene(s) on chr9q cooperates with the AML1-ETO (RUNX1-MTG8) fusion gene produced by t(8;21) in leukemogenesis. We recently mapped the commonly deleted region of del(9q) AML to less than 2.4 Mb and identified all the genes mapping to this region. Extensive sequence analysis of these genes failed to reveal obvious mutations in del(9q) AML samples although the expression of a number of these genes appeared low specifically in del(9q) samples (Genes Chr and Ca 44, 279–291). To help determine the critical affected genes in the del(9q) CDR we used an siRNA expression library directed against all del(9q) CDR gene transcripts in an in vitro complementation assay with AML1-ETO. Inducible expression of AML1-ETO in U937 cells (U937T-A/E, Mol Cell Biol 21, 5577–5590) leads to cell cycle arrest and apoptosis. We introduced the del(9q) CDR siRNA library into U937T-A/E cells with the goal of finding siRNAs that rescued these cells after induction of AML1-ETO expression. The most commonly recovered siRNA using flanking PCR primers was directed against TLE1. Transducin-like enhancer of split (TLE) 1 and TLE4, are two members of the Groucho family of co-repressors that map adjacent to the del(9q) CDR. We subsequently demonstrated that knockdown of either TLE1 or TLE4 with specific siRNAs were able to overcome the induced cell cycle arrest in U937T-A/E cells. We further demonstrate that knockdown of either TLE in the AML1-ETO+ Kasumi-1 cell line increased cell division, increased expression of cyclin D1 and Ki67 and reduced the G0/G1 fraction, while expression of TLE1 or TLE4 in Kasumi-1 cells induced cell cycle arrest and apoptosis. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs are known to inhibit NFKB as well as Wnt signaling, two pathways implicated leukemic stem cell expansion. TLE inhibition may represent an important cooperating mutation with AML11-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity and more directly evaluate the role of TLE inhibition in leukemogenesis.

Deletion of the long arm of chromosome 9, del(9q), is one of the most common mutations associated with t(8;21) AML. Up to 50% of del(9q) AML is seen in association with t(8;21). The Runx1-ETO (AML1-ETO) fusion gene produced by t(8;21) is insufficient for leukemogenesis and loss of a critical gene(s) on chr9q apparently cooperates in leukemogenesis. We recently identified two members of the Groucho family of co-repressors, Transducin-like enhancer of split (TLE) 1 and TLE4, as candidate tumor suppressor genes, based on our mapping of the commonly deleted region in del(9q) AML. These proteins are known to inhibit Wnt signaling which has been implicated in hematopoietic stem cell renewal and they interact with hematopoietic transcription factors such as Runx1 and Pu.1. We have demonstrated that the expression of these two genes is specifically repressed in both del(9q) and t(8;21) AML samples. Knockdown of TLE1 or TLE4 mRNA using specific small interfering RNAs (siRNA) activates Wnt signaling in 293T cells as measured by TOPFLASH activity, while forced expression of TLE1 or 4 inhibits TOPFLASH. We show that siRNA against TLE4 using a specific siRNA increased cell cycle progression and cell division, while over-expression of TLE1 or TLE4 slowed cell cycle progression and lead to a pronounced growth disadvantage in THP-1 and HL60 myeloid cell lines. In addition to these effects on cell proliferation, these genes also affected myeloid cell differentiation. Over-expression of either TLE1 or TLE4 in monocytic THP-1 and promyelocytic HL-60 cell lines initiated myeloid differentiation as monitored by CD11b expression. These two genes have different effects on more terminal myeloid differentiation as TLE4, but not TLE1, was able to induce more terminal differentiation of THP-1 cells into monocytes as measured by CD14 expression, while TLE1 was able to induce the granulocytic marker CD15 in HL-60 cells. Furthermore, lentiviral delivery of siRNAs for either TLE1 or TLE4 to HL-60 cells inhibited induction of granulocytic and monocytic differentiation with all-trans retinoic acid (ATRA) or 1,25-dihydroxycholechalciferol (Vit.D3). TLE1 siRNA inhibited ATRA and Vit.D3 induced CD11b expression by more than 75%. While TLE4 siRNA decreased the induction of monocytic CD14 after Vit.D3 by 40%, TLE1 siRNA almost completely abrogated induction of CD14 with Vit.D3 or granulocytic CD15 with ATRA in HL-60 cells. Ex vivo culture of siRNA infected CD34 sorted human cord blood (CB) cells in semi-liquid media with differentiation inducing cytokines for seven days exhibited a decrease in the percentage of early CD33+ myeloid cells by 15% and 70% relative to controls in TLE1 and TLE4 siRNA infected CB cells, respectively. These results indicate that inhibition of TLE activity can promote cell cycle progression and inhibit myeloid differentiation. This study is the first to demonstrate a potential role for the TLEs in leukemogenesis and also indicates the TLEs may have non-redundant functions in myeloid differentiation. Understanding how various mutations work cooperatively to produce a malignant phenotype is one of the great challenges in oncology. The TLEs may represent an important cooperating mutation with Runx1-ETO in AML that links Wnt signaling and core binding protein transcription factors. Studies are currently underway to demonstrate this cooperativity.

Deletions of 9q are recurring cytogenetic abnormalities in acute myeloid leukaemia (AML). In approximately one-third of cases del(9q) occurs in association with t(8;21). We have previously identified a 2.4Mb region located on 9q21.32–21.33 which is deleted in cases of del(9q) AML - the del(9q) commonly deleted region (CDR). This region encodes 11 genes which we have also previously shown not to be mutated in del(9q) AML. In order to further investigate the role of these genes in AML and in particular to elucidate the pathogenesis of del(9q) AML we have examined the expression of these genes in AML. RNA was extracted from the bone marrow or peripheral blood of patients with AML at the time of diagnosis. Patient samples from the following cytogenetic subgroups were included in this study: (1) del(9q) AML (n=8) - this includes 3 patients with associated t(8;21); (2) t(8;21) but no del(9q) (n=15); (3) Normal karyotype (n=6); (4) Complex Karyotype (n=6). Taqman assays were designed for 9 of the 11 genes located within the del(9q) CDR: FRMD3; ENSG00000148057; UBQLN1; GKAP42; Q9UF54; Q8N2B1; Q9H9A7; SLC28A3; NTRK2. For the other 2 genes within the region Taqman assays could not be performed because of uniformly low expression levels (Q8IZ41) and lack of specificity of primer-design (HNRPK). CD34-purified progenitors from normal individuals were used as controls. It was found that 6 of the 9 genes were significantly down-regulated in del(9q) AML (p