Opyrchal, Mateusz

Abstract 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

Attenuated Edmonston lineage measles virus (MV-Edm) vaccine strains can preferentially infect and lyse a wide variety of cancer cells. Oncolytic MV-Edm derivatives are genetically engineered to express the human carcinoembryonic antigen (MV-CEA virus) or the human sodium iodide symporter (MV-NIS virus) and are currently being tested in clinical trials against ovarian cancer, glioblastoma multiforme, multiple myeloma, mesothelioma, head and neck cancer, breast cancer and malignant peripheral nerve sheath tumors. This review describes the basic and preclinical data that facilitated the clinical translation of MV-Edm strains, and summarizes the clinical results of this oncolytic platform to date. Furthermore, we discuss the latest clinically relevant MV-Edm vector developments and creative strategies for future translational steps.

Abstract Background: During tumor progression, activation of Aurora A kinase (AURKA) is associated with epithelial to mesenchymal transition (EMT) reprogramming and expansion of a subpopulation of tumor initiating cells harboring a CD44+/CD24low/- phenotype [D'Assoro, Oncogene 2014]. These cells are characterized by their capacity to self-renew, resist drug therapies, and promote distant metastases. In ER+ breast cancer (BC) models, activation of AURKA is associated with down-regulation of ERα expression and resistance to endocrine therapy. Alisertib, a selective inhibitor of AURKA, can reverse EMT and restore tumor ERα expression and sensitivity to endocrine therapy [Opyrchal, PLoS One 2014]. As a single agent in HR+ advanced BC, alisertib was associated with a 6-month clinical benefit rate of 54% and median PFS of 7.9 months [Melichar, Lancet Oncol 2015]. The objectives of this phase I trial were to determine the maximum-tolerated dose (MTD) and evaluate the toxicities and clinical activity of alisertib with fulvestrant in patients (pts) with HR+ advanced BC. Methods: In this standard 3+3 dose-escalation phase I study, pts were assigned to two different oral doses of alisertib (40-50 mg BID on days 1-3, 8-10, 15-17 q 28-day cycle) in combination with standard dose fulvestrant (500 mg IM on day 1 and 15 of cycle 1 and then day 1 q 28-day cycle thereafter). Eligibility included HR+ advanced BC, postmenopausal status, measurable disease or nonmeasurable bone disease by RECIST v1.1, ECOG performance status ≤ 1, unlimited prior endocrine therapies, and ≤ 2 chemotherapy regimens in the metastatic setting. Results: Ten pts enrolled September 2014 - April 2015, and 9 were evaluable for the primary endpoint (one excluded due to ineligibility). The median pt age was 59 (range 48, 73). Prior endocrine therapies included AI (9, 100%), fulvestrant (6, 67%), and everolimus/exemestane (5, 56%). Eight pts (89%) had prior chemotherapy. A median of 4 cycles of therapy have been administered (range 1+, 9+). There were no severe (grade 3+) toxicities reported during cycle 1 at either dose level, thus the MTD was not reached. The cycle 1 grade 1/2 adverse events regardless of attribution were fatigue (6, 67%), neutropenia (5, 56%), anemia (5, 56%), leukopenia (4, 44%), diarrhea (3, 33%), nausea (3, 33%), and mucositis (1, 11%). As of June 3, 2015, 2 pts have discontinued treatment due to disease progression, and 7 remain on treatment with stable disease (Table). One pt with bone only disease had a near CR on PET scan. Dose LevelAlisertib Dose (BID)Treatment Cycles≥ Grade 3 Toxicity, All CyclesProgression-Free Survival (days)1 (n=3)40 mg4, 7+, 9+ 117, 170+, 223+2 (n=6)50 mg1+, 2, 3+, 3+, 4+, 5+grade 4 neutropenia (1 pt)28+, 56, 56+, 57+, 112+, 116++ indicates patients still receiving treatment Conclusion: Alisertib in combination with fulvestrant was well-tolerated. The recommended phase II dose is 50 mg twice daily on days 1-3, 8-10, and 15-17 q 28-day cycle with standard dose fulvestrant. Promising antitumor activity was observed. Correlative tissue evaluation of AURKA expression and other EMT biomarkers is underway. Funding: This work was funded by Takeda Oncology and supported by NIH Grant K12 CA90628 [TCH]. Citation Format: Haddad TC, D'Assoro AB, Suman VJ, Opyrchal M, Goetz MP, Ingle JN. A phase I trial to evaluate the safety of the addition of alisertib to fulvestrant in hormone receptor positive (HR+), advanced breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-04.

Abstract BACKGROUND: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis in patients with breast cancer. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A in breast cancer cells will lead to increased sensitivity to chemotherapy. RESULTS: Increased Aurora-A expression, as assessed by IHC staining, in patients with locally advanced disease was associated with worse prognosis. We show that in ER+ cell line, MCF7, activating MAPK pathway through overexpression of Raf1 leads to increase in Aurora-A activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to untransfected cells in MTT assays. This effect is even more exacerbated with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by specific inhibitor, Alisertib. This was further explored in triple negative (MDA-MB-231) or Her-2/neu expressing (SUM149) cell lines. These cells express higher levels of Aurora-A. Inhibition of Aurora-A activity in combination with paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are looking at a role of Aurora-A in activating SMAD5 and its role in resistance to chemotherapy. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. CONCLUSIONS: Inhibition of Aurora-A by a specific inhibitor, MLN8237, resulted in decreased SMAD5 expression and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be very useful in developing more effective treatments for breast cancer patients in neo-adjuvant, adjuvant and metastatic settings. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-07.

Tumor lysis syndrome (TLS) presenting in absence of chemotherapy is a rare occurrence. One of the true oncological emergencies, it can lead to significant morbidity and mortality. TLS is a phenomena usually associated with tumor cell death after treatment. The etiology of the spontaneous TLS is not well understood, which complicates the diagnosis. TLS is well known to oncologists but physicians outside of this specialty have little or no experience with this condition. Early recognition and treatment are the keys to limiting the sequela of the condition. Spontaneous tumor lysis syndrome is rare but presents added risks to the patient because of the potential for delayed diagnosis and no benefit of pretreatment. Diagnosis may be further delayed because this may be the first symptom of underlying malignancy. Therefore, it is imperative that all clinicians are familiar with the syndrome to assure timely recognition.