Myxoma Virus Targets Primary Human Leukemic Stem and Progenitor Cells While Sparing Normal Hematopoietic Stem and Progenitor Cells.

Abstract 14

High dose chemotherapy followed by autologous blood and marrow transplantation (ABMT) has been used to treat patients with acute myeloid leukemia (AML), but leukemia relapse rates remain high. One reason is the potential contamination of marrow with leukemic stem and progenitor cells (LSPCs). Purging autologous hematopoietic grafts of LSPCs prior to transplant serves as a viable strategy for increasing transplant efficacy in these cases; however, previous attempts using cytotoxic agents and cell culture techniques have generally resulted in loss of normal stem and progenitor cell numbers and/or functionality. Oncolytic poxviruses, such as myxoma virus (MYXV), are promising new instruments in targeting human cancer. MYXV has normal tropism towards European rabbits (Oryctolagus cuniculus) while remaining nonpathogenic for all other vertebrate species tested including humans and mice. Despite this host specificity, we have shown that MYXV is capable of infecting and killing a wide variety of human cancer cell lines. In light of these observations, we investigated whether MYXV could specifically target and eliminate LSPCs from primary AML using an ex vivo purging technique as assessed using both in vitro and in vivo functional analyses. Using a MYXV construct that expresses GFP upon cell infection, we observed GFP+ cells in leukemia exposed to MYXV at a concentration of 10 MOI over a 3-day period. No GFP expression was observed in normal bone marrow (BM) or mock (vehicle only) treated controls. GFP+ AML cells also began to undergo apoptosis shown by positive Annexin V staining. For myxoma to be a viable therapeutic for leukemia, it must not only target primary leukemia but also spare normal hematopoietic stem and progenitor cells (HSPCs). To test normal progenitor cell function following exposure to MYXV, normal BM cells were incubated with and without MYXV and tested for colony forming cell (CFC) content. Following incubation with MYXV, we observed differentiated colonies forming after 14 days indicating that the CFC potential of normal HSPCs was not adversely affected by MYXV. The frequency of the different colonies formed was also similar between mock and MYXV treated groups. When AML cells were mock treated pleomorphic colonies formed consistent with AML-colony forming units (AML-CFUs). Conversely, when exposed to MYXV, AML cells did not form recognizable AML-CFU colonies and instead remained heterodispersed suggesting impairment of progenitor cell function in vitro. To assess functional effects of MYXV on leukemia engraftment, sublethally irradiated NOG mice were transplanted with either mock treated primary AML (n=7) or primary AML pre-treated with MYXV for 3 hours (n=10). After 8 weeks, the percentage of engrafted mice was 100% after mock treated AML transplant but dropped to 10% after MYXV treatment. Significantly lower mean engraftment was observed in the group that received MYXV treated AML in comparison to mock treated samples (4.5% vs. 24% respectively; p < 0.05). Moreover, we show susceptibility of a primary AML specimen harboring an activating internal tandem duplication (ITD) mutation in FLT3, which represents an aggressive malignancy well-known for insensitivity to conventional chemotherapy. In animals showing leukemia engraftment by FACS, PCR was positive for the FLT3 ITD mutation. However, molecular remissions were evident in mice receiving MYXV treated samples. Efficacy against this leukemia signifies opportunity for disease eradication in an otherwise grim clinical setting. Finally, to assess functional effects of MYXV on normal HSPC engraftment, sublethally irradiated NOG mice were transplanted with either mock treated normal BM (n=10) or MYXV treated BM (n=9). After 8 weeks, there was no difference in the numbers of mice that engrafted between mock treated or MYXV treated groups (70% vs. 78% respectively; p = 0.72). There was also no difference in mean levels of engraftment per animal (1% vs. 2%; p = 0.41) suggesting that MYXV does not adversely affect the in vivo engraftment potential of normal HSPCs. In these studies, primary human LSPCs were targeted by MYXV purging, while normal human HSPCs showed no response maintaining both in vitro and in vivo functional potential. Given this demonstrated efficacy and safety, ex vivo purging of autologous hematopoietic grafts with MYXV may be feasible in cancer patients undergoing high dose chemotherapy followed by ABMT.