Acute lymphoblastic leukemia (ALL) in adult patients is often resistant to current therapy, making the development of novel therapeutic agents paramount. We investigated whether mTOR inhibitors (MTIs), a class of signal transduction inhibitors, would be effective in primary human ALL. Lymphoblasts from adult patients with precursor B ALL were cultured on bone marrow stroma and were treated with CCI-779, a second generation MTI. Treated cells showed a dramatic decrease in cell proliferation and an increase in apoptotic cells, compared to untreated cells. We also assessed the effect of CCI-779 in a NOD/SCID xenograft model. We treated a total of 68 mice generated from the same patient samples with CCI-779 after establishment of disease. Animals treated with CCI-779 showed a decrease in peripheralblood blasts and in splenomegaly. In dramatic contrast, untreated animals continued to show expansion of human ALL. We performed immunoblots to validate the inhibition of the mTOR signaling intermediate phospho-S6 in human ALL, finding down-regulation of this target in xenografted human ALL exposed to CCI-779. We conclude that MTIs can inhibit the growth of adult human ALL and deserve close examination as therapeutic agents against a disease that is often not curable with current therapy. IntroductionWhile children with precursor B-cell acute lymphoblastic leukemia (ALL) are often cured, children with relapsed ALL and adults with ALL usually succumb to their disease with current therapy. Even with aggressive therapy, these patient groups have 5-year diseasefree survival rates of only 28% to 39%. 1 Thus, the development of novel therapeutic agents is crucial.One potential class of novel therapeutics is mTOR inhibitors (MTIs). MTIs are a class of signal transduction inhibitors with anticancer activity that were initially developed as immunosuppressive agents. [2][3][4][5] Rapamycin, a macrocyclic lactone produced by Streptomyces hydroscopicus, 6 was the first MTI to be used in a clinical setting. Rapamycin is well tolerated in humans. 7 MTIs also have been shown to be active against a wide variety of tumor types. [8][9][10] We have previously shown that rapamycin induces apoptosis in precursor B ALL lines in vitro and has in vivo activity in transgenic mice with pre-B leukemia/lymphoma. 11 Second generation MTIs, CCI-779 and RAD-001, are currently in phase 1 to phase 3 clinical trials in patients with various cancers, 12-15 but preclinical studies have not previously been performed in primary human ALL.Preclinical testing of chemotherapeutic agents often involves using transformed tumor lines and transgenic mouse models. While these are valuable tools, they may not be representative of human disease. More clinically relevant data may be obtained from systems using primary human ALL cells. Two of these systems, bone marrow stroma-supported culture 16 and xenografting human ALL in nonobese diabetic/severe combined immunodeficient (NOD/ SCID) mice, have recently been developed. 17 Both of these systems allow for direct testing...

Primary normal and leukemic human precursor B cells can be cultured on bone marrow stromal cell layer for at least 3-4 weeks [1][2][3][4][5]. Studies using this model have provided important insights into the cell pathways that promote the expansion of human precursor B cells. For example, expansion of normal or leukemic human B cells does not require interleukin-7 (IL-7) [2, 6], a cytokine necessary for the expansion of murine precursor B cells. The rate of expansion is dictated, in part, by a balance of apoptosis and cell division. Apoptosis in both normal and leukemic precursor B cells is inhibited by direct contact with stromal cells [7,8] and is mediated by vascular cellular adhesion molecule-1, α4β1 integrins, and multiple antiapoptotic proteins [9,10]. Although the rate of apoptosis is similar among different samples of normal human precursor B cells, primary leukemic precursor B cells have variable rates of apoptosis; decreased apoptotic rate in culture is an independent predictor of poor clinical outcome [11].

The transcription factor E2A can promote precursor B cell expansion, promote G 1 cell cycle progression, and induce the expressions of multiple G 1 -phase cyclins. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated the 1-kb promoter of cyclin D3, which contains two E boxes. However, deletion of the E boxes did not disrupt the transactivation by E2A, raising the possibility of indirect activation via another transcription factor or binding of E2A to non-E-box DNA elements. To distinguish between these two possibilities, promoter occupancy was examined using the DamID approach. A fusion construct composed of E2A and the Escherichia coli DNA adenosine methyltransferase (E47Dam) was subcloned in lentivirus vectors and used to transduce precursor B-cell and myeloid progenitor cell lines. In both cell types, specific adenosine methylation was identified at the cyclin D3 promoter. Chromatin immunoprecipitation analysis confirmed the DamID findings and localized the binding to within 1 kb of the two E boxes. The methylation by E47Dam was not disrupted by mutations in the E2A portion that block DNA binding. We conclude that E2A can be recruited to the cyclin D3 promoter independently of E boxes or E2A DNA binding activity.E2A is essential to normal B-cell development. B cells, even at the earliest detectable precursor-B-cell developmental stage, do not develop in mice that lack E2A (2, 68). Heterozygote murine fetuses that express only one copy of E2A have half the number of B cells, suggesting that the level of E2A may dictate the number of B cells (68). E2A function is intrinsic to B cells, since transfer of E2A null bone marrow cells into wild-type mice fails to rescue B-cell development while ectopic expression of E2A in E2A null mice rescues B-cell development (67).E2A is frequently mutated in precursor-B-cell lymphoblastic leukemia. Acute lymphoblastic leukemia (ALL) is the most common neoplasm in childhood (65). Approximately 6% of pediatric ALLs have chromosomal translocations involving the E2A gene (29). These translocations represent the secondmost-frequent translocation in ALL and are almost always associated with ALL of precursor-B-cell origin. The most common E2A translocation, t(1;19), is associated with poor response to older standard risk ALL therapies. Even with the current ALL therapies, the t(1:19) translocation is sufficient to decrease the prognosis from low risk (Ͼ90% 4-year event-free survival) to standard risk (approximately 80% 4-year eventfree survival).Normal E2A regulates cell cycle progression, an important contributor of cell accumulation that is frequently disrupted during carcinogenesis. Hence, mutations in E2A may contribute to abnormal B-cell accumulation by disrupting normal regulation of cell cycle progression by E2A. The nature of this regulation is unclear. Numerous studies support the current model that E2A inhibits cell cycle progression (19,40,41).However, other st...

The transcription factor E2A is required for very early B cell development. The exact mechanism by which E2A promotes B cell development is unclear and cannot be explained by the known E2A targets, components of the pre-B cell receptor and cyclin dependent kinase inhibitors, indicating additional pathways and targets remain to be identified. We had previously reported that E2A can promote precursor B cell expansion, promote G1 cell cycle progression, and induce the expressions of multiple G1 phase cyclins including cyclin D3, suggesting that E2A induction of these genes may contribute to early B cell development. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated a luciferase reporter plasmid containing the 1kb promoter of cyclin D3 that contains two consensus E2A binding sites (E-boxes); however, deletion of the E-boxes did not disrupt the transactivation by E2A. We hypothesized three possible mechanisms: 1) indirect activation of cyclin D3 via another transcription factor, 2) binding of E2A to cryptic non-E-boxes, or 3) recruitment of E2A to the promoter via interaction with other DNA binding factor. To test the first possibility, promoter occupancy was examined using the DamID approach. In this approach, a fusion protein consisting of E. coli DNA adenosine methyltransferase (DAM) and a transcription factor of interest is expressed at low levels, resulting in specific methylation of adenosine residues within 2–5 kb of the transcription factor target sites. A fusion construct composed of E2A and DAM (E47Dam), was subcloned in lentiviral vectors, and used to transduce precursor B cell lines. The methylated adenosine residues were detected using a sensitive ligation-mediated PCR (LM-PCR) assay that required only 1 ug of genomic DNA and can detect methylation even if only 3% of the cells express E47Dam; no methylated adenosines were detected in control cells, indicating that all methylated residues resulted from E47Dam. Specific adenosine methylation was identified at the IgH intronic enhancer, a known E2A target site, but not at the non-target sites, CD19, HPRT, and GAPDH promoters. Specific methylation was detected at the cyclin D3 promoter but not 10 kb down-stream, despite similar concentrations of E-boxes at both sites. Chromatin immunoprecipitation analysis confirmed the DamID findings and further localized the binding to within 1 kb of the two E-boxes in the cyclin D3 promoter. To distinguish between the two remaining mechanisms (cryptic non-E-boxes versus recruitment via other DNA binding factors), two point mutations were introduced into E47Dam that disrupted its DNA binding activity. The mutated E47Dam continued to methylate at the cyclin D3 promoter. We conclude that E2A can be recruited to the cyclin D3 promoter, independent of E-boxes or E2A DNA binding activity. Our findings raise the possibility that some direct E2A target genes may lack functional E-boxes. Furthermore, mutated E2A, lacking an E2A DNA binding domain, that is seen in 6% of pediatric ALLs may still activate a subset of E2A target genes. Finally, our application of lentiviral vectors and LM-PCR to the DamID approach should permit analysis of primary human precursor B cells, despite the limitations in cell number and transduction efficiency.

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While children with precursor B cell acute lymphoblastic leukemia (ALL) are often cured, adults with ALL usually succumb to their disease. Thus, the development of novel therapeutic agents is paramount. mTOR inhibitors (MTI) are a class of signal transduction inhibitors developed as immunosuppressive agents. We have previously shown the ability of MTI to inhibit growth and induce apoptosis in precursor B ALL cell lines and primary murine pre-B leukemia. To determine if this finding could be translated to clinical therapy, we explored if MTIs would be similarly effective in three primary human adult ALL samples. We have tested the MTIs rapamycin and CCI-779 in these models with similar results, but the results utilizing CCI-779 are presented below. Stromal culture. ALL blasts were maintained in vitro on irradiated bone marrow stromal cells. Cells normally can be maintained for several weeks in these conditions. Cells were either untreated or treated with CCI-779 at 100ng/ml. Long-term cultures were assessed for the effect of MTI on cell proliferation and short term (48hr) cultures were assessed for induction of apoptosis. Treated cells showed a dramatic decrease in cell proliferation (6–24 fold compared to untreated) and a 4–5 fold increase in apoptotic cells as detected by Annexin-V compared to untreated cells. NOD/SCID xenografts. To further evaluate the effect of MTI on ALL cells, patient samples were engrafted into NOD/SCID animals for analysis. Robust engraftment, expansion and repopulation in secondary hosts of ALL cells was seen in 78% of tested samples, demonstrating greater than 70% ALL in peripheral blood, bone marrow, and spleen in the majority of the mice. There was greater than 10 fold expansion of disease within the mouse. Engraftment was detected by flow cytometry for human CD19+/CD45+ cells. Engrafted animals with established disease (>5% peripheral blasts) were either not treated or treated with the MTI CCI-779. Currently we have treated 45 mice engrafted from three separate patient samples. Untreated animals continued to show expansion of human ALL cells. In dramatic contrast, animals treated with CCI-779 as a single agent showed a 4–30 fold decrease in peripheral blood blasts and a decrease in splenomegaly (p<.02). Our data show that both in vitro and in an in vivo model of established ALL, MTIs decrease proliferation of lymphoblasts and promote apoptosis of ALL cells. These results suggest that the mTOR signaling pathway is necessary for the survival of ALL cells and that MTIs should be analyzed as therapeutic agents for the therapy of ALL.