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. 2012 Oct;87(10):969-76.
doi: 10.1002/ajh.23283. Epub 2012 Jul 5.

Role of c-Myb in the survival of pre B-cell acute lymphoblastic leukemia and leukemogenesis

Purvaba J Sarvaiya  1 , Jason R Schwartz, Claudia P Hernandez, Paulo C Rodriguez, Wayne V Vedeckis
Affiliations

Role of c-Myb in the survival of pre B-cell acute lymphoblastic leukemia and leukemogenesis

Purvaba J Sarvaiya et al. Am J Hematol. 2012 Oct.

Abstract

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The current treatment protocol for ALL involves an intense chemotherapy regimen yielding cure rates of nearly 80%. However, new therapies need to be designed not only to increase the survival rate but also to combat the risk of severe therapy associated toxicities including secondary malignancies, growth problems, organ damage, and infertility. The c-Myb proto-oncogene is highly expressed in immature hematopoietic cells. In this study, we demonstrate that loss of c-Myb itself decreased the viability of these leukemic cells. Additionally, the inhibition of c-Myb caused a decrease in cell proliferation, significantly increased the number of cells in G(0) /G(1) phase of the cell cycle, increased the sensitivity of pre-B-ALL cells to cytotoxic agents in vitro, and significantly delayed disease onset in a mouse model of leukemia. Furthermore, we demonstrate that Bcl-2 is a target of c-Myb in pre-B-ALL cells. Our results identify c-Myb as a potential therapeutic target in pre-B-ALL and suggest that suppression of c-Myb levels or activity, in combination with currently used therapies and/or dose reduction, may lead to a decrease in toxicity and an increase in patient survival rates. Because c-Myb is aberrantly expressed in several other malignancies, targeting c-Myb will have broad clinical applications.

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Conflict of interest statement

Conflict of interest: Nothing to report.

Figures

Figure 1
Figure 1
Controllable knockdown of c-Myb in 697 pre-B-ALL cells A: c-Myb mRNA transcript measurements were performed via QRT-PCR with a probe and primer set specific for c-Myb transcripts on 697 cells stably transduced with a c-Myb specific shRNAmir lentivirus (697 sh-c-Myb) or with a nonsilencing control vector (697 sh-NSC). The data are normalized to endogenous 18S RNA levels. Total RNA was isolated following 48 hr of Doxy treatment. Three separate experiments were performed, and the values are the average ± SEM. ***, P < 0.001 for Doxy-treated samples vs. untreated samples. B: 697 cells stably transduced with a c-Myb specific shRNAmir lentivirus and with nonsilencing control vector were treated with 1 μg/ml of Doxy for 48 hr. Expression levels of c-Myb, TurboRFP, and GAPDH proteins were analyzed by immunoblotting. Blots representative of three independent experiments are shown.
Figure 2
Figure 2
The knockdown of c-Myb decreases [3H] thymidine incorporation and increases the number of cells in the G0/G1 phase of the cell cycle. A: 697 sh-c-Myb cells and 697 sh-NSC cells were treated with or without 1 μg/ml Doxy for 96 hr. Doxy in the media was replenished every 48 hr. 1 μCi [3H] thymidine per well was added for the last 24 hr of the 96-h assay and [3H] thymidine uptake was measured as described in Materials and Methods. Data are expressed as counts per minute (CPM) (mean ± SD) for at least three experiments. ***, P < 0.001 for Doxy-treated samples vs. untreated samples. B and C: 697 sh-c-Myb cells and 697 sh-NSC cells were treated with or without 1 μg/ml Doxy for 66 hr. Cells were stained with propidium iodide, and cell cycle analysis was performed. Error bars represent the standard error of the mean of three separate experiments. ***, P < 0.001 for Doxy-treated samples vs. untreated samples for the % of cells in G0/G1 phase of the cell cycle.
Figure 3
Figure 3
The knockdown of c-Myb reduces the viability and induces apoptosis in 697 pre-B-ALL cells. A and B: 697 sh-c-Myb cells and 697 sh-NSC cells were treated with or without 1 μg/ml Doxy for 84 hr. Cell viability was then measured using the Vybrant #4 apoptosis assay. Apoptotic and dead cells were identified by flow cytometry analysis of cells stained with YO-PRO1 and propidium iodide. A: Representative flow cytometry profiles are shown. Cells undergoing apoptosis are stained with YO-PRO1 but are impermeable to propidium iodide. Dead cells and cells in late apoptosis are permeable to both dyes. Viable cells are not stained by either dye. Apoptotic cells are shown in the bottom right quadrants and dead cells are shown in the upper right quandrants. B: Cumulative data demonstrate a significant decrease in cell viability when c-Myb is knocked down. Error bars represent the standard error of the mean of three separate experiments. **, P < 0.001 for Doxy-treated samples vs. untreated samples C: 697 sh-c-Myb cells and 697 sh-NSC cells were treated with or without 1 μg/ml Doxy for 48 hr. The doxy-treated cells were then exposed to 5 μM 6-MP and 150 nM VP-16 for 24 hr. Whole cell lysates were prepared, and the expression of c-Myb, PARP, and GAPDH were determined by immunoblotting. Blots representative of three independent experiments are shown.
Figure 4
Figure 4
c-Myb targets the antiapoptotic protein Bcl-2 in 697 pre-B-ALL cells A: Bcl-2 and c-Myc mRNA transcript measurements were performed via QRT-PCR on 697 cells stably transduced with a c-Myb specific shRNAmir lentivirus (697 sh-c-Myb) or with a nonsilencing control vector (697 sh-NSC). The data are normalized to endogenous 18S RNA levels. Total RNA was isolated following 48 hr of Doxy treatment. Three separate experiments were performed, and the values are the average ± SEM. ***, P <0.001 for Doxy-treated samples vs. untreated samples. B: 697 sh-c-Myb and 697 sh-NSC cells were treated with 1 μg/ml of Doxy for 48 hr. Expression levels of the Bcl-2 and GAPDH proteins were analyzed by immunoblotting. A blot that is representative of three independent experiments are shown.
Figure 5
Figure 5
Inhibition of c-Myb expression results in increased chemosensitivity in 697 pre-B-ALL cells. 697 sh-c-Myb cells and 697 sh-NSC cells were treated with or without 1 μg/ml Doxy for 48 hr. The cells were then treated with the indicated concentrations of 6-mercaptopurine (6-MP) or etoposide (VP-16) for 48 hr, or with Doxorubicin (DOXO) or Dexamethasone (Dex) for 24 hr. Cell viability was then measured using the Vybrant #4 apoptosis assay. The knockdown of c-Myb caused a significant increase in chemosensitivity to all the four drugs in 697 cells. Error bars represent the standard error of the mean of three separate experiments. **, P < 0.01, ***, P < 0.001 for Doxy-treated samples vs. untreated sample.
Figure 6
Figure 6
Loss of c-Myb delays the progression of leukemia in a mouse xeno-graft model of human leukemia. A: Sublethally irradiated NOD-SCID mice were injected with 1 × 107 697 sh-NSC cells. Seven days after injecting the cells, the mice were treated with Doxy or PBS three times a week. Kaplan-Meier survival curve revealed no significant difference in leukemia free survival in these mice. B: Sublethally irradiated NOD-SCID mice were injected with 1 × 107 697 sh-c-Myb cells. Seven days after injecting the cells, the mice were treated with Doxy or PBS three times a week. Kaplan-Meier survival curve revealed a significant increase in leukemia free survival of mice injected with 697 sh c-Myb cells treated with Doxy. C: Real-time PCR was performed to confirm the reduction of c-Myb expression in CD19+ ALL cells harvested from the bone marrow of mice which were injected with 697 sh-c-Myb cells treated with Doxy. Similarly, the unchanged levels of c-Myb expression were confirmed in CD19+ ALL cells collected from mice injected with 697 sh-c-Myb cells treated with PBS as well as 697 sh-NSC cells treated with PBS as well as Doxy. Error bars represent the standard error of the mean of three separate experiments. **, P <0.01 for Doxy-treated samples vs. untreated sample.
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References

    1. Pui CH. Recent research advances in childhood acute lymphoblastic leukemia. J Formos Med Assoc. 2010;109:777–787. - PubMed
    1. Pui C-H, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371:1030–1043. - PubMed
    1. Gurney JG, Severson RK, Davis S, Robison LL. Incidence of cancer in children in the United States. Sex-, race-, and 1-year age-specific rates by histologic type. Cancer. 1995;75:2186–2195. - PubMed
    1. Pui CH. Childhood Leukemias. Cambridge: Cambridge University Press; 2006. pp. 21–47.
    1. Raimondi SC, Behm FG, Roberson PK, et al. Cytogenetics of pre-B-cell acute lymphoblastic leukemia with emphasis on prognostic implications of the t(1;19) J Clin Oncol. 1990;8:1380–1388. - PubMed

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