Abstract
Acute myeloid leukemia (AML) is the most common human leukemia and is a major area of unmet medical need among hematologic malignancies. Progress has been made in identifying therapeutic targets and several approved therapies, but resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies. Oncolytic viruses (OV) are a promising new class of therapeutics that rely on tumor specific oncolysis and the generation of a potent adaptive anti-tumor immune response for efficacy. To investigate if our newly developed oncolytic herpes simplex viruses (oHSVs), designed to potentiate anti-leukemia immunity, effectively target primitive AML cells, we evaluated oHSV-VG161, which is engineered to express IL-12, IL-15 and the IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interaction. After screening several AML cell lines that expressed relatively high levels of a HSV entry receptor (HVEM), we demonstrated that VG161-infected OCIAML3 and MOLM13 cells significantly enhanced cell killing (IC 50: 0.4 & 1.8 multiplicity of infection (MOI) as compared to MV4-11 and U973 cells (IC 50: 3.0 & 9.5 MOI). These effects were 2-3 folds lower in control VG160-infected cells. We also observed that VG161-infected AML cells induced apoptosis in a dose-dependent manner (~50%) after 48 hours and cleaved PARP, Caspase-3 and Caspase-8 were increased in these cells, and to a lesser extent in control VG160-infected cells. Both VG160 and VG161 viruses replicated efficiently in OCIAML3 and MOLM13 cells in a timely, dose-dependent manner, evidenced by qPCR detection of HSV-1 ICP27 DNA copy numbers (>500-fold increase) over 48 hours of treatment. This result was supported by detection of protein expression of HSV-1 glycoprotein D in VG160 and VG161-infected cells (up to 40% of protein detected) by FACS analysis. Interestingly, IL-12 but not IL-15 protein expression was found in intracellular-stained VG161-infected OCIAML3 and MOLM13 cells in a dose-dependent manner (up to 13% of protein detected, P<0.01) but not in VG160-infected cells, as assessed by FACS analysis. Production of IL-12 was also detected in cultured media obtained from VG161-infected AML cells (up to 150 pg/mL) by ELISA. To investigate potential molecular mechanisms of VG161-mediated anti-leukemia response and specific signalling pathways, we have screened several potential candidates and found immune regulating genes, such as IRF3, IRF7, IRF9, NFkB and ISGs, as well as type I IFN to be highly increased in VG161-infected cells as compared to VG160-infected cells (2-4-folds, P<0.001) in a dose dependent manner over 48 hours of treatment, assayed by qRT-PCR. Western blot analysis demonstrated increased phosphorylation of p-STAT1 and its protein expression in VG161-infected cells compared to VG160 control cells (~2-fold). These results suggest that VG161 viruses expressing several engineered immunomodulatory transgenes, particularly IL-12, contribute to anti-leukemia responses by activating specific immune regulating pathways such as the JAK/STAT pathway. In addition, we detected an increase in both RNA and protein levels of PD-L1 in VG161-infected AML cells, suggesting the necessity of PD-L1 blocking peptide in the viral construct. To further investigate VG161's role in regulating innate and adaptive immune responses, we have examined the biological effects of VG160/VG161 in the presence of healthy peripheral blood mononuclear cells (PBMC) in both AML cell lines and primary AML patient cells in vitro. Most interestingly, VG160 or VG161-infected OCIAML3 and MOLM13 cells show enhanced cell killing when co-cultured with PBMC and this cell killing effect was greatly enhanced in VG161-infected cells as compared to VG160-infected cells, especially in the MOLM13 cell line (up to 90% killing). This observation was further supported when primitive AML patient cells were co-cultured with VG161 and PBMC as compared to VG160 control cells. Moreover, PD-L1 expression was highly increased in AML patient cells when cultured with VG161 as compared to VG160 (2.7-fold) and this was further enhanced when co-cultured with VG161 and PBMC. Thus, we have demonstrated that newly developed oHSVs engineered with several immunomodulatory transgenes effectively target primitive AML cells, suggesting a potential treatment strategy for AML.
Disclosures
No relevant conflicts of interest to declare.
Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir