Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing.

BACKGROUND: Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation. OBJECTIVES: To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence. METHODS: Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20. RESULTS: We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+  BRAF mutant MM cells. CONCLUSIONS: In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.

Oncolytic Viruses and Immune Checkpoint Inhibitors: The "Hot" New Power Couple.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer care and shown remarkable efficacy clinically. This efficacy is, however, limited to subsets of patients with significant infiltration of lymphocytes into the tumour microenvironment. To extend their efficacy to patients who fail to respond or achieve durable responses, it is now becoming evident that complex combinations of immunomodulatory agents may be required to extend efficacy to patients with immunologically "cold" tumours. Oncolytic viruses (OVs) have the capacity to selectively replicate within and kill tumour cells, resulting in the induction of immunogenic cell death and the augmentation of anti-tumour immunity, and have emerged as a promising modality for combination therapy to overcome the limitations seen with ICIs. Pre-clinical and clinical data have demonstrated that OVs can increase immune cell infiltration into the tumour and induce anti-tumour immunity, thus changing a "cold" tumour microenvironment that is commonly associated with poor response to ICIs, to a "hot" microenvironment which can render patients more susceptible to ICIs. Here, we review the major viral vector platforms used in OV clinical trials, their success when used as a monotherapy and when combined with adjuvant ICIs, as well as pre-clinical studies looking at the effectiveness of encoding OVs to deliver ICIs locally to the tumour microenvironment through transgene expression.

Engineering Adenoviral Vectors with Improved GBM Selectivity.

Glioblastoma (GBM) is the most common and aggressive adult brain cancer with an average survival rate of around 15 months in patients receiving standard treatment. Oncolytic adenovirus expressing therapeutic transgenes represent a promising alternative treatment for GBM. Of the many human adenoviral serotypes described to date, adenovirus 5 (HAdV-C5) has been the most utilised clinically and experimentally. However, the use of Ad5 as an anti-cancer agent may be hampered by naturally high seroprevalence rates to HAdV-C5 coupled with the infection of healthy cells via native receptors. To explore whether alternative natural adenoviral tropisms are better suited to GBM therapeutics, we pseudotyped an HAdV-C5-based platform using the fibre knob protein from alternative serotypes. We demonstrate that the adenoviral entry receptor coxsackie, adenovirus receptor (CAR) and CD46 are highly expressed by both GBM and healthy brain tissue, whereas Desmoglein 2 (DSG2) is expressed at a low level in GBM. We demonstrate that adenoviral pseudotypes, engaging CAR, CD46 and DSG2, effectively transduce GBM cells. However, the presence of these receptors on non-transformed cells presents the possibility of off-target effects and therapeutic transgene expression in healthy cells. To enhance the specificity of transgene expression to GBM, we assessed the potential for tumour-specific promoters hTERT and survivin to drive reporter gene expression selectively in GBM cell lines. We demonstrate tight GBM-specific transgene expression using these constructs, indicating that the combination of pseudotyping and tumour-specific promoter approaches may enable the development of efficacious therapies better suited to GBM.

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis.

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor-initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell-specific immune checkpoint, which is responsible for NK cell-associated poor outcomes in many cancers.

Transcriptional Changes in the Ovaries of Perch from Chernobyl.

Fish have been highly exposed to radiation in freshwater systems after the Chernobyl Nuclear Power Plant (NPP) accident in 1986 and in freshwater and marine systems after the more recent Fukushima NPP accident in 2011. In the years after the accident, the radioactivity levels rapidly declined due to radioactive decay and environmental processes, but chronic lower dose exposures persisted. To gain insights into the long-term effects of environmental low dose radiation on fish ovaries development, a high-throughput transcriptomic approach including a de novo assembly was applied to different gonad phenotypes of female perch: developed gonads from reference lakes, developed/irradiated from medium contaminated lake, and both developed/irradiated and undeveloped from more highly contaminated lakes. This is the most comprehensive analysis to date of the gene responses in wildlife reproductive system to radiation. Some gene responses that were modulated in irradiated gonads were found to be involved in biological processes including cell differentiation and proliferation (ggnb2, mod5, rergl), cytoskeleton organization (k1C18, mtpn), gonad development (nell2, tcp4), lipid metabolism (ldah, at11b, nltp), reproduction (cyb5, cyp17A, ovos), DNA damage repair (wdhd1, rad51, hus1), and epigenetic mechanisms (dmap1). Identification of these genes provides a better understanding of the underlying molecular mechanisms underpinning the development of the gonad phenotypes of wild perch and how fish may respond to chronic exposure to radiation in their natural environment, though causal attribution of gene responses remains unclear in the undeveloped gonads.