Synergistic Antitumor Interactions Between Obatoclax And Olaparib Or Chloroquine In Pancreatic Cancer Cells

Pancreatic cancer is the fourth leading cause of cancer related deaths in theUnited States with an overall5-year survival rate of6%. There has been littleimprovement in the prognosis for patients with pancreatic cancer over the past20years, partially due to the delay of diagnosis, with many cases being diagnosed at anadvanced stage. Since2005, the standard chemotherapeutic treatment of the disease isthe administration of gemcitabine, a nucleoside analog, combined with erlotinib, akinase inhibitor. However, its efficacy remains low, with a median survival of8.5months and1-year survival rate of35%. Therefore, new therapeutic approaches areurgently needed.A common cause for chemotherapy resistance arises from the overexpressionof the anti-apoptotic Bcl-2family proteins. Bcl-xL and Mcl-1are expressed in88%whereas Bcl-2is expressed in23%of invasive ductal carcinomas. Therefore targetingthese proteins could be an effective treatment for pancreatic cancer. Obatoclax(GX15-070) is a small molecule that binds to the BH3-binding site of Bcl-2, Bcl-xL,and Mcl-1. This pan-Bcl-2inhibitor has been reported to directly induce apoptosis ina variety of cultured cancer cell lines and primary patient samples through themitochondrial apoptotic pathway as well as non-apoptotic cell death. Recent studieshave found compelling evidence to suggest that the anti-apoptotic Bcl-2familyproteins can regulate DNA double-strand break repair independent of theirpro-survival functions.PARP1is a DNA binding protein involved in apoptosis as well as DNAsingle-and double-strand break repair, and has been becoming a popular therapeutictarget for many different malignancies. PARP inhibitors, such as Olaparib (AZD2281), have been demonstrated to have a strong synthetic lethal relationship with BRCA1/2deficiency. Further, the use of PARP inhibitors has recently been extended to tumorswith other defects in the HR DNA repair pathway, as well as in combination withchemotherapy drugs and chemoradiotherapy. Combining PARP inhibitors with agentsthat impair DNA damage repair to treat BRCA1/2wild-type tumors could broaden theclinical use of these promising PARP inhibitors.We hypothesized that using a pan-Bcl-2inhibitor may sensitize pancreaticcancer cells to PARP inhibitors，since both Bcl-2family proteins and PARP involve inapoptosis and DNA damage repair. To test this possibility, antitumor effects ofGX15-070and AZD2281(olaparib), alone or in combination, were determined byMTT assays and flow cytometry analyses in6clinically-relevant pancreatic cancercell lines, ASPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, and PANC-1, whichexpress variable levels of PARP1, Bcl-2, Bcl-xL, and Mcl-1determined by WesternBlots. Both drugs caused growth arrest, however, very limited apoptosis. Further, theindividual drug sensitivities did not appear to correlate with PARP1, Bcl-2, Bcl-xL orMcl-1protein levels.When AZD2281was combined simultaneously with clinically achievableconcentrations of GX15-070, additive-to-synergistic antitumor interactions weredetected by calculating the combination index values with the CalcuSyn software andby standard isobologram analyses. Simultaneous treatment also resulted in asignificantly increased percentage of dead cells compared to the individual drugtreatments determined by trypan blue exclusion and colony formation assays. ShRNAknockdown of PARP1in pancreatic cancer cells also significantly enhancedGX15-070-induced cell death. However, we did not detect cleavage of caspase-3orPARP1or increased sub-G1population in the pancreatic cancer cells post drugtreatments, indicating that the increase in cell death was through non-apoptoticmechanisms.Cell cycle analysis in the BxPC-3cells revealed a small significant increase in G2/M phase after treatment with AZD2281alone, which was further significantlyincreased by the addition of GX15-070and accompanied by a decrease in CDK1protein levels. In the PANC-1cells, we detected a small significant increase in Sphase after individual drug treatments, which was further significantly increased whenthe drugs were administered simultaneously and accompanied by a minor decrease inCDK2protein levels.These results further indicate that GX15-070potentiates thecytotoxic effects of AZD2281through non-classical mechanisms.Small decrease inBcl-2was detected after the combined drug treatment compared to vehicle controltreated cells, implicating that the non-apoptotic cell death induced by the drugs ispotentially due to decrease in Bcl-2protein levels.Surprisingly, in our in vivo studies, neither the individual nor the combineddrug treatments resulted in significant delay of externally measurable tumor growth innude mice indicated a lack of response to drug treatments. However, serum CA19-9levels (a pancreatic cancer biomarker that is used to monitor chemotherapy response)in the combination group were significantly lower compared to that for the othertreatment groups, indicating treatment response. H&Estaining revealed substantiallyincreased necrosis in the tumors for the drug treated groups compared to the vehiclecontrol group, especially for the combination group. Analogous to the results fromH&E taining, immunohistochemical staining showed substantially decreasedexpression of PCNA and CD34in the combination group compared to the othergroups. These results indicate that the two drugs cooperate in suppressingproliferation of pancreaticcancer cells and angiogenesis in the tumors.Taken together, our results demonstrate cooperative antitumor effects ofGX15-070and AZD2281on preclinical models of pancreatic cancer in vitro and invivo. Although we do not have direct evidence, we suspect that GX15-070predominantly targets the non-apoptotic functions of Bcl-2, Bcl-xL and/or Mcl-1topotentiate the cytotoxic effects of the PARP inhibitor AZD2281in pancreatic cancercells. Further studies are necessary to determine the molecular mechanisms of non-apoptotic cell death induced by the combination. Our novel findings suggest thatthere might be a clinical benefit for using the combination of GX15-070andAZD2281to treat pancreatic cancer.Based on the classical roles Bcl-2, Bcl-xL and Mcl-1play in the mitochondrialapoptosis pathway, GX15-070would be expected to cause apoptotic cell death.However, at clinically achievable concentrations of GX15-070we detected minimalapoptosis, indicating that other mechanisms must exist, preventingGX15-070-induced apoptosis in pancreatic cancer cells. While anti-apoptotic Bcl-2family proteins function to inhibit apoptosis, data indicate that they can also inhibitautophagy. It is conceivable that GX15-070induces autophagy which promotessurvival of pancreatic cancer cells. Thus，adding an autophagy inhibitor may increaseGX15-070-induced apoptosis of pancreatic cells.Chloroquine acts as a lysosomal pH inhibitor, therefore preventing the fusionof autophagosomes with lysosomes and inhibiting late stages of autophagy. Aschloroquine is a well-tolerated drug, its potential use as mono-orcombination-therapy for cancer treatment is highly attractive. Previous findings haveled to the initiation of multiple clinical trials combining autophagy inhibitors andchemotherapeutic agents for diverse cancer types. Chloroquine is currently beingtested in the clinic as monotherapy treatment for pancreatic cancer(http://www.clinicaltrials.gov: NCT01273805). Therefore, we chose chloroquine forour proposed studies.As expected, GX15-070indeed induced autophagy in the pancreatic cancercell lines, reflected by its dose-dependent induction of LC3B-II in the cells.Transmission electron microscopy analysis of GX15-070-treated pancreatic cellsshowed increased autophagosomes, further confirming the induction of autophagy byGX15-070. When combined simultaneously, chloroquine additively-to-synergisticallyenhanced GX15-070induced growth arrest determined by MTT assays and by usingthe CalcuSyn software. Further, the two agents cooperatively induced apoptotic cell death in the pancreatic cancer cell lines. The combined treatment significantlydecreased Bcl-2protein levels compared to individual drug treatments. This wasaccompanied by induction of γH2AX, a biomarker of DNA double-strand breaks.These results support the use of the combination of chloroquine and Bcl-2inhibitorsfor the treatment of pancreatic cancer.In summary, AZD2281or chloroquine synergistically enhances GX15-070cytotoxicity against pancreatic cancer cells in vitro. Furthermore, in an in vivoxenograft model, the combination of AZD2281and GX15-070caused substantiallyincreased tumor necrosis compared to either treatment alone. Our results suggest thatcotreatment of pancreatic cancer patients with GX15-070and AZD2281or CQ mayprovide clinical benefits.