Multiple myeloma is still uncurable. Myeloma cells become resistant to common drugs and patients eventually die of tumour progression. Therefore, new targets and drugs are urgently needed. NVP-BGT226 is a novel, orally bioavailable small-molecule inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin. Here, we show that NVP-BGT226 inhibits growth in common myeloma cell lines and primary myeloma cells at nanomolar concentrations in a time-dependent and dose-dependent manner. Western blots for the detection of caspase 3 cleavage and annexin-V-fluorescein isothiocyanate/propidium iodide assays revealed induction of apoptosis in common myeloma cells lines. Induction of apoptosis was accompanied by upregulation of proapoptotic Bim and a moderate upregulation of Mcl-1 and Bad and a downregulation of Bcl-2, Bax and Bcl-Xl. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the 5-bromo-2'-deoxyuridine assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, cyclin D2, pRb and cdc25a. NVP-BGT226 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1 in a time-dependent and dose-dependent manner. Furthermore, we show that the stimulatory effect of insulin-like growth factor 1, interleukin-6 and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BGT226. Overall, inhibition of phosphoinositol-3-kinase/mammalian target of rapamycin by NVP-BGT226 is highly effective, and NVP-BGT226 represents a potential new candidate for targeted therapy in multiple myeloma.

ORF virus (ORFV) is the causative agent of contagious ecthyma, a pustular dermatitis of small ruminants and humans. Even though the development of lesions caused by ORFV was extensively studied in animals, only limited knowledge exists about the lesion development in human skin. The aim of the present study was to evaluate a three-dimensional (3D) organotypic culture (OTC) as a human skin model for ORFV infection considering lesion development, replication of the virus, viral gene transcription and modulation of differentiation of human keratinocytes by ORFV. ORFV infection of OTC was performed using the ORFV isolate B029 derived from a human patient. The OTC sections showed a similar structure of stratified epidermal keratinocytes as human foreskin and a similar expression profile of the differentiation markers keratin 1 (K1), K10, and loricrin. Upon ORFV infection, OTCs exhibited histological cytopathic changes including hyperkeratosis and ballooning degeneration of the keratinocytes. ORFV persisted for 10 days and was located in keratinocytes of the outer epidermal layers. ORFV-specific early, intermediate and late genes were transcribed, but limited viral spread and restricted cell infection were noticed. ORFV infection resulted in downregulation of K1, K10, and loricrin at the transcriptional level without affecting proliferation as shown by PCNA or Ki-67 expression. In conclusion, OTC provides a suitable model to study the interaction of virus with human keratinocytes in a similar structural setting as human skin and reveals that ORFV infection downregulates several differentiation markers in the epidermis of the human skin, a hitherto unknown feature of dermal ORFV infection in man.

Orf virus (Parapoxvirus ovis, ORFV) is a dermatotropic virus causing pustular dermatitis in small ruminants and humans. We analysed isolated human primary keratinocytes (KC) and dermal fibroblasts (FB) for cell death and virus replication by infection with a patient‐derived ORFV isolate. ORFV infection was associated with rapid induction of cell death in KC allowing for considerable virus removal. Upon infection with ORFV, KC and FB harboured intracytoplasmic ORFV and showed viral protein presence; however, missing virus spread indicated an abortive infection. Upon ORFV exposure, KC but not FB secreted the pro‐inflammatory cytokine interleukin (IL)‐6. ORFV infection enhanced the frequency of KC expressing intercellular adhesion molecule (ICAM)‐1 which was independent of IL‐6. Interestingly, ORFV inhibited ICAM‐1 up‐regulation on infected but not on non‐infected KC. Even interferon‐γ, a potent inducer of ICAM‐1, up‐regulated ICAM‐1 only on non‐infected KC. Transfer of ORFV‐free supernatant from infected to non‐infected KC induced ICAM‐1 on non‐infected KC pointing to the involvement of soluble mediator(s). Similarly as in KC, in FB interference with ICAM‐1 up‐regulation by ORFV infection was also observed. In conclusion, we shed light on epidermal and dermal defense mechanisms to ORFV infection and point to a novel ICAM‐1‐related immune evasion mechanism of ORFV in human skin.

We describe the repurposing and optimization of the TK-positive (thymidine kinase) vaccinia virus strain ACAM1000/ACAM2000™ as an oncolytic virus. This virus strain has been widely used as a smallpox vaccine and was also used safely in our recent clinical trial in patients with advanced solid tumors and Acute Myeloid Leukemia (AML). The vaccinia virus was amplified in CV1 cells and named CAL1. CAL1 induced remarkable oncolysis in various human and mouse cancer cells and preferentially amplified in cancer cells, supporting the use of this strain as an oncolytic virus. However, the therapeutic potential of CAL1, as demonstrated with other oncolytic viruses, is severely restricted by the patients’ immune system. Thus, to develop a clinically relevant oncolytic virotherapy agent, we generated a new off-the-shelf therapeutic called Supernova1 (SNV1) by loading CAL1 virus into allogeneic adipose-derived mesenchymal stem cells (AD-MSC). Culturing the CAL1-infected stem cells allows the expression of virally encoded proteins and viral amplification prior to cryopreservation. We found that the CAL1 virus loaded into AD-MSC was resistant to humoral inactivation. Importantly, the virus-loaded stem cells (SNV1) released larger number of infectious viral particles and virally encoded proteins, leading to augmented therapeutic efficacy in vitro and in animal tumor models.

Background: Oncolytic virotherapy is a promising immuno-oncology approach that has not realized its potential due to rapid elimination by humoral immunity mediated by complement and neutralizing antibodies. We propose to use an adipose-derived mesenchymal stemcell-based platform,where the virus can be protected and amplified and potentiated inside the stem cells in order to minimize the clearance by anti-viral immunity. ACAM2000, the smallpox vaccine currently licensed in the U.S., is a clonal derivative of Dryvax® with reduced virulence and a well-documented safety profile in humans. This vaccinia virus strain can potentially be used as an oncolytic virus for cancer treatment. In this study, we evaluate the ability of ACAM2000 to (1) selectively kill cancer cells, (2) to be genetically modified without affecting its natural tumor selectivity, and to (3) determine if a stem cell-based platform can protect the virus from inactivation and potentiate its anti-tumor effects. Methods: ACAM2000 was amplified in CV1 cells and named CAL1. CAL1 was tested for its ability to replicate and selectively kill various human prostate cancer cell lines in vitro and in vivo. Additionally, CAL1 was loaded into adipose-derived mesenchymal stem cells to generate a new therapeutic agent called SuperNova1 (SNV1). Both CAL1 and SNV1 were tested for their ability to kill cancer cells in the presence of active complement and neutralizing antibodies in cell culture as well as in mice. Furthermore, CAL1 was used as the backbone to generate derivative CAL2 viruses using CRISPR/Cas9 technology to insert the gene encoding the fluorescent protein TurboFP into the intergenic locus between ORF-157 and ORF-158 of CAL1 without disrupting any existing CAL1 ORFs. Results: We showed that in vitro CAL1 preferentially infected, amplified in and lysed tumor cells and was also able to cause tumor regression in vivo without signs of toxicity. Furthermore, we demonstrated that the backbone of CAL1 can be used to engineer recombinant viruses, CAL2, that carry therapeutic genes without additionally attenuating the ability of the virus to amplify or kill tumor cells. SNV1 significantly enhanced protection of CAL1 virus from clearance by the immune system, leading to higher therapeutic efficacy. Furthermore, SNV1 provided instantly active viral particles for immediate infection and simultaneous release of therapeutic proteins in the injected tumors. Conclusions: CAL1 could be used as an oncolytic agent. We show here that a major advantage of using a cell-based platform to deliver and potentiate oncolytic vaccinia virus is the prevention of viral inactivation by the humoral immune system resulting in enhanced oncolytic viral therapy. Citation Format: Duong H. Nguyen, Thomas Herrmann, Ashley Alamillo, Forrest Neuharth, Alberto Gomez, Ivelina Minev, Barbara Härtl, Laura Schneider, Boris Minev, Dobrin Draganov, Antonio F. Santidrian. CAL1 vaccinia virus as oncolytic agent and potential use of cell-based platform to enhance its therapeutic effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6542.