Anna Z. Castleton

Human mesenchymal stromal cells deliver systemic oncolytic measles virus to treat acute lymphoblastic leukemia in the presence of humoral immunity

Clinical trials of oncolytic attenuated measles virus (MV) are ongoing, but successful systemic delivery in immune individuals remains a major challenge. We demonstrated high-titer anti-MV antibody in 16 adults with acute lymphoblastic leukemia (ALL) following treatments including numerous immunosuppressive drugs. To resolve this challenge, human bone marrow-derived mesenchymal stromal cells (BM-MSCs) were used to efficiently deliver MV in a systemic xenograft model of precursor B-lineage-ALL. BM-MSCs were successfully loaded with MV ex vivo, and MV was amplified intracellularly, without toxicity. Live cell confocal imaging demonstrated a viral hand-off between BM-MSCs and ALL targets in the presence of antibody. In a murine model of disseminated ALL, successful MV treatment (judged by bioluminescence quantification and survival) was completely abrogated by passive immunization with high-titer human anti-MV antibody. Importantly, no such abrogation was seen in immunized mice receiving MV delivered by BM-MSCs. These data support the use of BM-MSCs as cellular carriers for MV in patients with ALL.

Differential Cytopathology and Kinetics of Measles Oncolysis in Two Primary B-cell Malignancies Provides Mechanistic Insights

Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.

Allogeneic stem cell transplantation as treatment for myelofibrosis

Idiopathic myelofibrosis (IMF) is a clonal disorder resulting from the proliferation of aberrant hematopoietic stem cells. Conventional treatment is unsatisfactory, and with the exception of supportive blood transfusions, none of the standard therapies have been shown to confer a survival advantage. Allogeneic stem cell transplantation represents the only treatment modality with proven curative potential. Myeloablative conditioning regimens are associated with high transplant-related mortality, particularly in the elderly, making most patients with IMF ineligible for this treatment. Strategies using reduced intensity conditioning regimes have allowed application of allogeneic transplantation to a broader range of patients and a number of recent reports have demonstrated potential efficacy.

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.

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.

Genome gains at chromosome 21q21/22 segment leads to co-amplification of Down Syndrome Critical Regions and known oncogenes in a case of donor cell-derived acute myeloid leukaemia following allogeneic sex mismatched umbilical cord blood transplantation for chronic myeloid leukaemia

Donor cell-derived leukaemia (DCL) following umbilical cord blood transplantation (UCBT) is a rare phenomenon with only a handful of cases appearing in the literature. Potential mechanisms for DCL development post-haematopoietic cell transplantation include occult pre-leukaemic clones in donor cells, immune surveillance defect(s), therapy-related stromal abnormalities, excess cytokine stimulation, and DNA replication/repair errors associated with post-transplant expansion (Ruiz-Argüelles et al, 2007). We agree with the view that a common mechanism is not likely. Although case numbers are small, no consistent chromosome abnormality has been identified thus far, with normal karyotype in approximately half the cases, and a mixture of numerical and/or complex structural aberrations in the remainder. We performed detailed analysis on patient and cord blood samples from a 32-year-old female with chronic myeloid leukaemia and a history of acute megakaryoblastic transformation (AMKL), who developed DCL following UCBT. Cytogenetic analysis at presentation identified t(9;22) (q34q11) and a marker inv(7)(q?22q?35) in all dividing cells (Fig. 1A). Following transformation to AMKL and the attainment of morphological remission with induction chemotherapy, the patient underwent reduced intensity conditioned single antigen mismatched UCBT from a male donor. Despite achieving complete molecular response, and full donor chimaerism [200 nuclei showing male signal pattern by XY fluorescent in situ hybridization (FISH)] at 1 month post-UCBT, bone marrow (BM) examination at 17 months revealed tri-lineage dysplasia, erythroid hyperplasia (53% of cells) and increased blasts (16% of total cells; 33% of nonerythroid cells). Immunophenotyping confirmed a clonal population of myeloblasts (CD34 30%, CD13 73%, CD33 73%, cMPO 46%, TdT 0%, CD7 45%, CD13:34 30%, CD7:33 30%). All cells were BCR/ABL1 negative by nested polymerase chain reaction (PCR) and of male origin by XY FISH. The patient died from overwhelming sepsis following the diagnosis of an evolving donor cell-derived acute myeloid leukaemia (AML). Analysis of BM aspirate unveiled an abnormal karyotype 44,XY,-7, der(17)t(17;21)(p13?p/q?),-21 confirmed by FISH (Fig. 1B). Genome-wide oligonucleotide array comparative genomic hybridization (aCGH) analysis identified loss of chromosome 7, and partial losses and gains of chromosome 21, whilst chromosome 17 showed no changes (Fig. 2). Genomic imbalances at the 21q21/q22 region involved gene rich sequences including:

65. Oncolytic Measles Virus Differentially Affects Mitochondrial Biogenesis in Transformed versus Non-Transformed BM-Derived MSCs

Vaccine strain measles virus (MV) is oncolytic in numerous models of malignancy. The mechanism behind the selectivity of MV for transformed cells is poorly understood. To investigate further, an established step-wise model of cellular transformation was used; in which progressive oncogenic hits were stably and additively expressed in human bone marrow derived mesenchymal stromal cells following retroviral transfer of human telomerase reverse transcriptase (hTERT), human papilloma virus16 E6 and E7 (3H), SV40 small T antigen (4+V), finally, H-RAS (5H) (Funes at al, 2007). The most highly transformed cells (5H) were more permissive to oncolytic MV infection than any of the less transformed counterparts, with significantly greater viral titres. MV-induced cell-death increased progressively with progressive transformation. This was not explained by any differences in MV receptors CD46, SLAM or Nectin-4 expression. Investigation of anti-viral type 1 IFN response in this model 24 and 48 hours post MV infection (hpi) by ELISA demonstrated a robust induction of IFNβ (to a lesser extent IFNα) in hTERT cells, which was significantly and progressively reduced in 3H, 4+V and 5H according to level of transformation, suggesting that defective IFN pathway is a potential mechanism for the enhanced MV permissiveness observed in transformed cells. Examination of integrity of the RLR signalling pathway, which triggers IFNα/β production, revealed that expression levels of RIG-I, MDA5 and MAVS, determined by RQ-PCR at 6, 12, 24 and 48 hpi, were lowest in 5H and highest in hTERT cells, proposing a role for the RLR pathway in MV-mediated oncolysis. To gain insight into the differential effects of MV infection in transformed versus non-transformed cells, metabolic effects post MV infection were investigated. A significant increase in oxygen consumption rate (OCR) was observed 24 hpi, proportional to the stage of transformation, followed by a steep decline at 48 hpi. This increase was abrogated in the presence of fusion inhibitory peptide (FIP), implicating the role of syncytia formation, the cytopathic effect of MV infection, in MV-mediated oncolysis in vitro. A similar pattern was observed in ATP levels post MV infection; highest at 24hpi in 4+V and 5H and dropping off dramatically at 48hpi when cell viability is compromised. To further characterize the consistent increase in OCR and ATP levels upon MV infection of the more transformed cell lines, we sought to assess mitochondrial biogenesis using the fluorescent probes TMRM, Calcein AM and Hoechst. Preliminary results indicated significantly increasing numbers of mitochondria per cell at 24 hpi principally in 4+V (16%) and 5H (~29%) cells compared to their uninfected counterparts potentially explaining the observed high levels of OCR and ATP. Altogether, our data suggests that altered mitochondrial numbers and functions correlate with the degree of MV permissiveness and thus MV-induced cell death. Mitochondrial mass was also seen to be increased with MV infection. However, this effect was not specific to transformed cells. Metabolomics and further evaluations will be crucial to comprehensively assess differences in metabolism between cells in this model and to understand the role of bioenergetics in the differential levels of MV-mediated oncolysis.

621. Fusion Between Neutrophils (PMN) and Target Cells Mediate Cytotoxicity During Measles Virus (MV) Oncolysis – A Novel Mechanism

We have previously shown that PMNs play a role in MV-mediated oncolysis (Grote et al) and that oncolytic vaccine strain of MV (as opposed to wild type) stimulates release of numerous cytokines with potential anti-tumor effects (Zhang et al). Recent data from our laboratory indicated that normal healthy PMNs were capable of specific killing of 35-40% of infected Jurkat target cells. On the basis that MV-induced cell-cell fusion amplifies IFN-alpha (α)/beta (β) production in infected cells, we sought to determine whether fusion between infected target cells and normal human PMNs specifically mediates cytotoxicity. First, we showed that PMN-mediated MV killing completely disappeared in the presence of FIP (fusion inhibitory peptide). Next, we examined the role of cell-cell fusion on PMN degranulation, reactive oxygen species (ROS) production and IFN production. We carried out all experiments with and without FIP. Jurkat cells were infected with MVNSe or mock-infected and incubated with PMNs isolated from healthy donors at 8:1 E:T ratio. At 24 hours, the cells were collected. PMN degranulation was determined by increase in cell surface expression of CD66b, CD63 and CD35 and ROS generation was determined by flow cytometry. IFN α and β production in the supernatant was quantified by ELISA. During co-culture of infected target cells and PMNs, IFN α (3000-5000pg/ml) and β (70-90pg/ml) were produced. No IFN α and β was produced by infected or uninfected Jurkats without PMNs present, confirming PMNs as the source. This was highly significantly abrogated in the presence of FIP (α – 200-300pg/ml; β – 0-3pg/ml). PMN degranulation and ROS generation followed the same pattern with abrogation in the presence of FIP, although the differences were not statistically significant. We confirmed that the α or β IFN was not directly responsible for cytotoxicity, since addition of the same quantity of exogenous IFN directly to Jurkat cells did not lead to cell death. Hence, we sought downstream cytotoxic effectors and evaluated TRAIL, which we know from our previous work, can be released from pre-formed granules in response to MV-infection of PMNs. The conditions that generated high levels of IFN also produced significant levels of soluble TRAIL (800-1500pg/ml), which was also abrogated in the presence of FIP (190-650pg/ml). Our data suggests infected target cells stimulate PMNs to display a cytotoxic effector phenotype, directly killing infected target cells. High levels of type I IFNs, are generated which are not in themselves responsible for the cytotoxicity. None of this occurs without target cell-neutrophil fusion as it is abrogated by FIP and does not occur in infected target cells alone or infected PMNs alone. This is a novel mechanism of stimulation of innate immunity by an oncolytic virus.

430. MVhGCSF Is Therapeutic In-Vivo B Cell Malignancies With Neutrophils (PMNs) Playing Different Roles

Measles virus (MV) has been shown to be oncolytic in various tumor models and is being evaluated in various Phase I clinical trials. The mechanisms by which MV kills cancer cells still remain obscure. We aimed to enhance the therapeutic efficacy of MV by expressing the human granulocyte colony stimulating factor (hGCSF), as an additional transcription unit of MV. We showed that MVhGCSF is therapeutic in two different in-vivo subcutaneous model of B cell malignancy, Raji (Burkitts lymphoma) and Nalm-6 (acute lymphoblastic leukemia). In the Raji model, MVhGCSF treatment generated a highly significantly superior (p=0.0001) anti tumor effect by comparison to MVNSe and the MVhGCSF treated mice survived significantly longer than MVNSe treated mice (p=0.0098). In the Nalm-6 model, both MVhGCSF and MVNSe resulted in complete regression of the tumors in both groups with no survival advantage to MVhGCSF therapy. We then determined therapeutic efficacy of MVhGCSF in disseminated Nalm-6 xenografts as our previous data showed that approx. 60% of mice have complete regression of the tumors with IV delivered MV, offering a greater potential to observe any therapeutic benefit to MVhGCSF. Nalm-6 tumor cells expressing luciferase were used, in order to enable in-vivo imaging of disease progression by imaging. Mice were treated with MVNSe, MVhGCSF, or UV-MV control and an additional control of hGCSF alone, using pegylated hGCSF. Mice treated with the controls alone succumbed to leukemia the most quickly; in the group treated with GCSF alone, the median survival was 50 days compared to 75 days in the UV-MV treated group (p=0.012). As expected, 75% of the mice treated with MVNSe responded well and were alive at the end of the experiment. Surprisingly, mice receiving treatment with MVhGCSF had a median survival of 78.5 days compared to the MVNSe treated cohort where the median was not reached by the end of the experiment (p=0.0149). At humane end point, the spleen sizes and cellularity were similar across all the groups. hGCSF was detected at comparable levels in the serum of the hGCSF and MVhGCSF treated cohorts. Flow cytometric analysis of CD11b, Ly6G6C, NK and Mac3 cells did not show any difference in the PMN, macrophage and NK cell percentages between the different groups. Finally, to be certain that the less good outcome of mice treated with MVhGCSF did not also result from toxicity of high GCSF levels, IfnarKO * CD46Ge mice were injected IV with either MVNSe or MVhGCSF. They were monitored for 35 days after which spleen size, differential cell count (NK, macrophage, PMN percentages) and serum hGCSF levels were determined. There was no difference between the groups. The mice treated with MVhGCSF showed 200-800pg/ml of hGCSF in the serum at Day 35, but there was no toxicity. Taken together these data in the disseminated Nalm-6 model, suggests that any adverse effect of expressing hGCSF as an additional transcription unit in tumor bearing mice relates solely to promotion of tumor growth and not to toxicity of GCSF production.Our data suggest that a “one-size fits all” model of immune response to viral oncolysis is not appropriate and each tumor target will need full characterization for the potential of both direct and indirect, innate immune responses to generate benefit.