Protect Acute Lymphoblastic Leukemia Cells Research Articles

Abstract 1419: Adipocytes protect leukemia cells from L-asparaginase treatment

Abstract Obesity increases cancer incidence and mortality. This is also true for acute lymphoblastic leukemia (ALL). L-Asparaginase (ASNase), an important front-line drug in leukemia treatment, acts through the hydrolysis of asparagine (ASN) and glutamine (GLN) into aspartic acid and glutamic acid. As lymphoblastic leukemia cells are highly dependent on these two non-essential amino acids, ASNase leads to decreased protein synthesis, cell cycle arrest, and apoptosis. We have previously shown that obesity impairs ASNase treatment in C57BL/J mice transplanted with syngeneic leukemia cells. The present study was designed to elucidate how obesity protects ALL cells from this drug. The effects of adipocytes on ALL ASNase resistance were examined in vitro using co-cultures with 3T3-L1 adipocytes and ALL cells, separated by TransWells. Differentiated 3T3-L1 adipocytes significantly protected ALL cells from a 3-day treatment with ASNase, compared to undifferentiated 3T3-L1 fibroblasts or no feeder layer (viable cells after ASNase treatment: 3.1 ± 1.2 fold greater than no feeder layer, 2.1 ± 0.6 greater than fibroblast feeder layer, p<0.05, n=6). Since ASNase acts through the hydrolysis of both ASN and GLN, we next determined whether the protection afforded by adipocytes was through the secretion of one or both of these amino acids. Seven human leukemia cell lines (RCH-ACV, SEM, BV-173, Nalm 6, K562, SD-1, and RS4;11) were tested for their ability to proliferate in media lacking either of these two amino acids. Compared to complete media, six had significantly decreased proliferation in media lacking GLN (n=4, p<.05), while only one (RS4;11) showed dependence on ASN. The GLN-dependent SD-1's and ASN-dependent RS4;11's were plated in TransWells over undifferentiated 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and no feeder layer in media lacking both amino acids. Differentiated 3T3-L1 adipocytes significantly protected both cell lines from amino acid starvation (RS4;11: 3.6 ± 0.4 fold greater than no feeder, 2.0 ± 0.3 fold great than fibroblasts, p<.05, n=3; SD-1: 4.2 ± 2.4 fold greater than feeder, 4.0 ± 1.0 fold greater than fibroblasts, p<.05, n=3). Furthermore, we determined that adipocytes secrete both amino acids over a 72 hour period (media concentration of ASN after 24, 48, and 72 hours: 34 uM, 166 uM, 369 uM; GLN: 0, 42, 85 uM, respectively). Most importantly GLN and ASN depleted media decreased mTOR signaling (measured by p70s6k phosphorylation), conditioning of this media by adipocytes for 48 hours reversed this effect. The current data demonstrate that adipocytes directly protect leukemia cells from ASNase treatment. We have demonstrated that adipocytes increase leukemia survival in media starved of ASN and/or GLN, secrete ASN and GLN, and enhance mTOR signaling in amino acid starved leukemia cells. Thus, obesity may impair the effects of ASNase on leukemia cells through adipose tissue secretion of both ASN and GLN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1419.

Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase

Because of their low asparagine synthetase (ASNS) expression and asparagine biosynthesis, acute lymphoblastic leukemia (ALL) cells are exquisitely sensitive to asparagine depletion. Consequently, asparaginase is a major component of ALL therapy, but the mechanisms regulating the susceptibility of leukemic cells to this agent are unclear. In 288 children with ALL, cellular ASNS expression was more likely to be high in T-lineage ALL and low in B-lineage ALL with TEL-AML1 or hyperdiploidy. However, ASNS expression levels in bone marrow-derived mesenchymal cells (MSCs), which form the microenvironment where leukemic cells grow, were on average 20 times higher than those in ALL cells. MSCs protected ALL cells from asparaginase cytotoxicity in coculture experiments. This protective effect correlated with levels of ASNS expression: downregulation by RNA interference decreased the capacity of MSCs to protect ALL cells from asparaginase, whereas enforced ASNS expression conferred enhanced protection. Asparagine secretion by MSCs was directly related to their ASNS expression levels, suggesting a mechanism - increased concentrations of asparagine in the leukemic cell microenvironment - for the protective effects we observed. These results provide what we believe to be a new basis for understanding asparaginase resistance in ALL and indicate that MSC niches in the bone marrow can form a safe haven for leukemic cells.

Mesenchymal Cells Determine the Response of Acute Lymphoblastic Leukemia Cells to L-Asparaginase.

Owing to their low expression of asparagine synthetase (ASNS), acute lymphoblastic leukemia (ALL) cells have low asparagine biosynthesis and are exquisitely sensitive to asparagine depletion caused by L-asparaginase. Differences in susceptibility to L-asparaginase have been attributed to the varying levels of ASNS mRNA in leukemic cells, but recent studies have challenged this concept. We found that among leukemic cells from 288 children with ALL analyzed by Affymetrix U133A GeneChip, ASNS levels were higher in patients with T-lineage ALL (P <0.001), and lower in patients with TEL-AML1 (P = 0.033) or hyperdiploid >50 chromosomes (P <0.001) B-lineage ALL. However, ASNS expression was not significantly related to response to remission induction therapy as determined by minimal residual disease measurements on day 46 of treatment.ALL cells grow in direct contact with bone marrow mesenchymal cells, which form the microenvironmental niches essential for their expansion. We observed that ASNS levels in mesenchymal cells were, on average, 20 times higher than those expressed by ALL cells by GeneChip analysis, real-time PCR and Western blotting with an anti-ASNS specific monoclonal antibody (gift of Dr. M. Kilberg, U. of Florida). When ALL cell lines (380, REH, RS4;11) were exposed to L-asparaginase in the presence of mesenchymal cells, cytotoxicity significantly decreased. To test whether the protective effect of mesenchymal cells was related to their ASNS expression, we used RNA interference (RNAi) to stably downregulate ASNS expression. This profoundly diminished their capacity to protect ALL cells from L-asparaginase cytotoxicity. We then investigated whether enforced expression of ASNS in mesenchymal cells using a MSCV retroviral vector could augment their protective capacity. Overexpression of ASNS significantly augmented the capacity of mesenchymal cells to protect ALL cells from L-asparaginase cytotoxicity. ASNS expression in mesenchymal cells was related not only to their capacity to protect ALL cells lines but also primary ALL cells obtained from 5 patients with newly diagnosed ALL.We found that mesenchymal cells secreted asparagine and that levels of asparagine in culture supernatants collected from mesenchymal cells after 24 hours of culture were directly related to levels of ASNS expression in the cells; asparagine in supernatants of mesenchymal cells treated with the RNAi target sequence was nearly undetectable. In line with these results, the protective effects of mesenchymal cells were also detectable when ALL cells were placed on a microporous membrane that prevented contact with mesenchymal, and when they were cultured with mesenchymal cell-conditioned medium. By contrast, addition of a mixture of mesenchymal cell-derived cytokines (IL-1 alpha and beta, IL-3, IL-6, IL-7, IL-11, SCF and Flt3 ligand) instead of mesenchymal cell-conditioned medium had no effect on L-asparaginase cytotoxicity.These results reveal an unexpected mechanism of drug resistance in ALL and indicate that microenvironmental niches can form a safe haven for leukemia cells, thus sustaining minimal residual disease. The role of mesenchymal cells in the response to other anti-leukemic drugs requires further investigation. A better understanding of the molecular mechanisms involved in the interaction between ALL cells and the bone marrow microenvironment may ultimately lead to innovative ways to enhance anti-leukemia therapy.

PI3-Kinase Inhibition Decreases the Proliferation of ALL Cells, Potentiates mTOR Inhibition, and Abrogates Growth Factor-Mediated Reversal of mTOR Inhibition.

Biologically targeted cancer agents, including signal transduction inhibitors, have shown great promise in treating hematologic malignancies. However, used as single agents, these drugs may not be curative secondary to innate or acquired cellular resistance. Thus, acute lymphoblastic leukemia (ALL) and other cancer cells may become resistant to rapamycin, an mTOR inhibitor (MTI), following extended exposure to the drug. A strategy to overcome such resistance is to combine targeted agents, and thereby inhibit multiple targets simultaneously. Previously, we have shown activity of MTI in models of both human and murine ALL. In mouse models, treatment of ALL with MTI prolongs survival but may not cure disease. IL-7, a lymphoid growth factor important in the regulation of progenitor B cell development and proliferation, can reverse the inhibitory effects of MTI on human and murine pre-B ALL cells. We wished to further explore the mechanisms by which IL-7-mediated signaling protects ALL cells from the inhibitory effects of MTI, through the investigation of modulators of growth factor signaling in ALL. Thus, we have evaluated the impact of LY294002, an inhibitor of phosphatidyl inositol-3 kinase (PI3K). PI3K is a critical signaling molecule in cell survival and proliferation, with one of its central roles being signal transduction from growth factor receptors to the activation of AKT (an upstream regulator of mTOR). PI3K/AKT pathway over-activation has been implicated in many different cancers. Treatment of ALL cell lines with the PI3K inhibitor LY294002 markedly decreased cell proliferation in a dose-dependent manner. More importantly, the inhibitory effects of LY294002 were additive or synergistic with the inhibitory effects of MTI, and prevented the ability of IL-7 to reverse the inhibitory effects of rapamycin. Treatment of pre-B ALL cell lines with 2.5 μM LY294002 resulted in decreased proliferation to 20–45% of baseline as compared to untreated cells, whereas treatment with a higher dose (5 μM) reduced cell proliferation to 10–20%. Combinations of LY294002 and rapamycin, even at low doses, inhibited cell proliferation to a greater degree than each drug individually. Co-treatment with 2.5 μM LY294002 and low dose rapamycin (1 ng/ml) resulted in profound inhibition of proliferation to <=5%, compared to 20–30% with rapamycin alone. Furthermore, co-treatment with low-dose LY294002 and low-dose rapamycin resulted in greater inhibition than even higher doses of each of these agents individually. While the addition of IL-7 (1 U/ml) to rapamycin-treated cells resulted in the reversal of rapamycin-mediated cell inhibition, the further addition of 2.5 μM LY294002 significantly antagonized this growth factor rescue of MTI-treated ALL cells. The blockade by LY294002 of the IL-7 effect was most apparent in ALL cell lines that were IL-7 dependent, with cell proliferation reduced to <20%. However, the effects were still significant in IL-7 independent cell lines, with proliferation reduced to 20–60%. Similar results were seen using human ALL cell lines. These data suggest that the PI3K signaling pathway serves as a potential rescue pathway from mTOR inhibition, mediating the ability of growth factors to rescue cells from rapamycin;PI3K itself is a therapeutic target for ALL; andcombination therapy with MTI and PI3K inhibitors may be more active than either agent alone.

Release of eicosanoid by rat gastric mucosa and its pharmacological modification

Ferroptosis has recently been identified as a mode of programmed cell death. However, little is yet known about the signaling mechanism. Here, we report that lipoxygenases (LOX) contribute to the regulation of RSL3-induced ferroptosis in acute lymphoblastic leukemia (ALL) cells. We show that the glutathione (GSH) peroxidase 4 (GPX4) inhibitor RSL3 triggers lipid peroxidation, production of reactive oxygen species (ROS) and cell death in ALL cells. All these events are impeded in the presence of Ferrostatin-1 (Fer-1), a small-molecule inhibitor of lipid peroxidation. Also, lipid peroxidation and ROS production precede the induction of cell death, underscoring their contribution to cell death upon exposure to RSL3. Importantly, LOX inhibitors, including the selective 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA), protect ALL cells from RSL3-stimulated lipid peroxidation, ROS generation and cell death, indicating that LOX contribute to ferroptosis. RSL3 triggers lipid peroxidation and cell death also in FAS-associated Death Domain (FADD)-deficient cells which are resistant to death receptor-induced apoptosis indicating that the induction of ferroptosis may bypass apoptosis resistance. By providing new insights into the molecular regulation of ferroptosis, our study contributes to the development of novel treatment strategies to reactivate programmed cell death in ALL.

Inhaled frusemide displays protecting activity against anaphylactic crisis of the guinea-pig

Ferroptosis has recently been identified as a mode of programmed cell death. However, little is yet known about the signaling mechanism. Here, we report that lipoxygenases (LOX) contribute to the regulation of RSL3-induced ferroptosis in acute lymphoblastic leukemia (ALL) cells. We show that the glutathione (GSH) peroxidase 4 (GPX4) inhibitor RSL3 triggers lipid peroxidation, production of reactive oxygen species (ROS) and cell death in ALL cells. All these events are impeded in the presence of Ferrostatin-1 (Fer-1), a small-molecule inhibitor of lipid peroxidation. Also, lipid peroxidation and ROS production precede the induction of cell death, underscoring their contribution to cell death upon exposure to RSL3. Importantly, LOX inhibitors, including the selective 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA), protect ALL cells from RSL3-stimulated lipid peroxidation, ROS generation and cell death, indicating that LOX contribute to ferroptosis. RSL3 triggers lipid peroxidation and cell death also in FAS-associated Death Domain (FADD)-deficient cells which are resistant to death receptor-induced apoptosis indicating that the induction of ferroptosis may bypass apoptosis resistance. By providing new insights into the molecular regulation of ferroptosis, our study contributes to the development of novel treatment strategies to reactivate programmed cell death in ALL.