The effect of calcium electroporation on viability, phenotype and function of melanoma conditioned macrophages

Electroporation in combination with chemotherapy is an established treatment used on solid malignancies that results in enhanced chemotherapeutic uptake. Recent advances have begun to transition to the use of non-toxic compounds, such as calcium, in lieu of chemotherapy, which can also induce tumour cell death. While the effect of treatment on tumour cell death has been well characterized and has been shown to induce an immunogenic form of cell death, the effect of treatment on intratumoural immune cells has not been investigated. Here we present data showing the effect of calcium electroporation on immune cells, using melanoma-conditioned bone marrow-derived macrophages. Similar to tumour cells, macrophage cell membranes are susceptible to poration following treatment and subsequently reseal. Macrophages are less susceptible to calcium electroporation induced cell death in comparison to B16F10 melanoma cells. However treatment with electroporation with or without bleomycin or calcium was shown to affect macrophage phenotype and function. Coculture of calcium electroporated macrophages revealed that both the capacity of macrophages to stimulate and direct T cell responses are affected following exposure to treatment. We conclude that calcium electroporation has the potential to boost the immunogenic capacity of exposed tumour associated macrophages, and further research is warranted to determine if calcium electroporation can be optimised to generate systemic anti-cancer immune responses.

Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.

P.042 Raloxifene sensitizes glioblastoma cells to hypoxia-induced death through inhibition of stress granule dissolution

Background: Glioblastoma (GBM) is the most common primary malignant brain tumour. Despite aggressive therapy, median survival is only 14 months. Death typically results from treatment failure and local recurrence. The GBM microenvironment is highly hypoxic, which correlates with treatment resistance. Cytoplasmic RNA stress granules (SGs) form in response to hypoxic stress and act as sights of mRNA triage, allowing preferential translation of pro-survival mRNA during stress. We hypothesize that SGs may play a role in hypoxia-induced resistance to therapy, and may be targetable by chemotherapeutics to improve outcomes. Methods: We screened 1280 approved compounds to identify drugs that inhibited formation or dissolution of SGs in U251 glioma cells. Raloxifene inhibited SG dissolution in a dose dependent manner. We treated cells with raloxifene and incubated them in hypoxia, and then measured rates of cell death using cell counting and Presto blue. Results: Cell death rates were synergistically higher in cells treated with the combination of raloxifene and hypoxia compared to either treatment alone. Conclusions: Raloxifene inhibits the dissolution of SGs in glioma cells, and combination treatment results in synergistic tumour cell death. Taken together, this provides evidence that inhibition of SG dissolution may be a viable target for future GBM chemotherapeutics.