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...
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