PKCLEUKEMOGENESIS · Analysis of AP-1 activation in T cells; new targets against T cell leukemias

Final Activity Report Summary - PKCLEUKEMOGENESIS (Analysis of AP-1 activation in T cells. New targets against T cell leukemias)

Until recently, there were many doubts about the existence of an efficient immune response able to cure cancer in the clinic. Many possibilities are coming into focus for this type of therapy; nevertheless, two major questions remain open: 1) how much can the immune system be stimulated to reject established tumours; and 2) how can we generate an efficient immune response against cancer Tumour associated antigens (TAA) are at best weak immunogens. Most of the efforts now are directed at genetic engineering of effector cells, i.e. Dendritic cells (DC). Another possibility, albeit less explored, is genetically modifying the tumour cells to make them strong immunogens. One example would be to use tumor cells undergoing apoptosis to induce a satisfactory immune response against tumour cells. Extracellular-regulated kinase 5 (ERK5), which we have shown that is present in normal and leukemic T cells, shares the TEY activation motif with other ERKs. Other structural features are unique, such as the large regulatory C terminus that controls its nucleo-cytoplasmic shuttling. Recent observations from different laboratories suggest that ERK5 plays an important role in oncogenesis, the mechanism of which remains poorly characterised. Our results show that ERK5 mediates the activation of the anti-apoptotic transcription factor NF-?B in human and mouse leukemic T cells. Reducing ERK5 levels with a specific small hairpin RNA (shERK5) decreases nuclear accumulation of the NF-?B p65 subunit, and conversely, ectopic activation of ERK5 leads to constitutive nuclear localisation of p65 and increases its ability to transactivate specific reporter genes. Importantly, TNFa induces apoptosis in shERK5-treated but not control leukemic T cells. Moreover, ERK5 knockdown by small hairpin RNA (shERK5) reduces cell viability and sensitizes cells to Fas-induced apoptosis and blocks the palliative effects of phorbol ester in Fas-treated cells. Finally, the T lymphoma cell line EL-4, upon expression of shERK5, proliferates in vitro but fails to induce subcutaneous tumours in mice. These data suggest that ERK5 is essential for survival of both human and mouse leukemic T cells and thus represents a promising target for therapeutic intervention in this type of malignancy. EL4 shERK5 cells also failed to induce tumours when injected in peritoneum, because they were eliminated in less than 72 h after injection. This short time for tumour cell clearance suggested that NK cells were at the origin of the immune response against these cells. Consistent with this, we observed a strong downregulation of MHC-I expression in EL4 shERK5 cells. This would explain why these cells were recognized as 'foreign' by NK cells. Accordingly, NK cells were responsible of EL-4 shERK5 eradication. Interestingly, these cells were far better NK cell targets than other syngenic MHC-I-deficient cells, i.e. RMA-S or Yac-1. This likely reflects the role of ERK5 in NF-kB activation, which would normally block apoptosis after death receptor engagement. We wondered if the high level of mortality observed in EL4 shERK5 might also affect EL-4 wt cell survival. Coinjection of EL4 shERK5 and wt cells significantly reduced wt cell survival in the peritoneum, suggesting that EL4 shERK5 cells induce an immune response against EL4 wt cells. To explore this possibility, we vaccinated syngenic mice with EL-4 shERK5 cells; this led to a significant reduction in tumour development by EL4 wt cells in 80 % of the mice and totally prevented tumour development in 20 % of them. Taken together, these results indicate that EL4 shERK5 cells are ideal candidates for immunotherapy. In summary, our results establish the basis of a new therapeutically approach in cancer treatment. In this context, we have started a collaboration with Dr Atanasio Pandiella, at the Centre de Investigacion del Cancer of Salamanca and Dr Jesus F. San Miguel, at the Hospital Universitario de Salamanca, to develop a clinical approach to downregulate ERK5 expression in human patients suffering from Chronic myeloma leukaemia (CML).

Data: CORDIS, © European Union