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Background:Therapies targeted towards the tumour vasculature can be exploited for the purpose of improving the systemic delivery of oncolytic viruses to tumours. Photodynamic therapy (PDT) is a clinically approved treatment for cancer that is known to induce potent effects on tumour vasculature. In this study, we examined the activity of PDT in combination with oncolytic vaccinia virus (OVV) against primary and metastatic tumours in mice.Methods:The effect of 2-[1-hexyloxyethyl-]-2-devinyl pyropheophorbide-a (HPPH)-sensitised-PDT on the efficacy of oncolytic virotherapy was investigated against subcutaneously implanted syngeneic murine NXS2 neuroblastoma and human FaDu head and neck squamous cell carcinoma xenografts in nude mice. Treatment efficacy was evaluated by monitoring tumour growth and survival. The effects of combination treatment on vascular function were examined using magnetic resonance imaging (MRI) and immunohistochemistry, whereas viral replication in tumour cells was analysed by a standard plaque assay. Normal tissue phototoxicity following PDT-OV treatment was studied using the mouse foot response assay.Results:Combination of PDT with OVV resulted in inhibition of primary and metastatic tumour growth compared with either monotherapy. PDT-induced vascular disruption resulted in higher intratumoural viral titres compared with the untreated tumours. Five days after delivery of OVV, there was a loss of blood flow to the interior of tumour that was associated with infiltration of neutrophils. Administration of OVV did not result in any additional photodynamic damage to normal mouse foot tissue.Conclusion:These results provide evidence into the usefulness of PDT as a means of enhancing intratumoural replication and therapeutic efficacy of OV.

Although the majority of breast cancers initially respond to the cytotoxic effects of chemotherapeutic agents, most breast cancer patients experience tumor relapse and ultimately die because of drug resistance. Breast cancer cells undergoing epithelial to mesenchymal transition (EMT) acquire a CD44+/CD24-/ALDH1+ cancer stem cell-like phenotype characterized by an increased capacity for tumor self-renewal, intrinsic drug resistance and high proclivity to develop distant metastases. We uncovered in human breast tumor xenografts a novel non-mitotic role of Aurora-A kinase in promoting breast cancer metastases through activation of EMT and expansion of breast tumor initiating cells (BTICs). In this study we characterized the role of the Aurora-A/SMAD5 oncogenic axis in the induction of chemoresistance. Breast cancer cells overexpressing Aurora-A showed resistance to conventional chemotherapeutic agents, while treatment with alisertib, a selective Aurora-A kinase inhibitor, restored chemosensitivity. Significantly, SMAD5 expression was required to induce chemoresistance and maintain a breast cancer stem cell-like phenotype, indicating that the Aurora-A/SMAD5 oncogenic axis promotes chemoresistance through activation of stemness signaling. Taken together, these findings identified a novel mechanism of drug resistance through aberrant activation of the non-canonical Aurora-A/SMAD5 oncogenic axis in breast cancer.

Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway at the level of MEK1 in breast cancer cells. This interaction may serve as a novel target as well as demonstrate by an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in relative levels of poMEK1/2 and poERK1/2 compared to untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced poERK and poMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. In situ proximity ligation and pull down assays demonstrated AURKA and MEK1/2 direct interaction. In vitro kinase assay showed direct phosphorylation of MEK1 by AURKA. Combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028) with early trend toward significance in survival in a BT549 xenograft breast cancer in vivo model. Conclusions: Our data shows a novel AURKA-MEK1 interaction in breast cancer cells. In depth in vivo analysis is ongoing. The results reveal a promising new strategy for the treatment TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Gandhi S, Gil M, Khoury T, Takabe K, Puzanov I, Gelman I, D'Assoro A, Opyrchal M. A novel interaction of AURKA with MAPK pathway in breast cancer cells as a potential therapeutic target [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-17.

Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway in wild type BRAF breast cancer cells as well as demonstrate an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative (ER-, PR-, HER2-) breast cancer cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in the levels of both pMEK1/2 and pERK1/2 compared to the untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. No differences in phosphorylation of MEK1/2 or ERK1/2 were observed in BRAFG464V triple negative MDA-MB-231 cells. Treatment with AURKA inhibitors resulted in downregulation of pAURKA and a significant increase in levels of total AURKA protein. The pull-down assay with Ras-binding domain coated agarose beads followed by western blot analysis with anti-pan-RAS Ab revealed no changes in active GTP-bound RAS in alisertib-treated MCF-7 cells compared to the untreated control. Consistently, no significant changes were observed in RAS-inducible phosphorylation of RAF1 activation site at Ser338 as demonstrated by western blot. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced pERK and pMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. Furthermore, combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028). Conclusions: Our data suggests that AURKA is a RAF1-independent negative regulator of MAPK activity in breast cancer cells. The in-depth analysis of the AURKA-MEK1/2 interaction is currently under investigation. The results reveal a promising new strategy for the treatment of wild type BRAF, TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin Gelman, Antonio D’Assoro, Mateusz Opyrchal. Inhibition of AURKA induces Raf1-independent activation of MAPK pathway in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2123. doi:10.1158/1538-7445.AM2017-2123

Background: Accumulating evidence indicates that breast cancer progression, resistance to therapies and development of metastasis are driven by cancer cells with stem cell-like characteristics. The increased numbers of cancer stem-like cells (CSCs) have been reported in triple negative breast cancers (TNBCs). Therefore, novel methods for targeting CSC population are needed. Aurora-A kinase (AURKA) has been shown to play a key role in acquisition of stem cell-like properties by breast cancer cells. Furthermore, the CSCs have been demonstrated to have increased sensitivity to AURKA specific inhibitor, MLN8237 (Alisertib). Our group has previously reported that oncolytic vaccinia virus (OVV) preferentially infects CSCs. Therefore we hypothesize that the combined treatment of OVV and AURKA inhibitor will result in overall enhanced anti-tumor activity due to improved elimination of CSCs. Results: We confirmed that AURKA overexpression in MCF-7 breast cancer cell line (MCF-7 AURKA) resulted in increased number of CSCs as shown by upregulation of CD44 antigen and decreases in expression of CD24 antigen. The MCF-7 and MCF-7 AURKA cells were subsequently infected with 0.1 pfu/cell of OVV expressing EGFP (OVV-EGFP). After 72h of incubation, viral replication was analyzed by fluorescent microscopy and standard plaque-forming assay. The analysis revealed over 2-fold increases in viral replication in MCF-7 AURKA cells as compared to the parental counterparts. The enhanced viral replication resulted in decreased viability of MCF-7 AURKA cells when compared to OVV-treated MCF-7 (64% vs 81%, p<0.031). The efficacy of combined treatment of OVV-EGFP and alisertib was next analyzed in MCF-7AURKA cells. The MTT assay analysis revealed 73% decreases in viability of cells treated with combination of oncolytic viortherapy and alisertib, whereas the effect of single treatment resulted in 34% and 52% reduction of viability for OVV-EGFP and alisertib, respectively (p <0.01 and p< 0.024). We next tested the efficiency of OVV-EGFP and Alisertib combination treatment against TNBC cell line, BT549. The cells treated with combination of OVV-EGFP and alisertib showed 63% decreases in cell viability compared to 25% and 46% reductions mediated by OVV-EGFP and alisertib, respectively (p<0.003 and p< 0.03). Conclusions: Combination therapy of alisertib and OVV has a significantly higher anti-cancer cell activity in vitro than either agent used alone, and it is particularly applicable for treatment of cells with CSC phenotype. In vivo studies are currently in progress. The combined treatment of alisertib and OVV represents novel therapeutic approach in treating breast cancer with increased numbers of CSCs, resistance to standard therapies and worse clinical outcomes. The results present a novel treatment approach with high translational potential. Citation Format: Malgorzata Gil, Marcin Komorowski, Danuta Kozbor, Mateusz Opyrchal. Concurrent use of alisertib and vaccinia virus demonstrate increased in vitro efficacy against breast cancer models with elevated numbers of cancer stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B35.

Background: Aurora-A (AURKA) belongs to the family of serine-threonine kinases that are crucial for cell cycle control. In particular, AURKA controls centrosome function, mitotic spindle formation, and proper chromosome segregation. In contrast to the well-defined function of AURKA in normal cells, the role of AURKA in malignant transformation and cancer progression is still under investigation. Here, we report that AURKA directly interacts with MEK1/2 kinase and induces chemotaxis of breast cancer cells. Results: We demonstrated that overexpression of AURKA in luminal ER+ MCF-7 cells (MCF-7AURKA) resulted in over 10-fold increase in the number of cells displaying chemotactic response upon stimulation with growth factors when compared to parental MCF-7 cells. Both AURKA inhibitor MLN8237 and MEK1/2 inhibitor PD0325901 reduced migratory properties of MCF-7AURKA cells. In contrast, treatment with the pan RAF inhibitor TAK-632 did not affect chemotactic response of MCF-7AURKA cells. We demonstrated colocalization of AURKA and MEK1/2 immunofluorescence signals in MCF-7AURKA and triple-negative BT549 cells. In situ proximity ligation assay (PLA) revealed the presence of AURKA-MEK1/2 complexes in MCF-7AURKA and BT549 cells, and the notion that AURKA and MEK1/2 formed cellular complexes was confirmed with co-immunoprecipitation experiments. In vitro kinase assays with recombinant AURKA and MEK1 proteins showed that AURKA kinase could directly phosphorylate MEK1. Incubation of MCF-7AURKA and BT549 cells with the AURKA-specific inhibitors MLN8237 or MK8745 resulted in over a 2-fold increase in the level of pERK1/2 compared to the untreated controls. Treatment with the pan RAF inhibitor TAK-632 did not diminish MLN8237-induced pERK1/2, whereas treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated MLN8237-induced activation of ERK1/2. Conclusions: Our data present a novel AURKA-MEK1/2 interaction that contributes to the increased chemotaxis of breast cancer cells. The results suggest that AURKA negatively regulates MEK1 through a novel, direct phosphorylation event, possibly leading to constitutive activation of MEK2 and increased chemotaxis. The combined treatment of AURKA and MEK1/2 inhibitors represents a new therapeutic approach to decrease metastatic potential of breast cancer cells and delay tumor progression. The in-depth analysis of the AURKA-MEK1/2 complexes as well as in vivo studies testing the efficacy of combining AURKA and MEK1/2 inhibitors on patient-derived xenografts are currently in progress. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin H. Gelman, Antonino B. D’Assoro, Evanthia Galanis, Mateusz Opyrchal. AURKA interaction with MEK1/2 complex and its role in promoting breast cancer cell metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A60.

Background: Triple negative breast cancer (TNBC) patients have poorer prognosis and there remains a lack of novel targeted therapies for their treatment. PIM2 (Proviral Integrations of Moloney virus 2) belongs to a family of three kinases that have been implicated in the survival and progression of hematologic malignancies and solid tumors. PIM2 has been linked to epithelial to mesenchymal transition in TNBC, which can lead to metastasis and chemotherapeutic resistance. We hypothesized that PIM2 may present as a therapeutic target in TNBC. Materials and Methods: The study involved both in vitro and in vivo studies involving a novel PIM2 inhibitor JP11646 (obtained from Jasco Pharmaceuticals). TNBC cell lines MDA-MB-231 and BT-549 were obtained for our in vitro studies. Cell viability was evaluated using MTT assay. Western Blot assay was used to evaluate relative protein expression. For in vivo studies, female SCID mice were inoculated in the mammary fat pads with 1 × 106 MDA-MB-231 cells. When tumor volumes reached 100 mm3, the mice were treated with JP11646 at the dosage 15mg/kg intraperitoneally for 2 consecutive days weekly for total of 4 weeks as determined from previous experiments. Control animals received vehicle only. The mice were euthanized once tumors reached ∼1,700 mm3. Results: BT-549 cells treated in vitro with 3 different available PIM kinase inhibitors AZD 1208, LGB321 and JP12641 showed only modest reduction in cell viability. However, treatment of both MDA-MB-231 and BT-549 with JP 11646 demonstrated significant reduction in cell viability with IC50 ranging from 40 to 71.6 nM. Treatment with JP11646 demonstrated a novel mechanism of action resulting in downregulation of PIM2 in both cell lines. Treatment with JP11646, but not other PIM kinase inhibitors, resulted in activation of apoptosis as measured by cleaved PARP (cPARP) levels. Anti-PIM2 siRNA treatment but not treatment with non-specific PIM kinase inhibitor AZD1208 resulted in cPARP induction. Inhibition of proteolysis by bortezomib resulted in preservation of PIM2 and inhibition of apoptosis as demonstrated by decreased cPARP levels after treatment with JP11646. PIM2 over-expressing clone of MDA-MB-231 cells showed enhanced proliferation and migration properties both in vitro and in vivo.Treatment of mice with orthotopically implanted MDA-MB-231 tumors with JP 11646 resulted in significant reduction in the tumor growth (p=0.0019) and increased overall survival (p=0.018) as compared to control mice. Conclusions: PIM2 upregulation in TNBC cell line resulted in more aggressive phenotype. JP11646, through novel mechanism of action resulting in degradation of PIM2, showed robust activity in TNBC cell lines both in vitro and in vivo. Further correlative studies in tumors harvested from in vivo experiments are ongoing. These results encourage further exploration of use of JP11646 as a targeted agent in treatment of TNBC. Citation Format: Mehta R, Kothai Guruswamy Sangameswaran D, Bezbatchenko K, Moore J, Gil M, Khoury T, Baldino C, Caserta J, Fetterly, Jr. G, Lee K, Adjei A, Opyrchal M. Preclinical efficacy of the novel PIM2 kinase inhibitor, JP11646 in triple negative breast cancer models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-10.