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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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Effect of rGel/BLyS on the growth of human ALL cells(A) Viability of US.7 cells treated for 3 days with different concentrations of rGel/BLyS or rGel as indicated. rGel/BLyS was added once on day 1. (B) Viability of Ph positive (TXL-2, P-2) or Ph-negative (US.7, US.7R) ALL cells after a 7-day incubation with 100 nM rGel/BLyS. rGel/BLyS was added on alternate days (n = 3). * p < 0.05, rGel/BLyS compared to rGel treated cells. (C–E) US.7 cells incubated with rGel/BLyS for 11 days. rGel/BLyS was added on alternate days with fresh medium (n=3) (C) Viability and viable cell numbers. * p < 0.05, rGel/BLyS compared to rGel-treated cells. (D, E) FACS analysis of rGel/BLyS treated samples for (D, marked area) FSC/SSC on gated live cells and for (E) Annexin V staining. All experiments were done in the presence of irradiated OP9 stroma.
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Figure 2: Effect of rGel/BLyS on the growth of human ALL cells(A) Viability of US.7 cells treated for 3 days with different concentrations of rGel/BLyS or rGel as indicated. rGel/BLyS was added once on day 1. (B) Viability of Ph positive (TXL-2, P-2) or Ph-negative (US.7, US.7R) ALL cells after a 7-day incubation with 100 nM rGel/BLyS. rGel/BLyS was added on alternate days (n = 3). * p < 0.05, rGel/BLyS compared to rGel treated cells. (C–E) US.7 cells incubated with rGel/BLyS for 11 days. rGel/BLyS was added on alternate days with fresh medium (n=3) (C) Viability and viable cell numbers. * p < 0.05, rGel/BLyS compared to rGel-treated cells. (D, E) FACS analysis of rGel/BLyS treated samples for (D, marked area) FSC/SSC on gated live cells and for (E) Annexin V staining. All experiments were done in the presence of irradiated OP9 stroma.

Mentions: We next examined the effect of rGel/BLyS on the growth of ALL cells. As shown in Figure 2A, rGel/BLyS induced apoptosis in a dose-dependent manner in US.7 cells after 3 days of treatment. Although higher concentrations of rGel/BLyS augmented the killing of US.7 cells, we chose the lowest dose of 100 nM for further studies because it has been shown that Gelonin causes toxicity to mammalian cells over an extended period, killing cells within 3–4 days by inhibiting protein synthesis.33 We also compared the sensitivity of different ALL cells to this toxin-BLyS conjugate. As shown in Figure 2B, 100 nM rGel/BLyS treatment decreased cell viability significantly in all treated cells as measured after 7 days of treatment. Extension of the US7 culture and treatment with rGel/BLyS for 11 days caused a further decline in the percentage of viable cells and inhibited cell proliferation (Figure 2C). The percentage of live gated cells as measured by FSC/SSC (Figure 2D) declined during this period and these cells underwent apoptosis, as confirmed by Annexin V staining (Figure 2E).


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)

Effect of rGel/BLyS on the growth of human ALL cells(A) Viability of US.7 cells treated for 3 days with different concentrations of rGel/BLyS or rGel as indicated. rGel/BLyS was added once on day 1. (B) Viability of Ph positive (TXL-2, P-2) or Ph-negative (US.7, US.7R) ALL cells after a 7-day incubation with 100 nM rGel/BLyS. rGel/BLyS was added on alternate days (n = 3). * p < 0.05, rGel/BLyS compared to rGel treated cells. (C–E) US.7 cells incubated with rGel/BLyS for 11 days. rGel/BLyS was added on alternate days with fresh medium (n=3) (C) Viability and viable cell numbers. * p < 0.05, rGel/BLyS compared to rGel-treated cells. (D, E) FACS analysis of rGel/BLyS treated samples for (D, marked area) FSC/SSC on gated live cells and for (E) Annexin V staining. All experiments were done in the presence of irradiated OP9 stroma.
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Related In: Results  -  Collection

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Figure 2: Effect of rGel/BLyS on the growth of human ALL cells(A) Viability of US.7 cells treated for 3 days with different concentrations of rGel/BLyS or rGel as indicated. rGel/BLyS was added once on day 1. (B) Viability of Ph positive (TXL-2, P-2) or Ph-negative (US.7, US.7R) ALL cells after a 7-day incubation with 100 nM rGel/BLyS. rGel/BLyS was added on alternate days (n = 3). * p < 0.05, rGel/BLyS compared to rGel treated cells. (C–E) US.7 cells incubated with rGel/BLyS for 11 days. rGel/BLyS was added on alternate days with fresh medium (n=3) (C) Viability and viable cell numbers. * p < 0.05, rGel/BLyS compared to rGel-treated cells. (D, E) FACS analysis of rGel/BLyS treated samples for (D, marked area) FSC/SSC on gated live cells and for (E) Annexin V staining. All experiments were done in the presence of irradiated OP9 stroma.
Mentions: We next examined the effect of rGel/BLyS on the growth of ALL cells. As shown in Figure 2A, rGel/BLyS induced apoptosis in a dose-dependent manner in US.7 cells after 3 days of treatment. Although higher concentrations of rGel/BLyS augmented the killing of US.7 cells, we chose the lowest dose of 100 nM for further studies because it has been shown that Gelonin causes toxicity to mammalian cells over an extended period, killing cells within 3–4 days by inhibiting protein synthesis.33 We also compared the sensitivity of different ALL cells to this toxin-BLyS conjugate. As shown in Figure 2B, 100 nM rGel/BLyS treatment decreased cell viability significantly in all treated cells as measured after 7 days of treatment. Extension of the US7 culture and treatment with rGel/BLyS for 11 days caused a further decline in the percentage of viable cells and inhibited cell proliferation (Figure 2C). The percentage of live gated cells as measured by FSC/SSC (Figure 2D) declined during this period and these cells underwent apoptosis, as confirmed by Annexin V staining (Figure 2E).

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