Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro

Abstract

Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

Figure 1. Inhibition of KG-1 Chloroma Formation after Treatment with MYXV

Human KG-1 leukemia cells were mock-treated or treated with live, UV-, or heat-inactivated vMyx-GFP, and then injected subcutaneously in immunocompromised NSG mice. (A) Overall chloroma formation was delayed in the cohort receiving live MYXV-treated KG-1 cells. Moreover, the rate of chloroma formation was slower in individual mice receiving live virus-treated cells. (B) Cumulative survival of animals receiving treated KG-1 leukemia cells. (C) Mean survival was significantly longer in animals receiving live MYXV treated KG-1 leukemia compared to those receiving inactivated virus or mock control treatment.

Figure 2. MYXV Inhibits Engraftment of Human Leukemia Cells in NSG mice

Human KG-1 or THP-1 leukemia cells were treated with live MYXV or mock control, and then transplanted intravenously into immunocompromised NSG mice. (A) Human leukemia engraftment was evaluated six weeks after injection by immunostaining mouse bone marrow for human CD45+ and HLA-ABC+ cells and analyzing by flow cytometry. The presence of double-positive human cells (CD45⁺/HLA-A,B,C⁺ in the upper right quadrant) indicated human leukemia engraftment in the mock-treated control. The absence of double-positive cells indicated no engraftment in the MYXV-treated sample. (B) Engraftment of KG-1 leukemia cells was effectively inhibited by treatment with MYXV. Percent KG-1 leukemia cell engraftment and the proportion of mice engrafted were both significantly reduced compared to mock treated control animals (P < 0.05). (C) THP-1 leukemia cells were completely purged by the MYXV treatment (P < 0.05).

Figure 3. KG-1 Leukemia Cell Function In Vitro is Not Affected by Treatment with MYXV

Human KG-1 or THP-1 cells were treated with live MYXV or mock control. (A) MYXV treatment reduced THP-1 leukemia cell proliferation in vitro; however, had no effect on KG-1 cell proliferation. (B) MYXV treatment reduced THP-1 viability as measured by the MTT assay, but did not reduce KG-1 viability. (C) THP-1 leukemia colony forming potential was impaired after MYXV treatment, while KG-1 leukemia colony forming potential was unchanged. (D) The percentage of THP-1 leukemia cells differentiating and adhering to the culture dish was significantly increased after MYXV treatment; whereas, no increase in adherent cells was observed in MYXV-treated KG-1 cells.

Figure 4. KG-1 Leukemia Cells are Non-Permissive for MYXV Infection and Replication In Vitro but are Competent for Virus Binding

(A) At 24 hours after MYXV treatment, THP-1 or KG-1 leukemia cells were evaluated for evidence of the early stages of MYXV infection (eGFP expression) by flow cytometry. The differentiating agent, PMA, was used in an attempt to augment permissiveness. (B) THP-1 or KG-1 leukemia cells were treated with MYXV and new infectious viral progeny production measured over time. Titers are expressed as log FFU/10⁶ leukemia cells. The differentiating agent, PMA, was used in an attempt to augment viral permissiveness. (C) THP-1 or KG-1 cells were exposed to MYXV for 1 hour, washed extensively and the remaining cell-associated viral protein assessed by Western blotting.

Figure 5. Virus Binding is Necessary for MYXV Purging of Leukemia Cells

(A) THP-1 or KG-1 cells were exposed to either: mock treatment, MYXV treatment, or MYXV + heparin treatment for 1 hour. The cells were washed extensively and the remaining cell-associated viral protein was assessed by Western blotting. (B) NSG mice were xenotransplanted with KG-1 cells treated ex vivo with KG-1+MYXV (ie virus-bound) or KG-1+Heparin+MYXV (ie virus-unbound) and then evaluated for human leukemia engraftment in the bone marrow after 8 weeks. A significantly decreased proportion of animals receiving MYXV treated KG-1 cells (KG-1+MYXV) showed leukemia engraftment compared to controls. Blocking virus binding with heparin abrogated the oncolytic effect of MYXV and restored KG-1 cell engraftment.