The effects of the clinically applicable chemotherapeutics on the immunogenicity of melanoma cell death.

During carcinogenesis, cancer cells that are efficiently recognised by the immune system are removed, while the variants capable of evading host defence systems form an aggressive tumour. Currently, it is becoming widely accepted that an effective anticancer therapy should reverse the immunoevasive and immunosuppressive effects elicited by the cancer cells and activate anticancer immune responses that will efficiently eliminate cancerous lesions and prevent relapses. Recently, increasing attention addressed the potential of a subtype of therapy-induced apoptotic cell death, which is associated with the activation of danger signalling pathways evoking emission of damage associated molecular patterns (DAMPs), referred to as ‘immunogenic cell death (ICD)’.

During my PhD studies I investigated the ICD-inducing potential (both in vitro and in vivo, whenever possible) of the two clinically relevant anti-melanoma therapies: melphalan and vemurafenib.

Vemurafenib is an inhibitor of BRAFV600E, a commonly mutated kinase within the MAPK pathway. This targeted agent has shown significant, but short-lasting clinical responses in patients with metastatic melanoma. In the first part of this thesis, I have demonstrated that the melanoma cell lines harbouring BRAFV600E mutation responded to vemurafenib with induction of caspase-dependent cell death and danger signalling activation. The latter one resulted in exposure of two DAMPs, ecto-CRT and ecto-HSP90, but failed to emit ATP. In contrast, vemurafenib was unable to induce these processes in two isogenic drug resistant cell lines. Trying to re-establish the danger signalling in vemurafenib-resistant cells, I showed that inhibition of MAPK pathway with MEK inhibitor, the downstream kinase activated by BRAF, re-ignited cell death and danger signalling in the these cells. The MAPK pathway-inhibition induced DAMPs were functional as only melanoma cells exposing death-induced danger signals were phagocytosed and induced DAMPs-related DC maturation. Recent research from the laboratory of Dr. Guido Kroemer and our group has highlighted that ICD-associated danger signalling is modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. Accordingly, I observed that the vemurafenib-resistant cells displayed higher basal and drug-induced autophagy, therefore autophagy abrogation was particularly efficacious in boosting cell death and danger signalling in vemurafenib-resistant cells upon blockage of MEK. Nevertheless, only autophagy inhibition was insufficient to re-establish vemurafenib-resistant cells sensitivity to this treatment.

The other investigated anti-melanoma therapeutic, Melphalan (Mel), is a (loco-)regionally applied drug. It is given to the patients with extremities localized melanoma within the Mel-based isolated limb perfusion (Mel-ILP). I have showed that in human melanoma biopsies, Mel-ILP caused a pro-inflammatory microenvironment characterized by upregulation of IL1B, IL8 and IL6, which was associated with the rapid (1 h) release of IL6 and IL1β detectable in patients’ (loco-)regional sera. Additionally, the in vitro experiments have demonstrated that Mel is a mild inducer of danger signalling that resulted in caspase-dependent, ER stress-related HSP90 exposure (out of CRT, HSP70, ATP tested). Mel’s limited capacity to induce prompt and a molecularly diverse set of DAMPs has been established to rely on Mel’s moderate ER stress-inducing abilities depending on Ca2+ release. In parallel, Mel-treated human melanoma cells elicited induction of semi-mature DCs and moderate T cell activation. Moreover, in vivo prophylactic immunization showed that Mel-treated cancer cells were able to stimulate a CD8+ T cell-dependent protective anti-tumour response, although to a lesser extent than an established inducer of ICD. Interestingly, the Mel-induced moderate “anticancer vaccination effect” was strongly potentiated through combinatorial treatment with exogenously added recombinant CRT.

In conclusion, both studies indicate that the clinically applied anti-melanoma therapeutics, Mel and vemurafenib, induce danger signalling that can be further optimize with certain combinatorial approaches. Most probably the treatments’ limited ability to stimulate danger signalling is caused by the induction of restricted ER stress and stimulation of autophagy. Thus, the results obtained within my PhD studies indicated that the vemurafenib and Mel clinical effects might be improved by combination with clinically used autophagy inhibitors or Thapsigargin-related derivatives, i.e. G-202 or by the addition of rCRT, respectively. Additionally, other immunostimulatory therapies, like IDO-inhibitors, immunocheckpoint inhibitors or anticancer vaccines, might be considered to further strengthen the moderate immunogenic properties of the investigated anti-melanoma therapeutics.