Fonseca, Rafael Hinojal

Abstract The retinoblastoma 1 (RB1) gene is a frequent target of mutation or inactivation in neoplastic diseases. However, the current model for RB1-mediated malignancy requires inactivation of both alleles for tumor progression. Single–copy loss of RB1 has been shown to correlate with an approximate 50% reduction in RB1 mRNA expression in patients diagnosed with multiple myeloma and survival in this cohort of patients is significantly reduced. Using siRNA to reduce Rb protein levels to 50% and Rb-expressing adenovirus to restore RB1, variations in proliferation and cell cycle were observed. The KMS–11 adherent cell line with normal Rb protein expression was labeled with carboxyfluorescein succinimidyl ester (CFSE) for tracking of cellular division and RB1 siRNA added to silence 50% of protein expression. Transfection efficiency for siRNA was measured at 95% or greater. Flow cytometric analysis was performed at 72 hours to identify changes in proliferation between controls and Rb knock–down. Using CFSE proliferation analysis software, reduction of Rb protein levels by 50% caused an average increase in the proliferation index from 2.05 to 2.50. Additionally, reduction of Rb protein levels caused an increase in the percentage of cells in S–phase, from 24 to 29%. CFSE and cell cycle experiments were performed in triplicate. Conversely, addition of Rb–expressing adenovirus to MM.1R and U266 cell lines, with mono-allelic and bi-allelic loss of RB1 respectively, resulted in a decrease in proliferation assayed by CFSE and an average decrease in the percentage of cells in S-phase from 29 to 23% compared to controls. No changes in proliferation or cell cycle compared to untreated controls were observed following null adenovirus infection. Western blot and quantitative RT–PCR (qPCR) were used to confirm reduction or addition of RB1 to cell lines. Further, qPCR was used to identify potential activation of the interferon response following addition of siRNA and adenovirus. No changes in annexin V expression were observed following reduction or replacement of Rb as compared to controls. These results suggest the addition of proliferation advantages to tumors with single copy loss of RB1. For malignancies such as multiple myeloma characterized by deletion of 13q14 coupled with an extended period of development, the proliferation advantage associated with RB1 haploinsufficiency may contribute to decreased survival in the deletion 13 cohort.

Abstract Abstract 1846 Background The low frequency of TP53 (p53) alterations (<10%) in MM makes this tumor an ideal candidate for p53-activation therapies. Unfortunately, DNA alkylating agents (cyclophosphamide and melphalan), when used alone, cannot fully exploit p53 mediated apoptosis. It is currently unknown the extent to which DNA alkylating agents and p53 activating small molecules are activating functional p53 signaling to induce apoptosis in primary MM cells, because, p53 mainly induces cell cycle arrest/DNA repair or apoptosis in the DNA damage response. Emerging studies demonstrate that PARP activation and p53 poly(ADP-ribosyl)ation can negatively influence apoptosis induction but the exact molecular mechanisms are unknown. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 2.5 μM of melphalan, 10 μM of olaparib and 2.5 μM of nutlin-3a alone and/or combinations of melphalan with olparib and/or nutlin3a with olaprib for 24 to 72 hours before they were examined for cell death by the annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For combination studies, cells were incubated with 0.194–50 μM of melphalan and 0.625–40 μM of either olaparib or ABT-888 for 72 hours. p53 knock-down performed by lentiviral mediated shRNA. Vk*MYC mice with significant gammopathy (>20g/l on SPEP) used for in-vivo drug combination studies. Results Preliminary results support our principle hypothesis that, a PARP inhibitor (olaparib) enhanced Melphalan sensitivity in wild type p53 MM cell lines H929 and MM1S but not in p53 mutated or homozygously deleted cell lines U266 and KMS11, respectively. Moreover, we demonstrate that p53 knock-down decreased the synergistic effect of combining a PARP inhibitor with melphalan and Nutlin-3a. Again, combining a PARP inhibitor with the DNA alkylating agent melphalan induced clear PARP cleavage, a signature of apoptosis in wild type p53 MM cell lines H929 and MM1S. No significant PARP cleavage was observed after melphalan or olaparib treatment alone in H929 and MMIS. As expected, the p53 mutant cell line U266 did not show any PARP cleavage and p21 up-regulation after both drug treatments. Here we propose that the hyper-activated p53 from PARP inhibition along with DNA alkylating agents (melphalan) and/or p53 activating agents (nutlin-3a) result in better responses. Finally, in a murine xenograft model of human MM, olaparib potentiated melphalan activity in vivo, with significant reduction in M spike level. Conclusion Our studies indicate that DNA alkylating agents activate wild type p53, but leads to DNA repair and cell survival and this can be abolished by PARP inhibition in wild type p53 expressing MM cases. Moreover, our study also found that olaparib, and ABT-888 - PARP1 inhibitors currently in clinical trials - display different dose responses in MM cell lines and in the abilities of individual PARP inhibitors to sensitize MM cell lines to melphalan varied to a great extent in a cell context- and cell line specific manner. Disclosures: No relevant conflicts of interest to declare.