mTORC1 is essential for leukemia propagation but not stem cell self-renewal
01 Jun 2012-Journal of Clinical Investigation (American Society for Clinical Investigation)-Vol. 122, Iss: 6, pp 2114-2129
TL;DR: Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation, and it was demonstrated that the reactivation of m TORC1 in those cells restored their leukemia-initiating capacity.
Abstract: Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.
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TL;DR: Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications.
Abstract: Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.
8 citations
TL;DR: Contrary to expectation, the studies revealed that Hif-1α-deleted-leukemias displayed a faster disease progression after chemotherapy, and warrant caution when applying HIF inhibition in combination with chemotherapy in AML.
Abstract: Relapse of acute myeloid leukemia (AML) remains a significant clinical challenge due to limited therapeutic options and poor prognosis. Leukemic stem cells (LSCs) are the cellular units responsible for relapse in AML, and strategies that target LSCs are thus critical. One proposed potential strategy to this end is to break the quiescent state of LSCs, thereby sensitizing LSCs to conventional cytostatics. The hypoxia-inducible factor (HIF) pathway is a main driver of cellular quiescence and a potential therapeutic target, with precedence from both solid cancers and leukemias. Here, we used a conditional knockout Hif-1α mouse model together with a standard chemotherapy regimen to evaluate LSC targeting in AML. Contrary to expectation, our studies revealed that Hif-1α-deleted-leukemias displayed a faster disease progression after chemotherapy. Our studies thereby challenge the general notion of cancer stem cell sensitization by inhibition of the HIF pathway, and warrant caution when applying HIF inhibition in combination with chemotherapy in AML.
8 citations
Cites background from "mTORC1 is essential for leukemia pr..."
...12 j 112–121 j January 8, 2019 to extend also to MLL-AF9 leukemia (Hoshii et al., 2012)....
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TL;DR: A review of recent developments in the understanding of systemic mastocytosis pathogenesis and potential treatment strategies for patients with SM can be found in this paper, however, the overall survival of patients treated with midostaurin remains unsatisfactory.
Abstract: Mastocytosis is a type of myeloid neoplasm characterized by the clonal, neoplastic proliferation of morphologically and immunophenotypically abnormal mast cells that infiltrate one or more organ systems. Systemic mastocytosis (SM) is a more aggressive variant of mastocytosis with extracutaneous involvement, which might be associated with multi-organ dysfunction or failure and shortened survival. Over 80% of patients with SM carry the KIT D816V mutation. However, the KIT D816V mutation serves as a weak oncogene and appears to be a late event in the pathogenesis of mastocytosis. The management of SM is highly individualized and was largely palliative for patients without a targeted form of therapy in past decades. Targeted therapy with midostaurin, a multiple kinase inhibitor that inhibits KIT, has demonstrated efficacy in patients with advanced SM. This led to the recent approval of midostaurin by the United States Food and Drug Administration and European Medicines Agency. However, the overall survival of patients treated with midostaurin remains unsatisfactory. The identification of genetic and epigenetic alterations and understanding their interactions and the molecular mechanisms involved in mastocytosis is necessary to develop rationally targeted therapeutic strategies. This review briefly summarizes recent developments in the understanding of SM pathogenesis and potential treatment strategies for patients with SM.
7 citations
01 Jan 2014
TL;DR: In this article, a connection between mTOR and the Fanconi anemia pathway has been found, which is an important DNA repair mechanism that is associated with drug resistance of multiple types of cancer.
Abstract: mTOR is a serine/threonine kinase and plays a critical role in mammalian cell growth, survival, and metabolism. mTOR is present in two cellular complexes: mTORC1 and mTORC2. Dysregulation of the mTOR pathway has been related to tumorigenesis, poor prognosis and/or chemotherapy resistance in a variety of malignancies. Inhibition of mTORC1 by Rapamycin and its analogs has been explored to treat a number of tumors. However, the effectiveness of patient response is limited and not all patients respond. Second generation of mTOR inhibitors have recently been developed to target mTOR kinase activity and to suppress both mTORC1 and mTORC2. Dual mTORC1/mTORC2 inhibitors generally are more efficacious in preclinical studies and clinical trials. We and others have recently found that dual mTORC1/mTORC2 inhibitors sensitize T-cell acute lymphocytic leukemia and rhabdomyosarcoma cells to DNA damaging agents by suppression of expression of FANCD2 of the Fanconi anemia pathway, an important DNA repair mechanism that is associated with drug resistance of multiple types of cancer. This review will highlight mTOR and the Fanconi anemia pathway in cancer, with a particular attention to our newly discovered connection between mTOR and the Fanconi anemia pathway.
7 citations
TL;DR: In this paper, the authors show that nutrient concentrations in extracellular milieux affect signaling mediated by environmental sensor proteins, such as rapamycin (mTOR), and demonstrate that the mechanistic target of mTOR can affect signaling.
Abstract: Ample evidence indicates that nutrient concentrations in extracellular milieux affect signaling mediated by environmental sensor proteins. For instance, the mechanistic target of rapamycin (mTOR) i...
7 citations
Cites methods from "mTORC1 is essential for leukemia pr..."
...Initially, samples from Rosa26–Cre-ER mice that were Rptor, Rptor, and Rptor were analyzed in the short (;10 d) period while mice remained healthy after tamoxifen injections caused deletionof theRptor conditionalallele inmultiple cell types (46)....
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References
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TL;DR: Stem cell biology has come of age: Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine.
Abstract: Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.
8,999 citations
TL;DR: It is reported that mTOR forms a stoichiometric complex with raptor, an evolutionarily conserved protein with at least two roles in the mTOR pathway that through its association with mTOR regulates cell size in response to nutrient levels.
2,902 citations
"mTORC1 is essential for leukemia pr..." refers result in this paper
...Although mTORC1 reportedly controls cell size (cell growth) (34), we did not observe a decrease in the size of our Raptor-deficient AML cells (Figure 6B), and we found that the amount of protein per cell was comparable in control and Raptor-deficient AML stem cells (Figure 6, C and D)....
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TL;DR: It is shown that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolongedRapamycin treatment reduces the levels of m TORC2 below those needed to maintain Akt/PKB signaling.
2,621 citations
"mTORC1 is essential for leukemia pr..." refers background in this paper
...We found that Raptor deficiency did not affect the expression level of Rictor, an essential component of mTORC2, but it is possible that long-term inactivation of mTORC1 in Raptordeficient AML cells affects the assembly and activity of mTORC2, leading to inhibition of AKT, in the same manner as prolonged rapamycin treatment (49)....
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...On the other hand, prolonged rapamycin treatment is reported to suppress AKT via the disassembly of the mTORC2 complex in certain cell types (49)....
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TL;DR: The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation, and reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.
Abstract: Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.
2,423 citations
"mTORC1 is essential for leukemia pr..." refers background in this paper
...org Volume 122 Number 6 June 2012 (32, 33), neither Raptor deficiency nor rapamycin resulted in hyperphosphorylation of AKT (S473) (Figure 6A and Supplemental Fig-...
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TL;DR: Recent findings on the regulators and effectors of mTOR are highlighted and specific cases that serve as paradigms for the different modes of m TOR regulation and its control of translation are discussed.
Abstract: The process of translation requires substantial cellular resources. Cells have therefore evolved complex mechanisms to control overall protein synthesis as well as the translation of specific mRNAs that are crucial for cell growth and proliferation. At the heart of this process is the mammalian target of rapamycin (mTOR) signalling pathway, which senses and responds to nutrient availability, energy sufficiency, stress, hormones and mitogens to modulate protein synthesis. Here, we highlight recent findings on the regulators and effectors of mTOR and discuss specific cases that serve as paradigms for the different modes of mTOR regulation and its control of translation.
2,328 citations
"mTORC1 is essential for leukemia pr..." refers background in this paper
...These target molecules control cell growth (size) and proliferation by modifying protein translation (1)....
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