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Treatment of acute lymphoblastic leukemia with an rGel/BLyS fusion toxin.

Parameswaran R, Yu M, Lyu MA, Lim M, Rosenblum MG, Groffen J, Heisterkamp N - Leukemia (2012)

Bottom Line: Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection.Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice.Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Carcinogenesis, Division of Hematology/Oncology and The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.

ABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and a major cause of mortality from hematopoietic malignancies in adults. A substantial number of patients become drug resistant during chemotherapy, necessitating the development of alternative modes of treatment. rGel (recombinant Gelonin)/BlyS (B-lymphocyte stimulator) is a toxin-cytokine fusion protein used for selective killing of malignant B-cells expressing receptors for B-cell-activating factor (BAFF/BLyS) by receptor-targeted delivery of the toxin, Gelonin. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection. Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice. Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation. Thus, a combination treatment with the B-cell-specific fusion toxin rGel/BLyS and the mobilizing agent AMD3100 could be an effective alternative approach to chemotherapy for the treatment of primary and relapsed ALL.

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rGel/BLyS binds to human ALL cells(A) FACS analysis using anti-Gelonin antibodies for rGel (black) or cell surface rGel/BLyS (blue) and total rGel/BLyS (red) after incubation with 400 nM rGel/BLyS. Percentages indicate positivity for total rGel/BLyS (B) Immunohistochemistry for rGel or rGel/BLyS in US.7 cells. Note the apparent signal of rGel/BLyS as a perinuclear ring, due to the relatively large nucleus and small volume of cytoplasm in these cells. Green, Gelonin antibodies, blue, DAPI counter-stain for the nucleus. Images were captured using a Leica DIC analyzer, 200x 1.4–0.7, oil. (C) Detection of rGel or rGel/BLyS with anti-Gelonin antibody after pre-incubation of US.7 cells with the indicated concentrations of (I) recombinant human BAFF or (II) BAFF-R antibody; both then followed by incubation with 100 nM rGel/BLyS or (III) when BAFF-R Fc was added together with rGel/BLyS. (n=3). Controls; isotype (black) and positive control (no treatment, red); treated cells (blue).
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Figure 1: rGel/BLyS binds to human ALL cells(A) FACS analysis using anti-Gelonin antibodies for rGel (black) or cell surface rGel/BLyS (blue) and total rGel/BLyS (red) after incubation with 400 nM rGel/BLyS. Percentages indicate positivity for total rGel/BLyS (B) Immunohistochemistry for rGel or rGel/BLyS in US.7 cells. Note the apparent signal of rGel/BLyS as a perinuclear ring, due to the relatively large nucleus and small volume of cytoplasm in these cells. Green, Gelonin antibodies, blue, DAPI counter-stain for the nucleus. Images were captured using a Leica DIC analyzer, 200x 1.4–0.7, oil. (C) Detection of rGel or rGel/BLyS with anti-Gelonin antibody after pre-incubation of US.7 cells with the indicated concentrations of (I) recombinant human BAFF or (II) BAFF-R antibody; both then followed by incubation with 100 nM rGel/BLyS or (III) when BAFF-R Fc was added together with rGel/BLyS. (n=3). Controls; isotype (black) and positive control (no treatment, red); treated cells (blue).

Mentions: We used both Ph negative (US.7, US.7R) and Ph positive (TXL-2, BLQ-1) ALL cells to examine if the rGel/BLyS fusion protein is able to bind to the BAFF-R expressed by pre-B ALL cells. After a 2-hour incubation of ALL cells with rGel/BLyS fusion protein or with control rGel, we examined the presence of these proteins in and on the ALL cells using a Gelonin-specific antibody. As shown in Figure 1A, rGel-treated cells showed no staining with anti-Gelonin antibodies (black line) and little rGel/BLyS (blue line) was detected on the surface of the cells. However, a clear positive signal in permeabilized cells indicates that rGel/BLyS had been internalized (Figure 1A, red line). This confirmed that Gelonin is unable to cross the plasma membrane without conjugation to BLyS. We also demonstrated the intracellular presence of rGel/BLyS by fluorescent microscopy (Figure 1B). To investigate the specificity of rGel/BLyS binding to the BAFF-R on the pre-B ALL cells, US.7 cells were either pre-treated with recombinant BAFF, with antibodies to BAFF-R (2 hours before addition of rGel/BLyS) or with recombinant BAFF-R Fc (added together with rGel/BLyS). As evidenced by FACS analysis on permeabilized cells (Figure 1C), BAFF, BAFF-R antibody and BAFF-R Fc inhibited rGel/BLyS detection in US.7 cells in a dose- dependent manner. Thus, rGel/BLyS failed to bind to the BAFF-R when these receptors were pre-occupied with recombinant BAFF or BAFF-R antibody.


Treatment of acute lymphoblastic leukemia with an rGel/BLyS fusion toxin.

Parameswaran R, Yu M, Lyu MA, Lim M, Rosenblum MG, Groffen J, Heisterkamp N - Leukemia (2012)

rGel/BLyS binds to human ALL cells(A) FACS analysis using anti-Gelonin antibodies for rGel (black) or cell surface rGel/BLyS (blue) and total rGel/BLyS (red) after incubation with 400 nM rGel/BLyS. Percentages indicate positivity for total rGel/BLyS (B) Immunohistochemistry for rGel or rGel/BLyS in US.7 cells. Note the apparent signal of rGel/BLyS as a perinuclear ring, due to the relatively large nucleus and small volume of cytoplasm in these cells. Green, Gelonin antibodies, blue, DAPI counter-stain for the nucleus. Images were captured using a Leica DIC analyzer, 200x 1.4–0.7, oil. (C) Detection of rGel or rGel/BLyS with anti-Gelonin antibody after pre-incubation of US.7 cells with the indicated concentrations of (I) recombinant human BAFF or (II) BAFF-R antibody; both then followed by incubation with 100 nM rGel/BLyS or (III) when BAFF-R Fc was added together with rGel/BLyS. (n=3). Controls; isotype (black) and positive control (no treatment, red); treated cells (blue).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376225&req=5

Figure 1: rGel/BLyS binds to human ALL cells(A) FACS analysis using anti-Gelonin antibodies for rGel (black) or cell surface rGel/BLyS (blue) and total rGel/BLyS (red) after incubation with 400 nM rGel/BLyS. Percentages indicate positivity for total rGel/BLyS (B) Immunohistochemistry for rGel or rGel/BLyS in US.7 cells. Note the apparent signal of rGel/BLyS as a perinuclear ring, due to the relatively large nucleus and small volume of cytoplasm in these cells. Green, Gelonin antibodies, blue, DAPI counter-stain for the nucleus. Images were captured using a Leica DIC analyzer, 200x 1.4–0.7, oil. (C) Detection of rGel or rGel/BLyS with anti-Gelonin antibody after pre-incubation of US.7 cells with the indicated concentrations of (I) recombinant human BAFF or (II) BAFF-R antibody; both then followed by incubation with 100 nM rGel/BLyS or (III) when BAFF-R Fc was added together with rGel/BLyS. (n=3). Controls; isotype (black) and positive control (no treatment, red); treated cells (blue).
Mentions: We used both Ph negative (US.7, US.7R) and Ph positive (TXL-2, BLQ-1) ALL cells to examine if the rGel/BLyS fusion protein is able to bind to the BAFF-R expressed by pre-B ALL cells. After a 2-hour incubation of ALL cells with rGel/BLyS fusion protein or with control rGel, we examined the presence of these proteins in and on the ALL cells using a Gelonin-specific antibody. As shown in Figure 1A, rGel-treated cells showed no staining with anti-Gelonin antibodies (black line) and little rGel/BLyS (blue line) was detected on the surface of the cells. However, a clear positive signal in permeabilized cells indicates that rGel/BLyS had been internalized (Figure 1A, red line). This confirmed that Gelonin is unable to cross the plasma membrane without conjugation to BLyS. We also demonstrated the intracellular presence of rGel/BLyS by fluorescent microscopy (Figure 1B). To investigate the specificity of rGel/BLyS binding to the BAFF-R on the pre-B ALL cells, US.7 cells were either pre-treated with recombinant BAFF, with antibodies to BAFF-R (2 hours before addition of rGel/BLyS) or with recombinant BAFF-R Fc (added together with rGel/BLyS). As evidenced by FACS analysis on permeabilized cells (Figure 1C), BAFF, BAFF-R antibody and BAFF-R Fc inhibited rGel/BLyS detection in US.7 cells in a dose- dependent manner. Thus, rGel/BLyS failed to bind to the BAFF-R when these receptors were pre-occupied with recombinant BAFF or BAFF-R antibody.

Bottom Line: Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection.Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice.Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Carcinogenesis, Division of Hematology/Oncology and The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.

ABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and a major cause of mortality from hematopoietic malignancies in adults. A substantial number of patients become drug resistant during chemotherapy, necessitating the development of alternative modes of treatment. rGel (recombinant Gelonin)/BlyS (B-lymphocyte stimulator) is a toxin-cytokine fusion protein used for selective killing of malignant B-cells expressing receptors for B-cell-activating factor (BAFF/BLyS) by receptor-targeted delivery of the toxin, Gelonin. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection. Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice. Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation. Thus, a combination treatment with the B-cell-specific fusion toxin rGel/BLyS and the mobilizing agent AMD3100 could be an effective alternative approach to chemotherapy for the treatment of primary and relapsed ALL.

Show MeSH
Related in: MedlinePlus