cAMP-mediated regulation of proliferation and cell death in normal and malignant cells

In the project period 20111001-20150930 we have published 8 papers directly associated with the project. In the project related to the effect of cAMP on DNA damage?induced cell death in acute lymphoblastic leukeamina cells (ALL-cells) we showed that cAMP-signaling leads to reduced levels of DNA damage-induced p53 (and thereby of cell death) by promoting the interaction between p53 and its negative regulator HDM2 (EH Naderi et al, Neoplasia, 2011). This effect of cAMP was later shown to involve inhibition of DNA damage-induced acetylation of p53 via histone deacetylases (HDACs) (MM Kloster et al. Int J Oncol, 2013). We also showed that the inhibiting effect of cAMP on DNA damage-induced cell apoptosis involves activation of the NFkB signaling pathway (MM Kloster et al., Mol Cancer, 2011). We later compared the effect of cAMP on B-cell precursors (BCPs) isolated from the bone marrow of children with ALL, with BCPs from normal bone marrow donors, and we showed that cAMP reduced the DNA damage-induced death of malignant (ALL-cells), but not the normal BCPs (EH Naderi et al, Blood, 2013). This suggested an intrinsic difference in the pathways from cAMP to DNA damage-induced cell death in the malignant ALL cells. In this paper we couls also show that the response to cAMP varied according to the specific karyotype of ALL. In co-cultures between ALL cells and bone marrow-derived stromal cells, we could recently show that prostaglandine E2 (PGE2) from the stromal cells increased the levels of cAMP in the ALL-cells. In turn, the effect of PGE2 resulted in reduced levels of DNA damage-induced p53 and cell death in the ALL cells (EH.Naderi et al., Mol Cancer, 2015). In the same paper we could show that the cox-inhibitor indomethacin prevented the production of PGE2 from the stromal cells, and that is reduced the protective effect of cAMP on DNA damage-induced cell death ? suggesting that indomethacin might improve the conventional therapy of ALLs by enhancing the effects of DNA damaging agents. In our project linked to the effects of cAMP on multiple myeloma, we showed that cAMP has a direct cytotoxic effect on myeloma cells in culture. In a mouse model of myeloma, we showed that cAMP slowed down the growth of the myeloma cells in vivo when the mice were treated with the cAMP-stimulating agent forskolin (V.Follin-Arbelet et al, BMC Cancer, 2011). We could later show that cAMP-signaling killed the myeloma cells via JAK/STAT-mediated down regulation of the anti-apoptotic protein Mcl-1 (V.Follin-Arbelet et al., Cancer Lett, 2013). Recently we showed that forskolin acts in synergy with dexamethasone to kill myeloma cells ? both cell lines and primary cells from patients with multiple myeloma (V. Follin-Arbelet et al, Scient Rep, 2015). Our focus the last year has been to use our recently established xenograft model of ALL in NSG-mice to reveal the role of PGE2 in development of ALL in vivo. Hence, our hypothesis has been that the cox-inhibitor indomethacin will slow down the growth of ALL-cells in vivo possibly by restoring the levels of p53. We will also challenge the mice with DNA damaging cancer agents like doxorubicin, and we will explore whether indomethacin and/or PKA inhibitors will improve the effects of such agents on death of ALL cells in vivo. So far, we have prelimiary results supporting our hypothesis. Hence, based on three different experiments, we have now shown that indomethacin reduces the progression of ALL in NSG mice, and we have shown that the p53 levels in the leukemia cells are restored. We have a paper (E.Duthil et al.) in preparation.

Our primary goal is to understand the mechanisms involved in cAMP-mediated regulation of DNA damage responses in normal and malignant cells. This may give us new insight into the mechanisms that lead to cancer, and thereby provide us with new tools for im provement of conventional cancer therapy. We have two main projects: In project A we wish to unravel the effect of cAMP on regulation of DNA damage responses in tumour cells. The tumour suppressor p53 is a key factor in DNA damage responses, and we w ill use activation of p53 as a readout system. To do so we will use a combination of p53luc reporter mice and a mouse myeloma model to assess p53 activation in vivo in both tumour and tumour surrounding during tumour development and tumour treatment. We w ill elucidate how cAMP levels in acute lymphoblastic leukaemia (ALL) cells affect DNA damage responses. Our hypothesis is that ALL cells have high levels of cAMP, and that DNA damage-induced apoptosis can be enhanced by inhibiting the cAMP/PKA pathway. We will also explore the possibility that prevention of PGE2 from bone marrow-derived stem cells may modulate DNA damage responses in the ALL cells. In project B we recently discovered that cAMP directly kills both murine and human multiple myeloma c ells even at low concentrations of cAMP/PKA activating agents. By using a mouse myeloma model we will elucidate the mechanisms involved in cAMP-mediated killing of the cells in vitro and in vivo. As multiple myeloma develops in the bone marrow, we will el ucidate the role of PGE2-production from the bone marrow on prolferation and cell death of the myeloma cells.