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The mechanism of tumor-selective replication of oncolytic measles virus (MV) is poorly understood. Using a stepwise 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. The type 1 interferon (IFN) response to MV infection was significantly reduced and delayed, in accordance with the level of transformation. Consistently, we observed delayed and reduced signal transducer and activator of transcription (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. IFN-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 h after 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.

The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance., How the bone marrow microenvironment evolves during induction chemotherapy to facilitate acute lymphoblastic leukaemia (ALL) survival remains unclear. Here the authors show that adipocytes emergent during ALL therapy aid the survival of ALL cells through a restrictive effect on the ALL proteome.

The mechanism by which oncolytic measles virus (MV) kills cancer cells remains obscure. We previously showed that neutrophils are involved in MV-mediated tumor regressions and become activated, upon MV infection. In the present study, we attempted to enhance the neutrophil response toward MV-infected tumor targets by generating an oncolytic MV-expressing human granulocyte colony-stimulating factor (MVhGCSF). Evaluating the effects in two different models of B-cell malignancy, we showed that depletion of neutrophils abrogated the MV therapeutic effect in an in vivo Raji-but not Nalm-6 tumor model. Next, we compared MVhGCSF with the unmodified backbone virus MVNSe. MVhGCSF enhanced the oncolytic capacity of MV in the Raji model in vivo, whereas in the Nalm-6 model, the opposite was unexpectedly the case. This finding was recapitulated by exogenously administered hGCSF. MVhGCSF replicated within an MV-infectable CD46 transgenic mouse model with detectable serum levels of hGCSF but no toxicity. 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.

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 (

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.

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.

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.

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.

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.

Although humanised models of ALL are well described they are largely based on paediatric disease. The distinct nature of ALL in adults - evidenced by inferior outcome and different genetic landscape - points to a need for targeted study of this disease. Key to this is development of in vivo models of adult ALL. In this study we sought to establish the optimal modelling system for adult pre-B ALL using NOD/LtSz-scid IL2Rγ nullc (NSG) mouse xenotransplantation approaches. Our aims were to define the conditions for sensitive assaying of adult ALL LIC and low frequency clones. Pre-conditioning irradiation has been shown to negatively influence pre-B ALL engraftment. Studies of paediatric ALL suggest this may be due to reduced homing of ALL cells to the bone marrow (BM) resulting from aberrant CXCR4 receptor desensitisation induced by SDF-1 which is upregulated after irradiation. To investigate the impact of irradiation on engraftment of adult ALL, 5 - 10 million viable cells were injected intravenously (IV) into 6-8 week mice either unconditioned or (+24 hrs) following irradiation (2.5Gy). After 6 months, BM leukaemic engraftment (>0.1% hCD45+/19+) was assessed. Engraftment occurred in only 2/17 (11.8%) samples from non-irradiated recipients but in 11/14 samples (78.6%) after pre-conditioning irradiation (p = 0.0003). Comparison of 8 samples assayed by both models demonstrated that pre-conditioning irradiation was a pre-requisite for successful engraftment in 7/8 cases. Contrasting this overall finding was t(4;11) ALL where engraftment was higher in non-irradiated models (2/3). To determine homing capacity in adult ALL, SDF-1 (0, 10ng, 50ng, 125ng + 1ug) induced directional migration was investigated. A dose dependant response was observed with maximal migration (37±15 fold) of adult pre-B ALL cells at high (125ng/ul) concentrations of SDF-1 (n=5, non t(4;11) ALL). Hence, in contrast to paediatric ALL, adult ALL tumours do not appear to exhibit significant CXCR4 desensitisation to high levels of SDF-1 which may explain the positive effect of irradiation on adult ALL engraftment. Notably, t(4;11) positive tumours were relatively insensitive to SDF-1 induced migration (max fold change ∼2.5). CXCR4 cell surface receptor analysis revealed a trend towards low expression in t(4;11) cases (8.7±3.3% blasts) vs non t(4;11) (41 ±12% blasts) which may account for the differential SDF-1 induced in vitro migration patterns. Further modifications to the NSG xenotransplantation system were investigated: direct intra bone marrow (IBM) vs IV injection, 2.5Gy vs 2.0Gy and IBM +/- 2.5Gy irradiation. An IBM injection route, 2.5Gy irradiation and IBM + irradiation were all associated with an overall faster engraftment kinetic and time to disease manifestation. However, overall engraftment potential was not affected by these factors. Hence, the optimal xenotransplantation assay for adult ALL was defined as IBM delivery in 2.5Gy irradiated mice. This system enabled quantification of at least 1:1000 adult ALL LICs as assessed by limiting dilution transplantation (cell dose:103-106 ). Finally, we used the optimised xenotransplantation system to study 9 MRD positive ALL BM samples (8.33 x 10-2 – 2.64-4 positivity by BCR/ABL or I/g TCR based PCR quantification). MRD positive BM samples (unfractionated) were injected at a cell dose of 8.7 x104 – 6 x106/mouse and, strikingly, in the absence of cellular enrichment, MRD induced leukaemic engraftment (83.6%) was achieved in 1/9 samples after an extended modelling phase of 7 months and importantly gave rise to overt leukemia associated morbidity. Hence we establish proof of principle, that MRD + ALL can be modelled in vivo. In conclusion we have established the parameters of a functional system for in vivo study of adult ALL. Our findings highlight the key role of irradiation in the homing of adult ALL cells and also in their retention and repopulation, possibly mediated by SDF-1/CXCR4 interaction. Importantly, we found distinct in vivo growth requirements not only between adult ALL subtypes but also compared to paediatric disease underlining the importance of age/leukaemia specific investigation. Our optimised system should facilitate the study of LIC and pre-clinical disease modelling in adult ALL. Crucially we establish a strategy for assaying arguably the most informative populations (MRD) for understanding disease resistance and evolution of relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract 4300 Since the first allogeneic umbilical cord blood transplantation in 1988, there has been an increasing recognition and utilisation of cord blood as a source of haematopoietic stem cells. Donor cell leukaemia (DCL) following cord blood transplantation (CBT) remains a rarely reported phenomenon with only a handful of cases appearing in the literature to date. A plethora of possible mechanisms have been suggested for the development of DCL following CBT. In addition to the presence of undetected pre-leukaemic clones in the cord blood, chronic antigenic stimulation in an impaired tumour immune surveillance environment, perturbations of the host environment and premature ageing of the donor cells with associated chromosomal instability are among the proposed mechanisms for leukaemogenesis. Here we report a case of donor cell leukaemia in a 35-year-old female suffering from chronic myelogenous leukaemia. At 17 months following sex mismatched cord blood transplantation, donor cell derived BCR/ABL1 negative acute myeloid leukaemia (FAB M0) was diagnosed. In addition to the finding of monosomy 7, amplification of part of chromosome 21 containing the RUNX1, Down Syndrome Critical Region (DSCR) and ERG genes among others was identified by high-density array comparative genome hybridisation. The array findings were confirmed by qPCR and the RUNX1 amplification was mapped by FISH onto a marker chromosome, der(17)t(17;21)(p13;q?21). Importantly, these genetic changes were not found in the retrospectively tested cord blood cells using the same techniques. Whereas aberrations of chromosome 7 have been previously reported in cases of DCL post CBT, here we describe for the first time co-amplifications of several genes from the 21q22 chromosome region in conjunction with the finding of monosomy 7. RUNX1 amplifications are known to occur in acute leukaemia of both myeloid and lymphoid lineage. Gains of the 21q22 region with multiple copies of RUNX1 gene have been associated with therapy related AML and MDS, but thus far has not been reported in the context of DCL following CBT. Furthermore, the possibility that additional genomic imbalances may have contributed to the pathogenesis of DCL in this case is also raised. Co-amplification of genes from the DSCR at 21q22 in the presence of unmutated GATA1 and JAK3 genes may be relevant in this context, as located within this region are genes with powerful effects on differentiation (such as DYRK1A) and several genes whose over-expression has been linked with disturbed megakaryopoiesis and myeloproliferation including miR155, BACH1, RCAN1 (DSCR1), RUNX1 and ERG. In addition to the classical genomic imbalances such as monosomy 7 that typify the genetic makeup of secondary AML, this case highlights for the first time the potential cooperative role of oncogenic co-amplification in the pathogenesis of donor cell leukaemia. Disclosures: No relevant conflicts of interest to declare.