doi: 10.1182/blood-2008-03-147587. Epub 2008 Jul 28.
K-RasG12D-induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to gamma-secretase inhibitors
Affiliations
- PMID:18663146
- PMCID: PMC2569181
- DOI: 10.1182/blood-2008-03-147587
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K-RasG12D-induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to gamma-secretase inhibitors
Thomas Kindler et al. Blood..
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Abstract
To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-Ras(G12D) murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to gamma-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.
Figures
Mice transplanted with K-RasG12D–expressing bone marrow cells develop an aggressive T-cell disease. (A) Schematic illustration of the bone marrow transplantation strategy. (B) Kaplan-Meier survival plot of mice transplanted with BM cells derived fromK-RasG12D/+Mx1-Cre+ (KM) andK-RasG12D/+ (K) together with 2 × 105 helper cells or with 106 cells derived from the thymi of 2 different diseased animals (donors 9988 and 9980; n = 5 each). Cumulative survival was plotted against days after BMT. KM 5:1 (n = 17) and KM 1:1 (n = 22) developed an aggressive T-cell disease with a median survival of 107 and 114 days, respectively. Sixteen of 17 K-mice were healthy during observation; 1 mouse died of a spontaneous thymic lymphoma. (C) Hematologic and pathologic data of diseased mice. KM mice have increased weight of thymus and spleen, elevated weight blood cell counts, and mild thrombocytopenia. (D) Histopathologic sections of BM (first row, original magnification 40×, left, original magnification 100×, right; hematoxylin and eosin), spleen (bottom left, original magnification 40×; hematoxylin and eosin), and liver (bottom right, original magnification 60×; hematoxylin and eosin) from a representative mouse with T-ALL. Diseased mice show infiltration of lymphoblasts in BM and spleen with destruction of normal architecture as well as extramedullary hematopoiesis in liver. (E) Cre-mediated activation of the oncogenicK-Ras allele. PCR for WT and activated (Δ)K-Ras allele demonstrates the presence of the activatedK-Ras allele in thymus, bone marrow, and spleen of 3 representative patients with T-ALL. C indicates control DNA from an patient with KM+-induced MPD; MW, molecular weight marker.
Malignant T cells are CD4/CD8 double-positive and aberrantly express early markers of T-cell development. (A) Flow cytometric analysis of single-cell suspensions of BM, spleen, and thymus of a representative diseased mouse demonstrates a CD4/CD8 double-positive population in all 3 tissues, with some cells becoming single-positive for CD8. The percentages of cells are indicated in each quadrant. (B) Flow cytometric analysis of single-cell suspensions of the thymus from 2 diseased mice demonstrates variable phenotypes. Plots were gated on live cells and stained for CD4 and CD8 (top panel). The gated tumor cell population was analyzed for the expression of CD25 and CD44 (bottom panel). The percentages of cells of interest are indicated. (C) T-ALL cells expressRAG1, RAG2, andpre–T-α. Tumor and control cDNA was amplified using primer forRAG1, RAG2, pre–T-α, andGAPDH, electrophoresed through a 2% agarose gel and stained with ethidium bromide. M indicates molecular weight marker; 1, KM donor 1; and W, water.
T-cell disease is oligoclonal. Analysis of TCR-β rearrangement. Thymocyte/tumor cDNA was amplified using primer pairs specific for each of the 19 and 20 possible variable/constant region junctions at the murineTCR-β locus (A) andTCR-α (B) locus, respectively. The samples were electrophoresed through a 1.8% agarose gel and stained with ethidium bromide. Top line represents KM donor; middle and bottom lines, 2 different tumor samples.
Primary, oncogenic K-Ras–expressing mice have a severe block in differentiation during early T-cell development. (A) Decreased thymic cellularity in oncogenic K-Ras–expressing mice (KM). Total thymocyte number of 4-week-old KM mice is reduced (P = .025) compared with control mice (8 days after pI-pC induction). (B) Histopathologic sections of the thymus of a primary mouse expressing oncogenic K-Ras (left) compared with normal control (right), 8 days after pI-pC induction (original magnification 40×; hematoxylin and eosin) show a perturbed thymic architecture with an increase in stromal tissue in the former. Shown is 1 representative example. (C) Expression of oncogenic K-Ras is associated with a decrease of the DP population and an increase of DN1 cells. Single-cell suspension from thymi of 4-week-old KM (n = 7) and control (n = 7) mice, 8 days after pI-pC induction, were prepared and stained for CD4 and CD8. CD25 and CD44 expression was analyzed gating on DN, DP, CD8 single-positive (SP), and CD4 SP populations. Compared with normal controls, DP cells were significantly reduced (P = .027, shown as percentage of live cells andP = .012 for total cell numbers), whereas DN1 and CD8 SP populations were significantly increased. Shown is 1 representative experiment of 7 independent experiments (top panel), and bar diagrams represent combined results (bottom: left, percentage of live cells; right, total cell number). DN indicates double-negative; DP, double-positive; and SP, single-positive. (D) DN cells of KM mice show reduced expression of CD3, TCR-β, and TCR-γ/δ. Histograms were gated on the DN population of thymocytes derived from KM or control mice. Shown is a representative example of 6 for each experiment. (E) Primary thymocytes exhibit enhanced apoptosis. DN-gated cells were analyzed with 7AAD and annexin V staining. Data are a representative of 3 independent experiments.
Oncogenic K-Ras–driven T-cell disease is hypersensitive toward Notch1 inhibitor therapy. (A) Primary oncogenic K-Ras–expressing T-ALL cells and the human cell line CCRF-CEM were grown in RPMI media without cytokines and treated with the γ-secretase inhibitor XXI as indicated. Cell viability was measured after 48 hours, and the proportion of viable cells relative to the control (no inhibitor) was plotted. Experiments were performed in triplicate. Values are mean plus or minus SEM. (B) Inhibition of Notch1 signaling caused reduced expression of known Notch1 target genes. Primary cells were treated as indicated, and expression of Hes1, Hey1, and Dtx1 mRNA levels was determined by RQ-PCR. Each experiment was performed in duplicate. (C) Inhibition of Notch1 results in cleavage of PARP (top panel) and a decrease of ICN levels (middle panel). Equal loading was confirmed by measuring GAPDH protein levels. GSI indicates γ-secretase inhibitor. (D) The primary oncogenic K-Ras expressing T-ALL cell line 470 and the human cell line CCRF-CEM were grown in RPMI-media without cytokines and treated with rapamycin and PD98059 as indicated. Cell viability was measured after 48 hours, and the proportion of viable cells relative to the control (no inhibitor) was plotted. Experiments were performed in triplicate. Values are mean plus or minus SEM. (E) Primary 470 T-ALL cells were treated with GSI XXI or PD98059 for 6 hours as indicated. Protein lysates were extracted, and immunoblot analysis was performed using antiphosph-p42/44 and anti-ICN antibodies. Equal loading was confirmed by measuring p42/44 and GAPDH protein levels, respectively. GSI indicates γ-secretase inhibitor.
Comment in
- Oncogenic Kras and Notch-1 cooperate in T-cell acute lymphoblastic leukemia/lymphoma.Mansour MR.Mansour MR.Expert Rev Hematol. 2009 Apr;2(2):133-6. doi: 10.1586/ehm.09.3.Expert Rev Hematol. 2009.PMID:21083447
References
- Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991;349:117–127. - PubMed
- Herrmann C. Ras-effector interactions: after one decade. Curr Opin Struct Biol. 2003;13:122–129. - PubMed
- Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature. 1993;366:643–654. - PubMed
- Repasky GA, Chenette EJ, Der CJ. Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol. 2004;14:639–647. - PubMed
- Rodriguez-Viciana P, Warne PH, Dhand R, et al. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994;370:527–532. - PubMed
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