Abstract PR04: Exploiting oncogene-induced DNA replicative stress as synthetic lethal approach to target myeloma.
Authors: Cottini Francesca , Hideshima Teru , Richardson Paul , Anderson Kenneth , Tonon Giovanni 
Published: August 2015

Multiple myeloma (MM) cells have a complex genomic phenotype with abnormal karyotypes, chromosomal translocations, and several copy-number variations. MM cells present ongoing DNA damage which activates an ATM/ABL1-dependent DNA damage response (1). However, MM cells do not undergo apoptosis due to the concomitant loss of the Hippo coactivator YAP1(1). We have now explored potential mechanisms underlying DNA damage in MM cells to identify novel therapeutic strategies. We detected signs of prominent DNA replicative stress in MM cells, evidenced by 53BP1, RPA and RAD51 foci associated with activation of ATR and its downstream target CHK1. A subset of approximately 20% of MM patients has also genomic instability, along with deregulated expression of DNA replication genes, and ATR/BRCA and cell cycle pathways. These patients have an unfavorable prognosis versus patients without this signature (p&lt;0.0001). Notably, this patient subset has significantly higher expression levels of the c-MYC oncogene. Oncogenes, such as c-MYC, increase origin activation and trigger an increased numbers of stalled and collapsed replication forks (2), resulting in replicative stress. To assess the role of c-MYC in the onset of replicative stress and DNA damage in MM, we used gain- and loss-of function models. Specifically, we re-expressed c-MYC in U266 MM cells, which have low c-MYC levels and low levels of ongoing DNA damage; and we silenced c-MYC in H929 and MM.1S MM cells using shRNAs. Re-expression of c-MYC triggered replicative stress, as indicated by increased number of RAD51, RPA, and 53BP1 foci, together with activation of an ATR-dependent cascade; whereas c-MYC silencing decreased replicative stress and DNA damage markers. ATR, unlike ATM, is necessary to resolve DNA damage arising from replicative stress; indeed, compounds have been designed to specifically block ATR and thereby prevent DNA repair in tumor cells with ongoing replicative stress.

We therefore hypothesized that MM cells could be sensitive to replicative stress overload, and that ATR blockade would induce apoptosis in these cells. We silenced ATR using specific shRNAs in H929 and OPM-2 cells, and inhibited its kinase activity by a specific inhibitor VE-821. In both cases, we observed a growth inhibitory effect, associated with an increase in DNA double strand breaks. Moreover, cells overexpressing c-MYC were significantly more sensitive to VE-821 than controls; conversely, cells silenced for MYC were less responsive, suggesting the potential synthetic lethality activity in MM cells over-expressing MYC. MYC also increases reactive oxygen species (ROS) that mediate DNA damage. Indeed, MYC overexpression induced a strong increase in ROS in U266 cells; and treatment with the antioxidant reagent N-Acetylcysteine (NAC), which scavenges ROS by replenishing glutathione stores, reduced replicative stress markers. We then explored whether pharmacologically increasing ROS levels could represent a synthetic-lethal approach. To this end, we used piperlongumine (PL): MM cells were sensitive to PL treatment, while PBMCs were minimally affected. We then combined ATR inhibition and PL treatment, to fully exploit synthetic lethality to target MM cells. ATR downregulation with shRNAs combined with 1-2.5 μM PL triggered synergistic apoptosis. Similar synergic effects were obtained with VE-821 (1-2.5 μM) in combination with PL (1-2.5 μM) in both MM cell lines and primary MM cells.

In conclusion, our data suggest that replication stress is a common phenotype in a subset of MM patients as a result of intense oncogene activation, such c-MYC overexpression. Triggering additional replicative stress by ROS induction, along with simultaneously inhibiting tumor DNA repair abilities by blocking ATR, may represent a novel therapeutic strategy in MM patients presenting with particularly aggressive disease.

1. Cottini, F., et al. Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers. Nature medicine 20, 599-606 (2014).

2. Hills, S.A. & Diffley, J.F. DNA replication and oncogene-induced replicative stress. Current biology : CB 24, R435-444 (2014).

Citation Format: Francesca Cottini, Teru Hideshima, Paul Richardson, Kenneth Anderson, Giovanni Tonon. Exploiting oncogene-induced DNA replicative stress as synthetic lethal approach to target myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr PR04.