Oncolytic measles virus and mesenchymal stromal cells: A therapeutic model and a dissection of mechanisms of action

Live, attenuated measles virus (MV) has demonstrated tumour-specific replication and anti-tumour activity in murine models of hematological and non-hematological malignancy, leading to a number of published or on-going clinical trials. However, the potentially overriding issue of how to achieve successful administration of oncolytic measles virotherapy in patients with intact humoral immunity remains. I have examined mechanisms for optimizing delivery of oncolytic MV to acute lymphoblastic leukaemia (ALL) – a disseminated haematological malignancy. Anti MV antibody titres in adult patients with ALL were evaluated before and after leukaemia treatment regimens including high dose corticosteroids, cyclophosphamide and anti-B cell monoclonal antibodies. Antibody titres were unaffected by induction and intensification treatment protocols, suggesting the need for consideration of optimal virotherapy delivery strategies to achieve therapeutic success. Here, I demonstrate that human bone marrow-derived mesenchymal stromal cells (BM-MSCs) can be used effectively as virus delivery vehicles, permitting ex-vivo cellular virus loading and intracellular virus amplification, delivery of virus to distant sites of disease following systemic infusion, and virus hand-off to precursor B lineage ALL cell targets in the presence of pre-existing anti-MV antibodies. In vivo modelling using SCID mice bearing disseminated pre-B ALL xenografts demonstrated enhanced survival of passively immunized animals following IV treatment with BM-MSC-delivered MV versus naked MV or BM-MSCs alone. In order for vaccine MV to be safely and rationally utilized as a novel therapeutic for ALL, a detailed mechanistic understanding of how the virus exerts its oncolytic effect is paramount. In this thesis, I have utilized a previously characterized model of cellular transformation generated using human BM-MSCs to characterize the phenomenon of relative tumour cell specificity by oncolytic MV, in terms of infectivity, productivity and cell killing. Furthermore, this thesis begins to explore some of the potential mechanisms that confer vaccine MV its tumour-tropic and anti-cancer properties

Type-1 Interferon Responses Underlie Tumor-Selective Replication of Oncolytic Measles Virus

The mechanism of tumor selective replication of oncolytic measles virus (MV) is poorly understood. Using a step-wise model of cellular transformation, in which oncogenic hits were additively expressed in human bone marrow-derived mesenchymal stromal cells, we show that MV-induced oncolysis increased progressively with transformation. Type-1 interferon response to MV infection was significantly reduced and delayed, in accordance with the level of transformation. Consistently, we observed delayed and reduced STAT1 phosphorylation in the fully transformed cells. Pre-treatment with IFNβ restored resistance to MV-mediated oncolysis. Gene expression profiling to identify the genetic correlates of susceptibility to MV oncolysis revealed a dampened basal level of immune-related genes in the fully transformed cells compared to their normal counterparts. Interferon-induced transmembrane protein 1 (IFITM1) was the foremost basally downregulated immune gene. Stable IFITM1 overexpression in MV-susceptible cells resulted in a 50% increase in cell viability and a significant reduction in viral replication at 24 hours post MV infection. Overall, our data indicate that the basal reduction in functions of the type 1 IFN pathway is a major contributor to the oncolytic selectivity of MV. In particular, we have identified IFITM1 as a restriction factor for oncolytic MV, acting at early stages of infection

Attenuated, oncolytic, but not wild-type measles virus infection has pleiotropic effects on human neutrophil function.

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV

Mouse xenograft modeling of human adult acute lymphoblastic leukemia provides mechanistic insights into adult LIC biology.

The distinct nature of acute lymphoblastic leukemia (ALL) in adults, evidenced by inferior treatment outcome and different genetic landscape, mandates specific studies of disease-initiating mechanisms. In this study, we used NOD/LtSz-scid IL2Rγ null(c) (NSG) mouse xenotransplantation approaches to elucidate leukemia-initiating cell (LIC) biology in primary adult precursor B (pre-B) ALL to optimize disease modeling. In contrast with xenografting studies of pediatric ALL, we found that modification of the NSG host environment using preconditioning total body irradiation (TBI) was indispensable for efficient engraftment of adult non-t(4;11) pre-B ALL, whereas t(4;11) pre-B ALL was successfully reconstituted without this adaptation. Furthermore, TBI-based xenotransplantation of non-t(4;11) pre-B ALL enabled detection of a high frequency of LICs (<1:6900) and permitted frank leukemic engraftment from a remission sample containing drug-resistant minimal residual disease. Investigation of TBI-sensitive stromal-derived factor-1/chemokine receptor type 4 signaling revealed greater functional dependence of non-t(4;11) pre-B ALL on this niche-based interaction, providing a possible basis for the differential engraftment behavior. Thus, our studies establish the optimal conditions for experimental modeling of human adult pre-B ALL and demonstrate the critical protumorogenic role of microenvironment-derived SDF-1 in regulating adult pre-B LIC activity that may present a therapeutic opportunity