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Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

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Increased tumor cell engraftment in PAX5− MCL xenografted mice(A) PAX5− MCL cells or control cells were I.V. injected into NOD/SCID mice (n=22) at two different dosages (1×106 or 5×106 cells). After 8 weeks, the xenografted mice were sacrificed. Bone marrow (femurs and tibias) and the spleen were collected and stained for human leukocyte cells using an anti-CD45 antibody. The samples were then analyzed for GFP+ and CD45+ cells using FACS. (B) PAX5− MCL cells (3 × 106) or control cells were subcutaneously injected into NOD/SCID mice (n=9), and the tumor volumes were measured using a digital caliper after 4 weeks. (C) CT and PET imaging revealed that PAX5− MCL cells formed larger subcutaneous tumors in vivo. 18F-FDG was injected into mice 2.5 hours prior to PET and CT imaging. Representative animals for each experimental arm are displayed in white light (Top), on CT (Middle) and on PET (Bottom). The white arrows indicate a subcutaneous tumor; the dotted circles represent the area of the subcutaneous tumor. CT imaging scale bar: Hounsfield scale; PET imaging scale bar: %ID/g. (D)18F-FDG counts of common MCL dissemination organ sites from SP53 PAX5− or control subcutaneous xenografts. The %ID/g of each target organ was normalized to that of muscle tissue. The dotted line represents the percentage of 18F-FDG uptake relative to muscle (muscle = 100%). Sub-cu = subcutaneous; LN = lymph node. (E) PAX5− MCL cells were stained for PKH26 and subsequently seeded on a pre-established monolayer of HS5 bone marrow stromal cells. The arrows indicate PKH26+ cells. Scale bar, 200 µm. Lower panel: The adhesion of lymphoma cells was calculated by measuring the PKH26 dye intensity relative to the fluorescence of the inputs. Each value represents the mean ± S.D. (n=6).*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 3: Increased tumor cell engraftment in PAX5− MCL xenografted mice(A) PAX5− MCL cells or control cells were I.V. injected into NOD/SCID mice (n=22) at two different dosages (1×106 or 5×106 cells). After 8 weeks, the xenografted mice were sacrificed. Bone marrow (femurs and tibias) and the spleen were collected and stained for human leukocyte cells using an anti-CD45 antibody. The samples were then analyzed for GFP+ and CD45+ cells using FACS. (B) PAX5− MCL cells (3 × 106) or control cells were subcutaneously injected into NOD/SCID mice (n=9), and the tumor volumes were measured using a digital caliper after 4 weeks. (C) CT and PET imaging revealed that PAX5− MCL cells formed larger subcutaneous tumors in vivo. 18F-FDG was injected into mice 2.5 hours prior to PET and CT imaging. Representative animals for each experimental arm are displayed in white light (Top), on CT (Middle) and on PET (Bottom). The white arrows indicate a subcutaneous tumor; the dotted circles represent the area of the subcutaneous tumor. CT imaging scale bar: Hounsfield scale; PET imaging scale bar: %ID/g. (D)18F-FDG counts of common MCL dissemination organ sites from SP53 PAX5− or control subcutaneous xenografts. The %ID/g of each target organ was normalized to that of muscle tissue. The dotted line represents the percentage of 18F-FDG uptake relative to muscle (muscle = 100%). Sub-cu = subcutaneous; LN = lymph node. (E) PAX5− MCL cells were stained for PKH26 and subsequently seeded on a pre-established monolayer of HS5 bone marrow stromal cells. The arrows indicate PKH26+ cells. Scale bar, 200 µm. Lower panel: The adhesion of lymphoma cells was calculated by measuring the PKH26 dye intensity relative to the fluorescence of the inputs. Each value represents the mean ± S.D. (n=6).*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).

Mentions: We first transplanted PAX5− MCL cells or control cells into NOD/SCID mice via intravenous injection. After 6–8 weeks, we discovered significantly greater numbers of CD45+ and GFP+ cells from the PAX5− MCL xenografted mice than in the control mice (Figure 3a and Supplementary Figure 3a). The differences in cell engraftment were particularly large in the bone marrow regardless of the number of transplanted cells (Figure 3a). IHC analyses of frozen tissue sections also displayed greater numbers of CD45+ cells in bones from PAX5− MCL xenografted mice (Supplementary Figure 3b).


Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

Increased tumor cell engraftment in PAX5− MCL xenografted mice(A) PAX5− MCL cells or control cells were I.V. injected into NOD/SCID mice (n=22) at two different dosages (1×106 or 5×106 cells). After 8 weeks, the xenografted mice were sacrificed. Bone marrow (femurs and tibias) and the spleen were collected and stained for human leukocyte cells using an anti-CD45 antibody. The samples were then analyzed for GFP+ and CD45+ cells using FACS. (B) PAX5− MCL cells (3 × 106) or control cells were subcutaneously injected into NOD/SCID mice (n=9), and the tumor volumes were measured using a digital caliper after 4 weeks. (C) CT and PET imaging revealed that PAX5− MCL cells formed larger subcutaneous tumors in vivo. 18F-FDG was injected into mice 2.5 hours prior to PET and CT imaging. Representative animals for each experimental arm are displayed in white light (Top), on CT (Middle) and on PET (Bottom). The white arrows indicate a subcutaneous tumor; the dotted circles represent the area of the subcutaneous tumor. CT imaging scale bar: Hounsfield scale; PET imaging scale bar: %ID/g. (D)18F-FDG counts of common MCL dissemination organ sites from SP53 PAX5− or control subcutaneous xenografts. The %ID/g of each target organ was normalized to that of muscle tissue. The dotted line represents the percentage of 18F-FDG uptake relative to muscle (muscle = 100%). Sub-cu = subcutaneous; LN = lymph node. (E) PAX5− MCL cells were stained for PKH26 and subsequently seeded on a pre-established monolayer of HS5 bone marrow stromal cells. The arrows indicate PKH26+ cells. Scale bar, 200 µm. Lower panel: The adhesion of lymphoma cells was calculated by measuring the PKH26 dye intensity relative to the fluorescence of the inputs. Each value represents the mean ± S.D. (n=6).*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 3: Increased tumor cell engraftment in PAX5− MCL xenografted mice(A) PAX5− MCL cells or control cells were I.V. injected into NOD/SCID mice (n=22) at two different dosages (1×106 or 5×106 cells). After 8 weeks, the xenografted mice were sacrificed. Bone marrow (femurs and tibias) and the spleen were collected and stained for human leukocyte cells using an anti-CD45 antibody. The samples were then analyzed for GFP+ and CD45+ cells using FACS. (B) PAX5− MCL cells (3 × 106) or control cells were subcutaneously injected into NOD/SCID mice (n=9), and the tumor volumes were measured using a digital caliper after 4 weeks. (C) CT and PET imaging revealed that PAX5− MCL cells formed larger subcutaneous tumors in vivo. 18F-FDG was injected into mice 2.5 hours prior to PET and CT imaging. Representative animals for each experimental arm are displayed in white light (Top), on CT (Middle) and on PET (Bottom). The white arrows indicate a subcutaneous tumor; the dotted circles represent the area of the subcutaneous tumor. CT imaging scale bar: Hounsfield scale; PET imaging scale bar: %ID/g. (D)18F-FDG counts of common MCL dissemination organ sites from SP53 PAX5− or control subcutaneous xenografts. The %ID/g of each target organ was normalized to that of muscle tissue. The dotted line represents the percentage of 18F-FDG uptake relative to muscle (muscle = 100%). Sub-cu = subcutaneous; LN = lymph node. (E) PAX5− MCL cells were stained for PKH26 and subsequently seeded on a pre-established monolayer of HS5 bone marrow stromal cells. The arrows indicate PKH26+ cells. Scale bar, 200 µm. Lower panel: The adhesion of lymphoma cells was calculated by measuring the PKH26 dye intensity relative to the fluorescence of the inputs. Each value represents the mean ± S.D. (n=6).*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
Mentions: We first transplanted PAX5− MCL cells or control cells into NOD/SCID mice via intravenous injection. After 6–8 weeks, we discovered significantly greater numbers of CD45+ and GFP+ cells from the PAX5− MCL xenografted mice than in the control mice (Figure 3a and Supplementary Figure 3a). The differences in cell engraftment were particularly large in the bone marrow regardless of the number of transplanted cells (Figure 3a). IHC analyses of frozen tissue sections also displayed greater numbers of CD45+ cells in bones from PAX5− MCL xenografted mice (Supplementary Figure 3b).

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

Show MeSH
Related in: MedlinePlus