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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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Combination therapy using rGel/BLyS and AMD3100 results in significantly reduced tumor burden in bone marrow(A) Schematic overview of treatment. Mice (n = 5/group) were started on treatment 6 days after transplant with i.p. injections of either saline or rGel/BLyS (2.75 mg/kg) twice a week with the last treatment on day 33. Total amount of rGel/BLyS injected, 22 mg/kg. The combination treatment group additionally was treated with 10 mg/kg AMD3100 as shown. rGel/BLyS was injected 5 hours after administration of AMD3100. (B) Survival of treated mice. *p=0.0047 for AMD3100 + rGel/BLyS or rGel/BLyS versus control or AMD3100. (C) Percentage of TXL-2 ALL cells in rGel/BLyS treated mice, compared to that of rGel/BLyS + AMD3100 treated mice, as detected by FACS for human CD19 and CD10, at the time of sacrifice. Each symbol represents the percentage of CD19+, CD10+ cells in the live-gated population in individual mice. For BM and spleen *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS) and *p<0.001 for PB samples (rGel/BLyS and AMD3100 + rGel/BLyS group compared to control or AMD3100 alone treated group). (D) Percentage of US.7 ALL cells in rGel/BLyS treated mice compared to rGel/BLyS + AMD3100 treated mice (n=3/group) as detected by FACS analysis for human CD19 and CD10 positive cells. *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS). Note that different scales are used in the individual panels of (C) and (D).
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Figure 6: Combination therapy using rGel/BLyS and AMD3100 results in significantly reduced tumor burden in bone marrow(A) Schematic overview of treatment. Mice (n = 5/group) were started on treatment 6 days after transplant with i.p. injections of either saline or rGel/BLyS (2.75 mg/kg) twice a week with the last treatment on day 33. Total amount of rGel/BLyS injected, 22 mg/kg. The combination treatment group additionally was treated with 10 mg/kg AMD3100 as shown. rGel/BLyS was injected 5 hours after administration of AMD3100. (B) Survival of treated mice. *p=0.0047 for AMD3100 + rGel/BLyS or rGel/BLyS versus control or AMD3100. (C) Percentage of TXL-2 ALL cells in rGel/BLyS treated mice, compared to that of rGel/BLyS + AMD3100 treated mice, as detected by FACS for human CD19 and CD10, at the time of sacrifice. Each symbol represents the percentage of CD19+, CD10+ cells in the live-gated population in individual mice. For BM and spleen *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS) and *p<0.001 for PB samples (rGel/BLyS and AMD3100 + rGel/BLyS group compared to control or AMD3100 alone treated group). (D) Percentage of US.7 ALL cells in rGel/BLyS treated mice compared to rGel/BLyS + AMD3100 treated mice (n=3/group) as detected by FACS analysis for human CD19 and CD10 positive cells. *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS). Note that different scales are used in the individual panels of (C) and (D).

Mentions: We translated our in vitro observations to an in vivo experiment. For in vivo combination treatments, we used NSG mice transplanted with Ph-positive TXL-2 ALL cells to study the beneficial effect of AMD3100 in combination with rGel/BLyS in killing ALL cells. Mice were treated with PBS, AMD3100 alone, rGel/BLyS alone or rGel/BLyS + AMD3100. In the combination group, mice were first treated with rGel/BLyS alone to reduce the overall leukemia burden. When AMD3100 was added on to this treatment, we injected the AMD3100 5–6 hours before the rGel/BLyS was administered. This strategy was used to ensure the presence of circulating ALL cells at the time when the toxin would also be in the circulation. When the mice in the control and AMD3100 alone treated group lost more than 20% of their initial body weight on day 32 after transplant, they were sacrificed. Leukemic mice treated with the combination of rGel/BLyS+AMD3100 and rGel/BLyS alone did not show any weight loss or other symptoms of the disease at day 32. They were sacrificed on day 46 (Figure 6A, B), when the rGel/BLyS alone treated group had exhibited significant loss of body weight (Suppl. Figure 5A, B). FACS analysis was performed for the presence of human ALL cells in spleen, PB, BM, lungs, liver and kidney. As expected and consistent with the in vivo experiment with US.7 cells, there was a complete eradication of ALL cells from the circulation. However, in contrast to the results with US7-transplanted mice, in which we found high amounts of ALL cells in the bone marrow, mice transplanted with TXL-2 cells and treated with additional applications of rGel/BLyS had a reduction of the leukemia burden in the BM (Figure 6C). Moreover, this was reduced even further in the mice treated with both rGel/BLyS and AMD3100. Human ALL cell numbers in the spleen were also significantly lower in the rGel/BLyS + AMD3100 treated group (Figure 6C). To examine ALL metastasis in other organs, we performed FACS analysis on lungs and liver cells (Figure 6C, bottom row). There was little infiltration of leukemic cells in these peripheral organs. Also, splenomegaly was not present (Suppl. Figure 5C). rGel/BLyS in combination with AMD3100 appeared to be well-tolerated since there was no weight loss in the dual treatment group (Suppl. Figure 5B) or changes in physical appearance.


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)

Combination therapy using rGel/BLyS and AMD3100 results in significantly reduced tumor burden in bone marrow(A) Schematic overview of treatment. Mice (n = 5/group) were started on treatment 6 days after transplant with i.p. injections of either saline or rGel/BLyS (2.75 mg/kg) twice a week with the last treatment on day 33. Total amount of rGel/BLyS injected, 22 mg/kg. The combination treatment group additionally was treated with 10 mg/kg AMD3100 as shown. rGel/BLyS was injected 5 hours after administration of AMD3100. (B) Survival of treated mice. *p=0.0047 for AMD3100 + rGel/BLyS or rGel/BLyS versus control or AMD3100. (C) Percentage of TXL-2 ALL cells in rGel/BLyS treated mice, compared to that of rGel/BLyS + AMD3100 treated mice, as detected by FACS for human CD19 and CD10, at the time of sacrifice. Each symbol represents the percentage of CD19+, CD10+ cells in the live-gated population in individual mice. For BM and spleen *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS) and *p<0.001 for PB samples (rGel/BLyS and AMD3100 + rGel/BLyS group compared to control or AMD3100 alone treated group). (D) Percentage of US.7 ALL cells in rGel/BLyS treated mice compared to rGel/BLyS + AMD3100 treated mice (n=3/group) as detected by FACS analysis for human CD19 and CD10 positive cells. *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS). Note that different scales are used in the individual panels of (C) and (D).
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Figure 6: Combination therapy using rGel/BLyS and AMD3100 results in significantly reduced tumor burden in bone marrow(A) Schematic overview of treatment. Mice (n = 5/group) were started on treatment 6 days after transplant with i.p. injections of either saline or rGel/BLyS (2.75 mg/kg) twice a week with the last treatment on day 33. Total amount of rGel/BLyS injected, 22 mg/kg. The combination treatment group additionally was treated with 10 mg/kg AMD3100 as shown. rGel/BLyS was injected 5 hours after administration of AMD3100. (B) Survival of treated mice. *p=0.0047 for AMD3100 + rGel/BLyS or rGel/BLyS versus control or AMD3100. (C) Percentage of TXL-2 ALL cells in rGel/BLyS treated mice, compared to that of rGel/BLyS + AMD3100 treated mice, as detected by FACS for human CD19 and CD10, at the time of sacrifice. Each symbol represents the percentage of CD19+, CD10+ cells in the live-gated population in individual mice. For BM and spleen *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS) and *p<0.001 for PB samples (rGel/BLyS and AMD3100 + rGel/BLyS group compared to control or AMD3100 alone treated group). (D) Percentage of US.7 ALL cells in rGel/BLyS treated mice compared to rGel/BLyS + AMD3100 treated mice (n=3/group) as detected by FACS analysis for human CD19 and CD10 positive cells. *p<0.001 (rGel/BLyS compared to AMD3100 + rGel/BLyS). Note that different scales are used in the individual panels of (C) and (D).
Mentions: We translated our in vitro observations to an in vivo experiment. For in vivo combination treatments, we used NSG mice transplanted with Ph-positive TXL-2 ALL cells to study the beneficial effect of AMD3100 in combination with rGel/BLyS in killing ALL cells. Mice were treated with PBS, AMD3100 alone, rGel/BLyS alone or rGel/BLyS + AMD3100. In the combination group, mice were first treated with rGel/BLyS alone to reduce the overall leukemia burden. When AMD3100 was added on to this treatment, we injected the AMD3100 5–6 hours before the rGel/BLyS was administered. This strategy was used to ensure the presence of circulating ALL cells at the time when the toxin would also be in the circulation. When the mice in the control and AMD3100 alone treated group lost more than 20% of their initial body weight on day 32 after transplant, they were sacrificed. Leukemic mice treated with the combination of rGel/BLyS+AMD3100 and rGel/BLyS alone did not show any weight loss or other symptoms of the disease at day 32. They were sacrificed on day 46 (Figure 6A, B), when the rGel/BLyS alone treated group had exhibited significant loss of body weight (Suppl. Figure 5A, B). FACS analysis was performed for the presence of human ALL cells in spleen, PB, BM, lungs, liver and kidney. As expected and consistent with the in vivo experiment with US.7 cells, there was a complete eradication of ALL cells from the circulation. However, in contrast to the results with US7-transplanted mice, in which we found high amounts of ALL cells in the bone marrow, mice transplanted with TXL-2 cells and treated with additional applications of rGel/BLyS had a reduction of the leukemia burden in the BM (Figure 6C). Moreover, this was reduced even further in the mice treated with both rGel/BLyS and AMD3100. Human ALL cell numbers in the spleen were also significantly lower in the rGel/BLyS + AMD3100 treated group (Figure 6C). To examine ALL metastasis in other organs, we performed FACS analysis on lungs and liver cells (Figure 6C, bottom row). There was little infiltration of leukemic cells in these peripheral organs. Also, splenomegaly was not present (Suppl. Figure 5C). rGel/BLyS in combination with AMD3100 appeared to be well-tolerated since there was no weight loss in the dual treatment group (Suppl. Figure 5B) or changes in physical appearance.

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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Related in: MedlinePlus