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Myxoma virus targets primary human leukemic stem and progenitor cells while sparing normal hematopoietic stem and progenitor cells.

Kim M, Madlambayan GJ, Rahman MM, Smallwood SE, Meacham AM, Hosaka K, Scott EW, Cogle CR, McFadden G - Leukemia (2009)

View Article: PubMed Central - PubMed

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Regimens of high dose chemotherapy followed by autologous blood and marrow transplantation (ABMT) have been used to treat patients with acute myelogenous leukemia (AML), but leukemia relapse is frequent, likely due to marrow contamination with leukemic hematopoietic stem and progenitor cells (HSPCs) and residual disease in protective niches... Purging autologous hematopoietic grafts of contaminating cancer cells prior to transplant serves as a viable strategy for increasing transplant efficacy but would also require sparing normal HSPCs within the same hematopoietic cell graft... Thus, to determine whether oncolytic viruses can be used as a therapeutic modality for purging leukemia from autologous grafts, we evaluated MYXV in a pre-clinical model of AML and normal HSPC transplantation... Moreover, using identical ex vivo conditions, MYXV does not affect either in vitro colony forming potential or in vivo engraftment of normal HSPCs derived from healthy human bone marrow (BM)... These results show safety and efficacy of using oncolytic MYXV to purge AML HSPCs from autologous HSPC grafts intended for hematopoietic rescue... In these samples, 40.3 ± 5.0 % of AML cells were infected with the virus, which was significantly higher in comparison to normal BM-derived MNCs (p<0.05; Figure 1A)... Of the total AML CD34 population, we found that 65 ± 6% were infected with MYXV, which was significantly higher in comparison to normal BM CD34 cells (p<0.05)... Together, these data indicated that MYXV effectively infects AML in vitro, and impairs clonal proliferation... Based on the in vitro safety results showing normal colony forming potential from MYXV treated normal HSPCs, we next tested safety by in vivo repopulation assays... None of the immunocompromised NOG mice developed any evidence of viral replication in their skin or in any internal tissues, indicating that even severely immunodeficient hosts are non-permissive for MYXV infection... The data indicate that ex vivo treatment with MYXV does not reduce in vivo engraftment potential of normal BM-derived MNCs or CD34 HSPCs... This critical safety data supports the notion that MYXV would be a safe candidate for purging leukemic cells from autologous hematopoietic grafts, even in immunocompromised recipients... In the primary, high risk AML specimen tested, normal HSPC levels were extremely low, thus normal HSPC engraftment in immunocompromised mice did not occur, as expected... Taken together, the data indicate that MYXV incubation ex vivo significantly inhibited subsequent AML HSPC engraftment in vivo, resulting in phenotypic and molecular remissions of FLT3 ITD AML.

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MYXV treatment of normal and leukemic HSPCs in vitro(A) Normal and leukemic cells (FLT ITD+) were incubated with MYXV expressing GFP at 10 MOI. Flow cytometric analysis demonstrated that a small population of normal MNCs was infected while a significant proportion of AML cells were susceptible to MYXV infection. (B) Analysis of apoptotic events was examined and showed that cell death was mainly observed in the GFP+ MYXV infected cell population as shown by Annexin V staining. All gating was established using appropriate isotype controls. (C, D) Normal BM cells were incubated with MYXV expressing GFP at 10 MOI and assessed for colony forming potential. Various types of CFC colonies were formed and showed no GFP expression in either MYXV-treated (D) or mock-treated samples (C) indicating that MYXV was unable to infect normal CFCs. (E) The frequency of each colony type was enumerated following MYXV or mock treatment. Similar frequencies were observed between both cohorts suggesting that MYXV does not prevent normal CFC colony differentiation in vitro. Data are representative of repeat experiments and values represent mean ± standard deviation. (F) Mock-treated AML cells formed AML-CFU colonies. (G) MYXV-treated AML cells showed signs of infection (GFP+) and did not form colonies.
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Figure 1: MYXV treatment of normal and leukemic HSPCs in vitro(A) Normal and leukemic cells (FLT ITD+) were incubated with MYXV expressing GFP at 10 MOI. Flow cytometric analysis demonstrated that a small population of normal MNCs was infected while a significant proportion of AML cells were susceptible to MYXV infection. (B) Analysis of apoptotic events was examined and showed that cell death was mainly observed in the GFP+ MYXV infected cell population as shown by Annexin V staining. All gating was established using appropriate isotype controls. (C, D) Normal BM cells were incubated with MYXV expressing GFP at 10 MOI and assessed for colony forming potential. Various types of CFC colonies were formed and showed no GFP expression in either MYXV-treated (D) or mock-treated samples (C) indicating that MYXV was unable to infect normal CFCs. (E) The frequency of each colony type was enumerated following MYXV or mock treatment. Similar frequencies were observed between both cohorts suggesting that MYXV does not prevent normal CFC colony differentiation in vitro. Data are representative of repeat experiments and values represent mean ± standard deviation. (F) Mock-treated AML cells formed AML-CFU colonies. (G) MYXV-treated AML cells showed signs of infection (GFP+) and did not form colonies.

Mentions: Although there have been no case reports of human bone marrow failure despite widespread MYXV exposure, to document MYXV safety in the context of normal human HSPC function, we incubated BM-derived HSPCs from healthy donors (n=3) with a GFP expressing MYXV construct (vMYX-GFP) in vitro over a 3-day period at a high multiplicity of infection (MOI) of 10 per cell. After exposure to vMYX-GFP, BM-derived mononuclear cells (MNCs) were analyzed by flow cytometry to determine the extent of infectivity. Greater than 99.6 ± 0.2 % of BM MNCs were free of vMYX-GFP infection (Figure 1A). A small population of MNCs accounting for 0.5 ± 0.2 % of the total population was GFP-positive by this treatment, which was essentially indistinguishable from mock (vehicle only) treated cells and is typical of a nonpermissive infection by MYXV. No GFP expression was observed in normal CD34+ HSPC cells. On lineage subset analysis, CD15+ myeloid cells and CD19+ B lymphocytes also showed no signs of infection (data not shown).


Myxoma virus targets primary human leukemic stem and progenitor cells while sparing normal hematopoietic stem and progenitor cells.

Kim M, Madlambayan GJ, Rahman MM, Smallwood SE, Meacham AM, Hosaka K, Scott EW, Cogle CR, McFadden G - Leukemia (2009)

MYXV treatment of normal and leukemic HSPCs in vitro(A) Normal and leukemic cells (FLT ITD+) were incubated with MYXV expressing GFP at 10 MOI. Flow cytometric analysis demonstrated that a small population of normal MNCs was infected while a significant proportion of AML cells were susceptible to MYXV infection. (B) Analysis of apoptotic events was examined and showed that cell death was mainly observed in the GFP+ MYXV infected cell population as shown by Annexin V staining. All gating was established using appropriate isotype controls. (C, D) Normal BM cells were incubated with MYXV expressing GFP at 10 MOI and assessed for colony forming potential. Various types of CFC colonies were formed and showed no GFP expression in either MYXV-treated (D) or mock-treated samples (C) indicating that MYXV was unable to infect normal CFCs. (E) The frequency of each colony type was enumerated following MYXV or mock treatment. Similar frequencies were observed between both cohorts suggesting that MYXV does not prevent normal CFC colony differentiation in vitro. Data are representative of repeat experiments and values represent mean ± standard deviation. (F) Mock-treated AML cells formed AML-CFU colonies. (G) MYXV-treated AML cells showed signs of infection (GFP+) and did not form colonies.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: MYXV treatment of normal and leukemic HSPCs in vitro(A) Normal and leukemic cells (FLT ITD+) were incubated with MYXV expressing GFP at 10 MOI. Flow cytometric analysis demonstrated that a small population of normal MNCs was infected while a significant proportion of AML cells were susceptible to MYXV infection. (B) Analysis of apoptotic events was examined and showed that cell death was mainly observed in the GFP+ MYXV infected cell population as shown by Annexin V staining. All gating was established using appropriate isotype controls. (C, D) Normal BM cells were incubated with MYXV expressing GFP at 10 MOI and assessed for colony forming potential. Various types of CFC colonies were formed and showed no GFP expression in either MYXV-treated (D) or mock-treated samples (C) indicating that MYXV was unable to infect normal CFCs. (E) The frequency of each colony type was enumerated following MYXV or mock treatment. Similar frequencies were observed between both cohorts suggesting that MYXV does not prevent normal CFC colony differentiation in vitro. Data are representative of repeat experiments and values represent mean ± standard deviation. (F) Mock-treated AML cells formed AML-CFU colonies. (G) MYXV-treated AML cells showed signs of infection (GFP+) and did not form colonies.
Mentions: Although there have been no case reports of human bone marrow failure despite widespread MYXV exposure, to document MYXV safety in the context of normal human HSPC function, we incubated BM-derived HSPCs from healthy donors (n=3) with a GFP expressing MYXV construct (vMYX-GFP) in vitro over a 3-day period at a high multiplicity of infection (MOI) of 10 per cell. After exposure to vMYX-GFP, BM-derived mononuclear cells (MNCs) were analyzed by flow cytometry to determine the extent of infectivity. Greater than 99.6 ± 0.2 % of BM MNCs were free of vMYX-GFP infection (Figure 1A). A small population of MNCs accounting for 0.5 ± 0.2 % of the total population was GFP-positive by this treatment, which was essentially indistinguishable from mock (vehicle only) treated cells and is typical of a nonpermissive infection by MYXV. No GFP expression was observed in normal CD34+ HSPC cells. On lineage subset analysis, CD15+ myeloid cells and CD19+ B lymphocytes also showed no signs of infection (data not shown).

View Article: PubMed Central - PubMed

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Regimens of high dose chemotherapy followed by autologous blood and marrow transplantation (ABMT) have been used to treat patients with acute myelogenous leukemia (AML), but leukemia relapse is frequent, likely due to marrow contamination with leukemic hematopoietic stem and progenitor cells (HSPCs) and residual disease in protective niches... Purging autologous hematopoietic grafts of contaminating cancer cells prior to transplant serves as a viable strategy for increasing transplant efficacy but would also require sparing normal HSPCs within the same hematopoietic cell graft... Thus, to determine whether oncolytic viruses can be used as a therapeutic modality for purging leukemia from autologous grafts, we evaluated MYXV in a pre-clinical model of AML and normal HSPC transplantation... Moreover, using identical ex vivo conditions, MYXV does not affect either in vitro colony forming potential or in vivo engraftment of normal HSPCs derived from healthy human bone marrow (BM)... These results show safety and efficacy of using oncolytic MYXV to purge AML HSPCs from autologous HSPC grafts intended for hematopoietic rescue... In these samples, 40.3 ± 5.0 % of AML cells were infected with the virus, which was significantly higher in comparison to normal BM-derived MNCs (p<0.05; Figure 1A)... Of the total AML CD34 population, we found that 65 ± 6% were infected with MYXV, which was significantly higher in comparison to normal BM CD34 cells (p<0.05)... Together, these data indicated that MYXV effectively infects AML in vitro, and impairs clonal proliferation... Based on the in vitro safety results showing normal colony forming potential from MYXV treated normal HSPCs, we next tested safety by in vivo repopulation assays... None of the immunocompromised NOG mice developed any evidence of viral replication in their skin or in any internal tissues, indicating that even severely immunodeficient hosts are non-permissive for MYXV infection... The data indicate that ex vivo treatment with MYXV does not reduce in vivo engraftment potential of normal BM-derived MNCs or CD34 HSPCs... This critical safety data supports the notion that MYXV would be a safe candidate for purging leukemic cells from autologous hematopoietic grafts, even in immunocompromised recipients... In the primary, high risk AML specimen tested, normal HSPC levels were extremely low, thus normal HSPC engraftment in immunocompromised mice did not occur, as expected... Taken together, the data indicate that MYXV incubation ex vivo significantly inhibited subsequent AML HSPC engraftment in vivo, resulting in phenotypic and molecular remissions of FLT3 ITD AML.

Show MeSH
Related in: MedlinePlus