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Background: Whether axillary lymph node dissection (ALND) following sentinel lymph node biopsy (SLNB) could be spared in patients with initially clinically positive axilla after neoadjuvant chemotherapy (NAC) is still controversial even though recent studies indicate that axillary recurrence seems to be a rare event. Our aim is to find out whether omitting ALND could be oncologically safe in patients undergoing SLNB after NAC. Material and Methods: Of patients presented with c T1-4N1-3M0 disease, those undergoing SLNB after NAC were included in the prospective multicentre registry trial " MF18-03/BHWG" (ClinicalTrials.gov/NCT04250129). Cases with inflammatory breast cancer, distant metastases, pregnancy, bilateral breast cancer, or other cancers and those without adjuvant nodal radiotherapy were excluded from the study. The end points of the present report are the axillary nodal recurrence (AR) and locoregional recurrence (LRR) rates at a median follow-up more than 2 years, and determine factors associated with AR and LRR . The locoregional recurrences included ipsilateral, and contralateral axillary recurrences, infra-and supraclavicular recurrences, and recurrences in the mammaria interna region. Results: Between January 2018 to January 2021, 2358 patients with cN(+) disease, who became cN0 after NAC, and underwent SLNB, were analyzed. Median age was 47 (range, 21-86). Of those, the majority of patients had cT1-2 (80.5%) and N1 (80.3%) disease. Following NAC, half of the patients (50%) had breast conserving surgery, whereas the remaining half had mastectomy (50%). Of 2358 patients, 908 (38.5%) had ALND following SLN (ypN+, 85%) and 1450 (61.5%) underwent SLNB alone (ypN0, 72%). SLNB was performed by using the blue dye technique-alone in 66.6% of patients and by targeted axillary dissection in 659 patients (27.9%). Of those, 819 (34.8%) were HER2(+) and 373 (15.8%) were triple negative. The pCR rates for the axilla, breast and both for the axilla and breast were 50%, 35% and 28%, respectively. At a median follow-up time of 28 months (range, 12-62), the LRR, AR and isolated AR rates were 0.6% (n=14), 0.25% (n=6) and 0.13% (n=3), respectively. Furthermore, no significant difference could be found in LRR- and AR- rates between SLNB-alone and ALND groups regardless of the definitive nodal pathology (Table 1). Nodal recurrences were seen at a median of 12 months after the surgery. Of 6 cases with AR, 3 had synchronous local recurrences in breast, and 2 of them also had lung metastases in addition to local recurrence. All patients with AR were interestingly found to have HER2(+) or triple negative breast cancer at the initial diagnosis, and had residual invasive cancer in the breast surgical specimen. Logistic regression analyses revealed that patients with AR were significantly more likely to be younger than 45 (RR=7.81 ; 95% CI, 0.91-66.91) and have a cN2-3 (RR=4.1; 95% CI, 0.83-20.38), and non-luminal breast cancer (RR=12.47; 95% CI, 1.45-106.9) at the initial diagnosis (Table 2). Similarly, patients with LRR were more likely to present with cN2-3 disease (RR=3.09; 95% CI, 1.07-8.94) and non-luminal pathology (RR=6.27; 95%CI, 1.96-20.06) . Conclusion: This large prospective registry data also suggest that nodal recurrences can be detected at very low rates within 3 years after surgery in patients with clinically node-positive disease following NAC regardless of the extent of axillary surgery or nodal pathology as long as regional nodal radiation is provided. Since patients with early nodal recurrences have an agressive tumor biology with a potential of systemic recurrences, effective adjuvant systemic therapies should be considered in those with HER2(+) or triple negative residual breast cancer after surgery following adjuvant nodal radiation. Table 1. Local locoregoinal and systemic recurrences in cT1-4N1-3 patients with ypN0/ypN(+) diseases (n =2358) Table 2. Factors associated with axillary and locoregoinal recurrences (AR = axillary recurrences, LRR = locoregoinal recurrences, pCR = pathologic complete response) Citation Format: Neslihan Cabıoğlu, Hasan Karanlik, Mehmet Ali Gulcelik, Abdullah İgci, Mahmut Muslumanoglu, Havva Belma Kocer, Cihan Uras, Gokhan Giray Akgul, Mustafa Tukenmez, Serkan Ilgun, Didem Can Trabulus, Guldeniz Karadeniz Cakmak, Ahmet Dağ, Nilufer Yıldirim, Baha Zengel, Ebru Sen Oran, Kazim Senol, Halil Kara, Selman Emiroglu, M. Umit Ugurlu, Bulent Citgez, Yeliz Emine Ersoy, Atilla Celik, Ece Dilege, Yasemin Bolukbaşı, Niyazi Karaman, Gul Basaran, Aykut Soyder, Ayfer Kamali Polat, Gurhan Sakman, Serdar Ozbas, Ayse Altınok, Leyla Zer, Alper Akcan, Ibrahim Ali Ozemir, Levent Yeniay, N. Zafer Utkan, Lutfi Dogan, Mutlu Dogan, Mehmet Velidedeoglu, Beyza Ozcinar, Fazilet Erozgen, Abut Kebudi, Kemal Atahan, Vafa Valiyeva, Serdar Yormaz, Ali Sevinc, Cumhur Arici, Atilla Soran, Vahit Ozmen. PD15-01 AXILLARY NODAL RECURRENCE IS RARE IN PATIENTS WITH NODE-POSITIVE BREAST CANCER UNDERGOING SLNB FOLLOWING NEOADJUVANT CHEMOTHERAPY : EARLY RESULTS OF THE NEOSENTITURK-TRIAL/MF-18-03 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD15-01.

Background: BRCA1 and BRCA2 mutations are responsible for two thirds of hereditary BC. Germline genetic testing for BC susceptibility has evolved from a single-gene analysis to a multigene panel testing. Identification of a pathogenic mutation in BRCA and other panel represent a therapeutic opportunity today. Methods: We aimed to investigate clinical and pathologic characteristics of BC patients who were referred to our center between 2011-2019 and underwent BRCA1, BRCA2 or 26-gene inherited cancer panel testing based on NCCN criteria for hereditary breast/ovarian cancer testing. We analyzed the frequency of pathogenic mutations and its relationship with clinical and pathologic factors. Results: A total of 576 patients were identified. Among them 356 (63%) had their test in our university, 218 (38%) patients had their test at other centers. Sixty-six % (n:376) of patients had panel testing, 34% had only BRCA 1-2 mutation test. Median age was 42(22-87) and 5 patients were male. Ten % of patients had metastatic disease, 70% had early BC and 20% had locally advanced stage at the time of referral. The indication for genetic testing was family history in 169 (29%) patients, triple negative (TN) subtype in 100 (17%) patients and age Citation Format: Aysun Isiklar, Aykut Soyder, Kanay Yararbas, Cumhur Ekmekci, Hulya Yazici, Gul Basaran. Clinical and pathologic characteristics of Turkish breast cancer (BC) patients screened with BRCA1, BRCA2 or 26 - gene inherited cancer panel testing: Single institution experience [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-24.

Background: The MF07-01 trial is a multicenter phase III randomized controlled trial of treatment naive stage IV BC patients comparing loco-regional surgery (LRS) followed by appropriate systemic therapy (ST) versus ST alone. Aims: To evaluate the importance of loco-regional tumor burden and surgery on overall survival rate in patients with de novo stage IV breast cancer. Methods: At initial diagnosis patients were randomized 1:1 to LRS group or ST group. The surgery was a lumpectomy (L) or mastectomy (M) and sentinel lymph node biopsy (SLNB) ± axillary lymph node dissection (ALND). After surgery all patients received systemic treatment + endocrine treatment (ET) and Trastuzumab based on pathology results. The demographic, pathologic, and clinical characteristics of the patients were recorded. Results:274 patients were accrued; 138 in the LRS group and 136 in the ST group. The groups were comparable regarding age, BMI, HER2 neu, tumor type and size, histologic grade, and bone and visceral metastasis (all p>0.05). In the LRS group 36 patients (26%) had L+ALND, 92 patients (67%) had M+ALND and 10 patients (7%) had M+SLNB, respectively. The patients and tumor characteristicsPatients and Tumors Characteristics and Surgical TreatmentSurgerySystemic TherapyP ValueAge (mean /year±SD)51.8 ±12.651.5±13.6NSMedian follow-up (25%,75%)41.0 (24,54)37 (18,49) Tumor Size (%) T18.7 (12) NST252.2 (72) NST321.7 (30) NST417.4 (24) NSHistologic Grade (%) I4.4 (6)9.6 (10)NSII39.9 (55)31.7 (33)NSIII55.8 (77)58.9 (61)NS Surgical Treatment Lumpectomy+ ALND26 (36)--M + SLNB7 (10)--M + ALND67 (92)---SLNB17 (23)--ALND92.8 (128)--pN+89.1 (123)--30-day mortality1.4 (2)1.5 (2)0.98SLNB-Sentinel Lymph Node Biopsy; ALND-Axillary Lymph Node Dissection; M-Mastectomy The axillary positivity rate was 89.1%. There were 76 (55%) deaths in the LRS group and 101 (74%) in the ST group during the median 40 (20-51) month follow-up. Overall survival (OS) was 34% higher in the LRS group compared to the ST group (HR: 0.66, 95%CI 0.49-0.88: p = 0.005). Overall survival rate was higher in LN (+) (p=0.01), tumor size Conclusion: In this subgroup analysis, we observed that patients with high grade tumor, without skin or chest wall involvement and positive axilla who underwent surgery for primary breast tumor and axilla had better overall survival than ST in de novo stage IV breast. These results can be considered in clinical research design for stratification. Citation Format: Ozmen V, Ozbas S, Karanlik H, Muslumanoglu M, Igci A, Canturk Z, Utkan NZ, Ozaslan C, Evrensel T, Uras C, Aksaz E, Soyder A, Ugurlu UM, Col C, Cabioglu N, Bozkurt B, Sezgin E, Dagoglu T, Uzunkoy A, Dulger M, Koksal N, Cengiz O, Gulluoglu B, Unal B, Atalay C, Yildirim E, Erdem E, Salimoglu S, Sezer A, Koyuncu A, Gurleyik G, Alagol H, Ulufi N, Berberoglu U, Soran A. The importance of loco-regional tumor burden and surgery on survival in patients with de novo stage IV breast cancer; post-hoc analyses of protocol MF07-01 [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 P6-16-01.

Ewing sarcoma (ES) cells express high levels of the cell surface protein CD99, which is used for diagnosis of ES. More importantly, the inhibition of CD99 activity results in reduced growth of ES cells in vitro and in vivo. In an earlier publication, we have identified clofarabine and cladribine as selective inhibitors of CD99, which inhibited ES growth both in vitro and in vivo. Clofarabine and cladribine directly bound to CD99, inhibited its molecular interactions, and regulated CD99 specific intracellular signaling pathways. In order to gain further insight into how clofarabine may regulate cellular functions through inhibiting CD99, we utilized a phospho-kinase array to identify changes in phosphorylation levels of 43 proteins in response to clofarabine or CD99 antibody treatment. We identified ERK1/2-MSK1/2-CREB signaling axis as one of the signaling pathways downstream of CD99. These findings were validated in four different ES cell lines and in xenograft lysates that were harvested from clofarabine-treated mice compared to control mice. The phosphorylation events induced by clofarabine treatment was significantly diminished when CD99 was knocked down either by siRNA-mediated knockdown or CRISPR/Cas9-mediated genomic disruption. Time-course experiments on ES cells showed that clofarabine triggers a very rapid signaling cascade through CD99. Cytarabine, a structurally similar pyrimidine analog and an inhibitor of the EWS-FLI1 transcriptional activity that has been failed in clinical trials on ES patients, did not activate ERK1/2, MSK1/2 or CREB phosphorylation in ES cells. In order to test whether the observed changes in phosphorylation levels are required for cell death, we screened a kinase inhibitor small molecule library for their ability to rescue clofarabine-induced death of ES cells. We identified TG100-115 (PI3K inhibitor) and rebastinib (c-abl inhibitor) as the most potent kinase inhibitors that showed significant reversal of clofarabine-induced ES cell death, suggesting that clofarabine-induced death of ES cells requires specific phosphorylation cascades. We then discovered that CD99 can be co-immunoprecipitated with FGFR1 and clofarabine treatment of ES cells results in phosphorylation of FGFR1. In conclusion, we discovered a novel mechanism of action for clofarabine and cladribine in that their selective cytotoxic effect on ES is through inhibiting CD99 on the cell surface, and it is not dependent on their ability to inhibit DNA synthesis. CD99 inhibition resulted in increased phosphorylation of FGFR and two downstream signaling pathways (PI3K/Akt and MEK1/ERK1). Inhibition of PI3K pathway rescues clofarabine-induced ES cell cytotoxicity. These findings provide additional mechanistic support for repurposing clofarabine and cladribine for ES indication. Note: This abstract was not presented at the meeting. Citation Format: Haydar Celik, Levent Dusunceli, Anna Molotkova, David V. Allegakoen, Erin J. Conn, Jeff R. Petro, Jeffrey A. Torestky, Aykut Uren. CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2868.

The term epithelial to mesenchymal transition (EMT) and its reverse (MET) describe the dynamic and reversible metamorphosis of cells from a highly organized to a loose migratory phenotype through cytoskeletal reorganization causing cellular plasticity. EMT/MET are inherent to normal embryonal development and wound healing, but cancer cells hijack the underlying mechanisms to enable oscillations between proliferation, invasion and migration that cause metastasis, the major killer of cancer patients. Recently, fluctuations of the oncoprotein EWS-FLI1 were identified to drive EMT/MET in Ewing sarcoma. We previously demonstrated that, in presence of EWS-FLI1, transcriptional co-activators MRTFB and TAZ are largely blocked from associating with TEAD and its target genes keeping tumor cells in a poorly migratory, highly proliferative state. In contrast, under EWS-FLI-low conditions, MRTFB and TAZ associate with YAP/TEAD complexes on chromatin resulting in cytoskeletal target gene activation and phenotypic transition to a highly migratory and low proliferative state. We therefore hypothesized that pharmacologic inhibition of YAP/TAZ/TEAD protein interaction should prevent Ewing sarcoma cells from EMT and consequently interfere with their metastatic potential. Verteporfin is a small molecule safely used in the treatment of age-related macular degeneration. Independent of its photosensitizing activity exploited in ophthalmology, it is a YAP/TAZ pathway -blocking compound. In vitro treatment of EWS-FLI1-low Ewing sarcoma cells with verteporfin resulted in decreased YAP/TAZ/TEAD complex formation in the nanomolar range, reversal of the de-repression of a EWS-FLI1 controlled EMT transcriptional signature, and inhibition of tumor cell migration in a Boyden chamber assay. Upon orthotopic implantation of TC71 Ewing sarcoma cells into the tibial crest of SCID beige mice, intra-peritoneal treatment of mice with 25mg verteporfin/kg/day before and after amputation of the affected limb led to a drastic decrease of lung metastases without affecting primary tumor growth and without obvious general toxicity. Therefore, YAP/TAZ pathway blockade holds promise as a potential metastasis-preventive strategy in the treatment of Ewing sarcoma patients with primary localized disease. Citation Format: Lisa Bierbaumer, Anna M. Katschnig, Branka Radic-Sarikas, Jeffrey R. Petro, Karin Mühlbacher, Dave N. Aryee, Anna R. Pötsch, Sandra Högler, Lukas Kenner, Aykut Uren, Heinrich Kovar. Targeting YAP/TAZ pathway inhibits Ewing sarcoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1049.

Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy of unknown primary origin. ES cells express high levels of CD99, a cell surface protein routinely used as a marker antigen in the histologic diagnosis of ES. A substantial body of evidence makes CD99 an attractive therapeutic target for patients with ES. We identified two structurally similar FDA-approved nucleoside (purine) analogues, clofarabine and cladribine, as specific CD99 inhibitors in a Biacore screening experiment. In validation experiments, both drugs exhibited selective cytotoxicity toward ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A membrane-impermeable derivative of clofarabine, clofarabine-5′-triphosphate, showed a similar cytotoxicity on ES cell lines, suggesting inhibition of CD99 activity on cell surface as a likely mechanism by which the drug exerts its cytotoxic action without any effect on DNA metabolism. Clofrabine also inhibited the growth of three different ES xenografts in vivo. We performed a phosphokinase array to identify the mechanism behind how clofarabine may regulate cellular functions through inhibiting CD99. Mitogen- and stress-activated kinases 1 and 2 (MSK1/2) were the most significantly activated proteins by both CD99 antibody and clofarabine. Four of the 7 proteins with increased phosphorylation were related to MSK1/2, which were MSK1/2 itself, its substrates CREB and c-Jun, and its upstream kinase ERK1/2, thus representing the most likely intracellular pathway responsible for cell death due to CD99 inhibition. Clofarabine induced a significant increase in phosphorylation levels of MSK1/2 in cohort of ES xenografts, supporting its use as a potential pharmacodynamic marker of CD99 inhibition. We compared the activity of clofarabine with cytarabine, a structurally similar pyrimidine analog that has been identified as an inhibitor of EWS/FLI1 transcriptional activity but failed in clinical trials on ES patients. Cytarabine did not activate MSK1/2 phosphorylation in ES cells, suggesting that clofarabine may function through alternative mechanisms on ES cells that are different than cytarabine. Overall, our findings suggest that clofarabine directly binds to CD99 and inhibits its oncogenic activity through a novel mechanism in ES, and therefore it is a good candidate for early-phase clinical trials in children with ES. Citation Format: Haydar Çelik, Marika Sciandra, Maria Cristina Manara, Jeffrey Torestky, Katia Scotlandi, Aykut Üren. Inhibition of CD99 activity by clofarabine as a novel therapeutic for Ewing sarcoma involves a novel molecular mechanism that is different than cytarabine [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A06.

Ewing sarcoma (ES) is an aggressive malignancy of bone and soft tissue that affects predominantly children and young adults with a high propensity to metastasize and poor prognosis. ES cells express high levels of a cell surface protein CD99, which is routinely used as a marker antigen in the histological diagnosis of ES, and contributes to its pathogenesis. We performed a phospho-kinase array to uncover specific cellular responses evoked by CD99 inhibition in ES cells. The treatment of ES cells with CD99 antibody or a small molecule CD99 inhibitor (clofarabine) resulted in strong activation of mitogen- and stress-activated kinases 1 and 2 (MSK1/2) as well as its substrate CREB and its upstream kinase ERK1/2. These findings were further confirmed with western blot analysis in ES cells treated with either different CD99 antibodies or clofarabine. The treatment of osteosarcoma (OS) cells with clofarabine or the forced expression of CD99 in OS cells did not change their phosphorylation levels, suggesting that CD99-mediated ERK1/2-MSK1/2-CREB signaling cascade is specific to ES cells. Interestingly, knock-down of the CD99 protein in ES cells led to a significant decrease in ERK1/2 phosphorylation without any effect on the downstream phosphorylation of MSK1/2 and CREB. Cytarabine, a structurally similar pyrimidine analog that has been identified as an inhibitor of EWS/FLI1 transcriptional activity but failed in clinical trials on ES patients, did not activate ERK1/2-MSK1/2-CREB axis unlike clofarabine in ES cells. These findings suggest that clofarabine functions through alternative mechanisms on ES cells that are different than cytarabine. Clofarabine induced a significant increase in phosphorylation levels of MSK1/2 in a small cohort of TC-71 xenografts, supporting its use as a potential pharmacodynamic marker of CD99 inhibitor activity. In conclusion, our findings suggest that CD99 regulates ERK1/2-MSK1/2-CREB axis in ES, which represents an intracellular pathway that may be responsible for CD99's biological activities in ES. Citation Format: Haydar Celik, Erin J. Conn, David V. Allegakoen, Jeff R. Petro, Jeffrey A. Toretsky, Aykut Uren. CD99 regulates ERK1/2-MSK1/2-CREB axis in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3176.

Cervical carcinoma, the second leading cause of cancer deaths in women worldwide, is associated with human papillomavirus (HPV). HPV-infected individuals are at high risk for developing cervical carcinoma; however, the molecular mechanisms that lead to the progression of cervical cancer have not been established. We hypothesized that in a multistep carcinogenesis model, HPV provides the initial hit and activation of canonical Wnt pathway may serve as the second hit. To test this hypothesis, we evaluated the canonical Wnt pathway as a promoting factor of HPV-induced human keratinocyte transformation. In this in vitro experimental cervical carcinoma model, primary human keratinocytes immortalized by HPV were transformed by SV40 small-t (smt) antigen. We show that smt-transformed cells have high cytoplasmic β-catenin levels, a hallmark of activated canonical Wnt pathway, and that activation of this pathway by smt is mediated through its interaction with protein phosphatase-2A. Furthermore, inhibition of downstream signaling from β-catenin inhibited the smt-induced transformed phenotype. Wnt pathway activation transformed HPV-immortalized primary human keratinocytes even in the absence of smt. However, activation of the Wnt pathway in the absence of HPV was not sufficient to induce transformation. We also detected increased cytoplasmic and nuclear staining of β-catenin in invasive cervical carcinoma samples from 48 patients. We detected weak cytoplasmic and no nuclear staining of β-catenin in 18 cases of cervical dysplasia. Our results suggest that the transformation of HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve as a screening tool in HPV-positive populations to detect malignant progression.

Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy that affects predominantly children and adolescents with a high propensity to metastasize and poor prognosis. CD99 is a transmembrane cell surface protein that is highly expressed on ES cells, and routinely used as a marker for histological diagnosis of ES. We screened small molecule libraries for their binding to recombinant CD99 protein and subsequent selective inhibition of ES cell growth. We identified two structurally similar FDA-approved nucleoside analogues, clofarabine and cladribine that selectively inhibited the growth of ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A significant negative correlation was found in human cell lines between CD99 expression and IC50 values for clofarabine and cladribine. Both drugs inhibited CD99 dimerization and its interaction with downstream signaling components cyclophilin A and PKA-RIIα as well as led to reduced ROCK2 protein expression and migration in ES cells. A membrane-impermeable analog of clofarabine showed similar cytotoxicity in ES cells, suggesting that it can function through inhibiting CD99 alone without any effect on DNA metabolism. Clofarabine and cladribine led to a significant increase in hypodiploid DNA content of ES cells, which was diminished by suppression of CD99 expression. Both drugs drastically inhibited anchorage-independent growth of ES cells, but clofarabine was more effective in inhibiting ES xenografts. Finally, the screening of a set of chemotherapy drugs revealed a synergy for the combination of anti-CD99 drugs and dasatinib in ES cells, which may translate into increased survival and reduced toxicity. Overall, our findings suggest that clofarabine is a good candidate for early phase clinical trials in children with ES. Citation Format: Haydar Celik, Marika Sciandra, Bess Flashner, Elif Gelmez, Neslihan Kayraklıoğlu, David V. Allegakoen, Jeff R. Petro, Erin J. Conn, Sarah Hour, Jenny Han, Lalehan Oktay, Purushottam B. Tiwari, Mutlu Hayran, Maria Cristina Manara, Jeffrey A. Toretsky, Katia Scotlandi, Aykut Uren. Discovery of first-in-class small molecule CD99 inhibitors for targeted therapy of Ewing sarcoma [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 1933. doi:10.1158/1538-7445.AM2017-1933

One of the most significant challenges in creating more potent, less toxic treatments for patients is to identify new cancer therapeutic targets that distinguish the malignant from normal cells. EWS-FLI1 is a well-established Ewing sarcoma (ES) oncogene that has the potential to be an ideal therapeutic target by directly impacting malignant cells. We have previously reported the discovery and characterization of YK-4-279, an enantiomer-specific inhibitor of EWS-FLI1, which has been demonstrated to induce apoptosis, inhibit EWS-FLI1 transcription, block RNA helicase A co-immunoprecipitation with EWS-FLI1, and result in alternative splicing to mimic EWS-FLI1 knockdown. Continuous efforts in structure-guided medicinal chemistry has yielded TK-216, an analog of YK-4-279 inhibitor of EWS-FLI1, which is 3-4 fold more potent with excellent drug-like properties. TK-216 potently inhibits the proliferation of ES cells. Induces apoptosis in a dose -dependent manner as measured by caspase-3 activity in multiple ES cell lines with distinct translocation variants. The effects of TK-216 on alternative splicing (AS) were further validated using genes including ARID1A, CLK1, CASP3, PPFIBP1 and RUNX2. The splicing pattern was similar between TK-216 and YK-4-279. In addition to the in vitro activity of TK-216 , we show that TK-216 displays anti-tumor activity in a number of ES xenograft models. In summary, TK-216, a novel, first-in-class therapeutic which directly inhibits EWS-FLI1, offers a promising approach for the treatment of Ewing Sarcoma and is currently in Phase 1 clinical trials in patients with relapsed or refractory Ewing Sarcoma (clinicaltrials.gov - NCT02657005). Citation Format: Saravana P. Selvanathan, Eric Moseley, Garrett T. Graham, Katti Jessen, Brian Lannutti, Aykut Üren, Jeffrey A. Toretsky. TK-216: a novel, first-in-class, small molecule inhibitor of EWS-FLI1 in early clinical development, for the treatment of Ewing Sarcoma [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 694. doi:10.1158/1538-7445.AM2017-694

Ezrin is a member of the ezrin, radixin, moesin (ERM) protein family of membrane-cytoskeleton linkers. Ezrin has been implicated in many essential cellular functions including cell adhesion, motility, maintenance and determination of cell shape, cell proliferation and apoptosis, regulation of ion channels, morphogenesis and signal transduction. Ezrin promotes invasive and migratory capabilities of cancer cells. A high level of ezrin expression is associated with poor clinical outcome and metastatic behavior of pediatric solid tumors including osteosarcoma and rhabdomyosarcoma as well as multiple other tumor types. Ezrin, therefore, could be a promising molecular target for the prevention and treatment of cancer metastasis. We previously discovered two small molecule inhibitors, NSC305787 and NSC668394, which bind directly to ezrin and inhibit its activity in mediating the invasive phenotype of osteosarcoma cells in multiple in vitro and in vivo assays. In this study, we expand on our previous findings by demonstrating that NSC305787-treatment but not NSC668394 significantly reduces pulmonary metastasis in a genetically engineered mouse model of osteosarcoma. We assessed the pharmacokinetics of compounds in mice and demonstrated that NSC305787 has a more favorable pharmacokinetic profile compared with NSC668394. In order to uncover ezrin-mediated biological pathways that can be used for a specific pharmacodynamic marker(s) of response to ezrin inhibition, we profiled global gene expression in osteosarcoma cells after treatment with inhibitors. We identified several commonly up-regulated genes with functional relevance to integrated stress response, implicating that a common underlying mechanism may be shared by these compounds. We further validated the microarray data through extensive testing using real-time qPCR and verified the specificity of the transcriptional response using another novel ezrin inhibitor MMV667492 that we have identified recently from the MMV400 “Malaria Box” library. The effect of ezrin inhibitors on the expression of stress genes was recapitulated by siRNA-mediated depletion of ezrin. The up-regulation of stress genes was much weaker in cells with reduced ezrin levels compared to wild-type cells, indicating the specificity of the compounds on ezrin-mediated cellular responses. Analysis of the expression of stress genes in white blood cells and skin of NSC305787-treated mice demonstrated up-regulation of the DDIT4/REDD1, suggesting that DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of response to ezrin inhibition. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive, may have important functions regulating gene expression and inhibition of ezrin activity by NSC305787 in osteosarcoma could be an attractive therapy to prevent clinically significant metastasis. Citation Format: Haydar Çelik, Gülay Bulut, Jenny Han, Garrett T. Graham, Tsion Z. Minas, Erin J. Conn, Sung-Hyeok Hong, Gary T. Pauly, Mutlu Hayran, Xin Li, Metin Özdemirli, Ayşe Ayhan, Michelle A. Rudek, Jeffrey A. Toretsky, Aykut Üren. Ezrin inhibition up-regulates stress response gene expression and blocks osteosarcoma metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2446.

Alternative splicing has been implicated as an oncogenic process and provides both a categorization of cancer as well as an opportunity for more effective targeted treatments. Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing, leading to oncogenic protein isoforms. Many proteins that participate in altering splicing have regions of low complexity amino acids, such as serine-arginine (SR) repeats. These SR repeats cause these proteins to have unique biophysical properties leading to phase separation and putatively formation of speckles in the nucleoplasm. EWS-FLI1 has low complexity regions and multiple validated direct interactions with spliceosome components. In addition, our work shows that EWS-FLI1 has novel direct binding to mRNA. We hypothesize that EWS-FLI1, the Ewing sarcoma (ES) oncoprotein, modulates post-transcriptional gene regulation through both novel protein interactions (trans-acting factors) and by directly binding to transcribed mRNA (cis-elements). Our initial results led us to build a novel EWS-FLI1 protein interaction model that identifies both direct and indirect binding throughout the spliceosome. Specific protein interactions of EWS-FLI1 include a newly discovered direct binding partner, DDX5, which is part of the U1 subunit. In addition, direct interactions with both U2 and U5 were identified. We have validated a small molecule probe, YK-4-279, as an enantio-specific inhibitor of EWS-FLI1 that directly blocks its binding to both RHA and p68 (DDX5). Reduction of EWS-FLI1 and YK-4-279 treatment alters exon skipping and intron inclusion events identified from RNA-seq. We then identified splicing events that were similarly reverted by both EWS-FLI1 reduction and YK-4-279, including CLK1, PPFIBP1, and CASP3. In addition, YK-4-279 reverts alternative splicing changes seen in the presence of EWS-FLI1, which was not an effect of altering RNA pol II activity. Our recent unpublished work explores the biophysical interactions of EWS-FLI1 in nucleoplasm. Inhibition of EWS-FLI1 protein interactions by YK-4-279 appear to alter nuclear speckle formation and may, through this mechanism, lead to altered spliceosomal activity. To establish that EWS-FLI1 alternative splicing changes the function of a protein through expression of a different isoform, we selected TERT as an enzyme with an isoform shift in the presence of EWS-FLI1 . When EWS-FLI1 is reduced, a γ-isoform of hTERT is synthesized that lacks exon 11. The γ-isoform of hTERT is reported by others to have increased hTERT activity and we confirm this in cell-based assays following EWS-FLI1 reduction. The cellular and oncogenic implications of this enzymatic activity change is under investigation. To confirm the relevance of splicing modulated by EWS-FLI1 occurring in human tumors, we determined splicing patterns from 75 ES patients and matched splicing patterns for 10 genes in cell line models. Exon-specific expression loci in this comparison were statistically significant for their overlap. In addition, overall gene expression levels did not stratify for overall survival, however, principal component isoform specific analyses did segregate survivors. In metastatic patients, this was significant (p=0.05) while in localized patients it approached significance (p=0.1). This work illuminates potentially critical oncogenic splicing shifts as well as provide a potential stratification mechanism for ES patients. A splicing map specific to ES will define and inform potential functional aspects of alternative isoforms. The biophysical role of EWS-FLI1 in creating or maintaining speckles is an ongoing investigation. Assessment of aberrant splicing driven by EWS-FLI1 may inform oncogenesis and reciprocally, EWS-FLI1 activities may inform splicing mechanisms. Citation Format: Jeffrey Toretsky, Garrett Graham, Uta Dirksen, Hayriye Verda Erkizan, Elizabeth Lawlor, Aykut Uren, Saravana Selvanathan. Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A39.

Introduction: Previous reports of carefully selected patients presenting with stage IV breast cancer (BC) suggest that surgery on the primary tumor may result in improved survival, but this remains unproven. The MF07-01 trial is a phase III randomized controlled trial of BC women with distant metastases at presentation who receive loco-regional (LR) treatment for intact primary tumor compared with those who do not receive such treatment. Aim: The primary objective of the trial is to compare overall survival (OS) in women treated with or without initial LR resection prior to systemic therapy for de novo stage IV BC. Materials and Methods: At the discretion of the surgeon, LR treatments consisted of either mastectomy or breast conserving surgery with level I-II axillary clearance in clinically or sentinel lymph node positive patients. Radiation therapy to whole breast was required following breast conserving surgery. At the discretion of the medical oncologist standard systemic therapy of either endocrine treatment or chemotherapy (plus trastuzumab for HER2 +) was given to all patients either immediately after randomization (no surgery group) or after surgical resection of the intact primary tumor (surgery group). After consideration of previous retrospective studies, the assumed OS difference between the two groups was determined to be 18% (35% in LR treatment group versus 17% in no-LR treatment group). A 10% drop out rate including lost to follow up was assumed. By using a one sided log-rank test with a 95% confidence (α = 0.05) and a 90% power (β = 0.9), sample size calculation revealed that 271 patients were needed to be randomized. Results: There were 140 women in the surgery group and 138 in the no-surgery group. The mean follow up time was 21.1 ± 14.5 months. The mean age was 51.6 ± 13.2 years and the groups were comparable regarding age, BMI, ER/PR, Her 2, Triple negative, tumor type and size between the groups (all p>0.05). Metastatic patterns included bone only in 45.7%, organ except bone in 28.8%, and bone plus organ in 25.5%. There were a total of 86 (31%) deaths. At 54 months the survival rate was 35% in the surgery group and 31% in the no surgery group (p = 0.24). However, OS was statistically higher in bone only, ER/PR positive and patients younger than 50 years but was lower in the triple negative patients (p Conclusion: In early follow-up of this trial comparing surgery of the primary tumor with no surgery in stage IV BC at presentation OS was similar but there were important subgroup differences; in particular those with solitary bone metastasis have a significant survival benefit and patients with bone metastasis only have a trend toward improved survival with initial surgery. Further follow-up will expand on these important findings. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S2-03.

Ezrin is a member of the ezrin-radixin-moesin (ERM) family of actin-membrane linker proteins that play key roles in regulating cell shape, movement, adhesion and signal transduction pathways. The expression of ezrin is linked to the metastatic progression in several cancers including osteosarcoma (OS). We discovered a small molecule, NSC305787, that inhibits ezrin activity and metastatic phenotype both in vitro and in vivo. We hypothesized that the anti-metastatic effects of NSC308787 could be mediated through preventing specific protein-protein interactions involving ezrin. In this study, we used affinity pull-down coupled with mass spectrometry-based proteomic approach to unravel putative ezrin interactors that are competed away by NSC305787. We identified a number of candidate ezrin binding proteins that are associated with metastatic behavior and implicated in the regulation of stress granule dynamics and protein translation initiation. We selected DDX3, a DEAD-box RNA helicase, as a candidate for further analysis. We confirmed that ezrin directly binds to DDX3. Depletion of ezrin protein expression by RNA interference in several cancer cell lines resulted in substantial reduction in DDX3 protein levels without affecting its transcription, which suggested that ezrin is required for post-transcriptional maintenance of DDX3 in the cell. Paradoxically, recombinant ezrin specifically inhibited the RNA duplex unwinding activity and stimulated the ATPase activity of DDX3. Our data suggest that ezrin regulates the translation of mRNAs with 5′ secondary structure through DDX3, at least in part, through maintaining its intracellular protein level and/or modulating its unwinding and ATPase activities. Therefore, our findings suggest that a novel function of ezrin regarding the regulation of mRNA translation exists that is independent from its classical role as a cytoskeletal cross-linker protein at the plasma membrane. Citation Format: Haydar Celik, Kamal P. Sajwan, Amrita V. Pai, Ben J. Marsh, Yasemin Saygideger Kont, Said Rahim, Jenny Han, Tsion Minas, Jeffrey A. Toretsky, Aykut Uren. Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3269. doi:10.1158/1538-7445.AM2015-3269

The cytoskeletal cross linker protein ezrin is a member of the ezrin-radixin-moesin (ERM) family and plays important roles not only in cell motility, cell adhesion, and apoptosis, but also in various cell-signaling pathways. Ezrin interacts with EGFR in the cell membrane and involves in cell motility events, but little is known about the effects of this interaction on the EGFR signaling pathway. We investigated the role of Ezrin in EGFR signaling and nuclear trafficking in non-small cell lung cancer (NSCLC) cell lines. The ligand induced interaction between Ezrin and EGFR was evaluated by immunoprecipitation (IP) and immunofluorescence (IF) in H292 and A549 cells. Ezrin levels were reduced using siRNA in these two cell lines. Downstream signaling protein phosphorylation and nuclear localization of EGFR were detected after EGF treatment. Expressions of nuclear EGFR target genes were evaluated by qPCR. Endogenous Ezrin was found in a complex with EGFR in IP and IF. When Ezrin protein expression was inhibited, phosphorylation levels of EGFR at Y1068, Y1101 and Y845 were reduced as well as phosphorylation levels of downstream signaling pathway proteins ERK and STAT3. Cell fractionation revealed that EGFR nuclear translocation after EGF treatment significantly reduced in Ezrin-knockdown cells. Further, mRNA levels of EGFR target genes AuroraK-A, COX2, Cyclin D1 and iNOS were decreased in Ezrin-knockdown A549 cells. Small molecule ezrin inhibitors showed strong synergy with EGFR inhibitors in cytotoxicity assays. These results suggest that Ezrin has a role as an enhancer in the EGFR pathway and targeting ezrin may potentiate anti-EGFR based therapies in NSCLC. Citation Format: Yasemin Saygideger Kont, Haydar Celik, Hayriye V. Erkizan, Tsion Minas, Jenny Han, Jeffrey Toretsky, Aykut Uren. Ezrin enhances signaling and nuclear translocation of the epidermal growth factor receptor in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2015-54

The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH2 terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4– and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies. (Cancer Res 2006; 66(20): 9862-9)

RNA helicase A (RHA), a member of the DEXH box helicase family of proteins, is an integral component of protein complexes that regulate transcription and splicing. The EWS-FLI1 oncoprotein is expressed as a result of the chromosomal translocation t(11;22) that occurs in patients with the Ewing's sarcoma family of tumors (ESFT). Using phage display library screening, we identified an EWS-FLI1 binding peptide containing homology to RHA. ESFT cell lines and patient tumors highly expressed RHA. GST pull-down and ELISA assays showed that EWS-FLI1 specifically bound RHA fragment amino acids 630 to 1020, which contains the peptide region discovered by phage display. Endogenous RHA was identified in a protein complex with EWS-FLI1 in ESFT cell lines. Chromatin immunoprecipitation experiments showed both EWS-FLI1 and RHA bound to EWS-FLI1 target gene promoters. RHA stimulated the transcriptional activity of EWS-FLI1 regulated promoters, including Id2, in ESFT cells. In addition, RHA expression in mouse embryonic fibroblast cells stably transfected with EWS-FLI1 enhanced the anchorage-independent phenotype above that with EWS-FLI1 alone. These results suggest that RHA interacts with EWS-FLI1 as a transcriptional cofactor to enhance its function. (Cancer Res 2006; 66(11): 5574-81)

Ewing sarcoma is an aggressive neoplasm affecting bone and soft tissue occurring almost exclusively in children and young adults. It is characterized by a distinct chromosomal translocation involving EWS and FLI1 genes making targeted therapy feasible since the resulting oncoprotein, EWS-FLI1, is exclusively expressed in the cancerous cells. The fusion protein has been shown to confer its tumorigenic phenotype through aberrant transcriptional activity, splicing, and protein-protein interactions. Previous work from our lab has identified a small molecule inhibitor, YK-4-279 that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing Sarcoma cell lines. Uncertainty about the cell of origin for Ewing sarcoma contributes to the lack of in vivo models for the disease, which has hindered preclinical study efforts. Torchia et al. (Mol. and Cell. Bio. 2007. 27(22): 7918-7934) has generated a transgenic mouse model where expression of EWS-FLI1 was targeted to the bone marrow, spleen, and liver by crossing Rosa26-loxP-stop-loxP-EWS-FLI1 mice with mice that express cre recombinase under Mx1-promoter, which responds to pIpC. Expression of EWS-FLI1 in these tissues induce a rapid expansion of primitive myeloid progenitors leading to rapid leukemia development. We used this mouse model to assess the efficacy of YK-4-279 in disrupting oncogenic activity of EWS-FLI1 in vivo. The mice were injected with 1mg of pIpC at an age of one month to induce leukemia. Once an increase in white blood cell count is observed following pIpC injection, the mice were randomly assigned to a treatment or control groups. Treatment of the transgenic mice daily, five times a week, intraperitoneally with 75mg/kg YK-4-279 vs. vehicle led to improved overall survival. Median survival for control group was 10.5 days while for treatment group was 24 days (p=0.045). Mice treated with 75mg/kg YK-4-279 showed significant reduction of disease burden within two weeks as monitored by weekly White Blood Cell count (DMSO vs. 75mg/kg YK Week 1: p=0.023 and Week 2: p=0.004). Mice treated with 150mg/kg YK-4-279 showed even more reduction of disease burden where one week after treatment WBC for DMSO group increased on average by 6495 cells/ul of blood while for YK-4-279 group it decreased by 1278 cells/ul of blood. Two weeks after the treatment started, the WBC for DMSO group increased even more by an average of 17,500 cells/ul of blood while the disease was stabilized for 150mg/kg YK-4-279 treated group (DMSO vs. 150mg/kg YK-4-279 Week 1: p-value Citation Format: Tsion Z. Minas, Jenny Han, Sung-Hyeok Hong, Jeffrey Toretsky, Aykut Uren. YK-4-279 is effective in treating EWS-FLI1 induced myeloid/erythroid leukemia in a transgenic mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-197. doi:10.1158/1538-7445.AM2014-LB-197

Osteosarcoma (OS) is the malignant tumor of bone that is most common in children and adolescents. OS is characterized by a high propensity for metastasis, especially to lungs, which is the main cause of death. The membrane-cytoskeleton cross-linker ezrin plays a key role in the metastatic progression of OS. In our earlier studies, we discovered that a small molecule, NSC305787, directly binds to ezrin and inhibits its function related to metastatic phenotype. In order to gain a better insight into the molecular mechanisms of NSC30787 action, an affinity pull-down coupled mass spectrometry was conducted to find the putative ezrin-interacting proteins whose interaction with ezrin is decreased or lost in the presence of NSC305787. Detailed mass spectrometry analysis identified 78 proteins after filtering for the potential background contaminants. Out of 78 proteins, 9 proteins were known ezrin-binding proteins including ezrin, radixin, moesin, polyadenylate-binding protein 1 (PABP-1), Na(+)/H(+) exchange regulatory cofactor (NHERF1/EPB-50), calpains, epidermal growth factor receptor kinase substrate 8 (EPS8), protein kinase C ioto (PKC-ioto) and focal adhesion kinase (FAK). For the remaining 69 proteins, we immunoprecipitated ezrin from K7M2 OS cells, and then western blotted for 8 selected functionally relevant proteins in order to test the validity of our approach. Of these 8 candidate proteins, Co-IP experiments verified binding of ezrin to 5 novel binding partners including DDX3 RNA helicase, caprin-1, AEG-1/MTDH/LYRIC, Rho guanine nucleotide exchange factor 2 (GEFH1) and catenin delta-1 (p120-catenin). NSC305787 effectively reduced the binding of DDX3 RNA helicase, AEG-1/MTDH/LYRIC and GEFH1 to ezrin in Co-IP experiments. Direct binding of ezrin to DDX3 RNA helicase was also demonstrated by surface plasmon resonance analysis using Biacore instrument. In conclusion, our results suggest that NSC305787 can modulate ezrin function through blocking its interaction with crucial proteins involved in adhesion, migration, invasion and survival. We also identified novel ezrin interactors that are involved in different levels of mRNA metabolism suggesting a completely new molecular function of ezrin that is independent from its classic role as a membrane cytoskeletal cross-linker at the plasma membrane. Thus, these results highlight novel molecular mechanisms for ezrin in cancer metastasis. Citation Format: Haydar Celik, Jenny Han, Sung-Hyeok Hong, Gulay Bulut, Jeffrey Toretsky, Aykut Uren. NSC305787 inhibits specific protein–protein interactions involving ezrin in osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2014-3979

Ezrin is a member of the ERM (Ezrin, Radixin, Moesin) family of proteins that functions as a cross-linker between the plasma membrane and actin cytoskeleton, thus allowing the cell to regulate cytoskeletal dynamics in response to external stimuli. Ezrin plays important roles in cell motility, membrane trafficking, morphogenesis, adhesion, survival and apoptosis. Ezrin also participates in crucial signal transduction pathways. Overexpression of ezrin contributes to the aggressive cancer behavior in various tumor types especially by enhancing the metastatic phenotype. Thus, ezrin represents a promising molecular target for cancer therapy. In our earlier studies, we have established that NSC305787 can directly bind to ezrin and inhibit its activity, which results in reduced metastasis. Since NSC305787 shares significant structural similarities with some anti-malarial drugs, we tested 400 anti-malarial compounds including 200 drug-like compounds and 200 probe-like compounds for their potential to directly bind to ezrin protein. We used surface plasmon resonance technology (Biacore) to screen direct binding. In this initial screen, 12 compounds demonstrated binding to ezrin over background values and selected as primary hits. Detailed SPR analysis of primary hits resulted in 7 compounds with comparable KD values to that obtained with our lead compound NSC305787. Primary hits were also evaluated for inhibition of the cell motility of murine osteosarcoma (OS) cells K7M2 and K12 that express high and low levels of ezrin, respectively. Cell motility and chemotaxis assays were conducted in parallel with viability assays to rule out general toxicity. Selected anti-malarial compounds were effective in inhibiting the motility of OS cells with high ezrin compared to cells with low ezrin. In conclusion, several anti-malarial drugs may have new indications in cancer therapy by inhibiting ezrin. Citation Format: Haydar Celik, Abraham Kallarakal, Daisy Colon-Lopez, Jeffrey Toretsky, Jurgen Bosch, Aykut Uren. Evaluation of anti–malarial compounds as inhibitors of ezrin in osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2014-3987

The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in a majority of prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP and fusion-negative PC-3 tumors. Animals were treated with YK-4-279 and its effect on tumor size, lung metastasis and survival were observed. YK-4-279 treatment resulted in decreased tumor size in the LNCaP cohort only. A reduction in tumor metastasis to the lungs was observed in compound treated LNCaP animals with comparable tumor sizes. YK-4-279 also increased survival in LNCaP mice. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 was reduced as well. ETS fusion-negative PC-3 xenografts were unresponsive to the compound. YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. As part of this study, we also established that (S) -YK-4-279 is the active enantiomer in prostate cancer cells. (S) -YK-4-279 binds to ETV1 with comparable kinetics as the racemic mixture and inhibits ETV1 activity. (R) -YK-4-279 does not demonstrate ETV1 binding or inhibition. Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and should be further evaluated for its clinical applications in prostate cancer. Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Perrer Tosso, Haydar Celik, Yasemin Sayedigar-Kont, Milton Brown, Colm Morrissey, Jeffrey Toretsky, Aykut Üren. YK-4-279 is a small molecule inhibitor of ETV1 and inhibits metastasis in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2949. doi:10.1158/1538-7445.AM2014-2949

Chromosomal translocations involving the ETS family of transcription factors are found in a majority of prostate cancers, including the most clinically aggressive forms. These translocations produce a chimeric gene, which fuses the promoter region of an androgen responsive gene to the coding region of ETS factors, most frequently ETV1 or ERG. Over-expression of ETS factors in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously injected with LNCaP and PC-3 xenografts. Mice were treated with YK-4-279 and its effect on primary tumor size and lung metastasis was observed. YK-4-279 treatment resulted in reduced metastasis of the tumor from primary site to lungs. YK-4-279 also affected expression of ETV1 target genes such as MMP7 and MMP13. ETS fusion-negative PC-3 xenografts were unresponsive to YK-4-279. Our results demonstrate that YK-4-279 decreases tumor growth and inhibits metastasis in fusion-positive prostate cancer cell xenografts. Therefore, YK-4-279 may have an impact on preventing metastasis in prostate cancer patients, which is a leading cause of death. Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Yali Kong, Milton L. Brown, Colm Morrissey, Jeffrey A. Toretsky, Aykut Uren. YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5453. doi:10.1158/1538-7445.AM2013-5453

Metastatic castration-resistant prostate cancer (CRPC) has a poor prognosis and remains a significant therapeutic challenge. Gene fusions between the TMPRSS2 gene and members of the ETS family of transcription factors (ERG, ETV-1 ETV-4 and 5) have been found in prostate cancer (PCa). In this study we hypothesized that the overexpression of ERG in PCa promotes the expression of common genes and pathways in nuclear ERG positive hormone sensitive primary PCa and metastatic CRPC tumors. Our objective was to identify these common genes and pathways. Using LuCaP PCa xenografts developed at the University of Washington, specimens obtained at radical prostatectomy, and metastatic specimens obtained at rapid autopsy, 3 sets of tissue microarrays were made from 24 LuCaP PCa xenograft lines, 114 radical prostatectomies and 155 metastases from 50 autopsy patients who died from CRPC (with up to 4 metastases from each patient). Nuclear ERG expression was analyzed by immunohistochemistry (IHC). In addition, to characterize the molecular features of ERG expressing PCa, 24 LuCaP PCa xenografts, 24 radical prostatectomies, and 78 corresponding metastases were also assessed by Agilent gene expression analysis. Consistent with previously published FISH and qRT-PCR analyses, IHC revealed that 7 of 24 LuCaP xenograft lines were nuclear ERG positive. Fifty-one of 114 primary prostate cancers were nuclear ERG positive. However, among the 155 CRPC metastases from 50 autopsy patients, 15 of 155 metastases had nuclear ERG positivity. These 15 metastases represent 7 of 50 patients with at least 1 nuclear ERG positive metastasis. This suggests that nuclear ERG expression is less frequent in CRPC than in hormone sensitive primary prostate carcinomas. Furthermore, not all metastases in a given patient were nuclear ERG positive. In addition, gene expression data was generated from 24 LuCaP PCa xenografts, 24 radical prostatectomies and 78 CRPC metastases, which were grouped into ERG positive and ERG negative metastases based on nuclear ERG positivity in the same tissue specimen by IHC. The gene expression profiles of the ERG positive and ERG negative cancer tissues are being analyzed to identify ERG regulated genes and ERG regulated pathways of interest. This study is a comprehensive analysis of ERG expression and downstream effectors of ERG in xenograft models of PCa, primary PCa and CRPC. Our data suggest that ERG expression is less frequent in CRPC and nuclear ERG status can vary from site to site within a CRPC patient. The gene expression studies will further define the role of ERG in PCa. Citation Format: Martine Roudier, Ilsa Coleman, Xiaotun Zhang, Roger Coleman, Lisly Chéry, Lisha Brown, Bryce Lakely, Celestia Higano, Lawrence D. True, Paul H. Lange, Shiv Srivistava, Aykut Üren, Eva Corey, Robert L. Vessella, Peter S. Nelson, Colm Morrissey. Characterizing the molecular features of ERG positive tumors in primary and castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5122. doi:10.1158/1538-7445.AM2013-5122

Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic transformation. RHA is a DExH box RNA helicase family member. RHA has critical roles modulating transcription, splicing and translation. RHA also functions as a scaffolding protein in multi-protein complexes including BRCA1, DICER, EGFR, POL2R, TOPO2. Our previous work demonstrated that YK-4-279 inhibited the protein-protein interaction of RHA, with EWS-FLI1 and led to cellular apoptosis. The biochemical mechanism remained cryptic, so we investigated the effect of EWS-FLI1 upon the helicase activity of RHA. Methods: Full-length purified recombinant RHA from insect cells. Infrared-labeled double stranded RNA was used as a substrate to measure helicase activity of RHA. We included full-length recombinant EWS-FLI1 in to the helicase reaction to test the effect of EWS-FLI1. Results: RHA unwinds double stranded RNA in the presence of ATP with Km value of 5.322 nM. We also showed that RHA manifests reannealing activity of single stranded RNA to double stranded RNA in the absence of ATP. Unlike ADP, ATP and additionally the non-hyrolysable gamma-S-ATP prevent reannealing activity of RHA. A molecular modeling of RHA structure predicted the surface exposure of the region (between 823-832aa), which EWS-FLI1 interacts with. The function of this region is not well described but presumed to support helicase activity. EWS-FLI1 interferes RNA helicase activity of RHA in a dose dependent manner. Moreover, the reduction of RHA protein level exerted cytotoxic effect on Ewing Sarcoma cells. Decreased RHA protein level changed EWS-FLI1 and its target gene expression profile. IGFBP3, GRK5, p21, and PRR3 gene expression levels increased massively. RPLA1 and NR0B1 mRNA levels were also increased by moderately in these cells. The change in the profile may contribute to apoptosis. Conclusions: The purified recombinant RHA is very active in vitro helicase assays. We demonstrated that RHA's helicase activity might depend on ATP binding but not ATP hydrolysis. The protein partnering as well as posttranslational modification of RHA might modulate the helicase and/or reannealing activity of RHA, which may augment oncogenesis of EWS-FLI1. This is the first time that a protein partner reduces the helicase activity of RHA. Further experiments are ongoing that investigate whether YK-4-279 blocking of EWS-FLI1 binding to RHA will restore the helicase activity of RHA as part of it contribution to cell death. Citation Format: Hayriye Verda Erkizan, Kamal Sajwan, Maksymilian Chruszcz, Jeffrey Schneider, Sarah E. Gamble, Aykut Uren, Wladek Minor, Radhakrishnan Padmanabhan, Jeffrey Toretsky. EWS-FLI1 reduces RNA Helicase A activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2747. doi:10.1158/1538-7445.AM2013-2747

Our previous studies have demonstrated the association between Ezrin and the metastatic biology of pediatric sarcomas including osteosarcoma (OS) and rhabdomyosarcoma. Mechanistic studies exploring this association have shown that Ezrin expression enhances the survival of metastatic cells upon their arrival to the secondary metastatic site. In order to better understand this role in metastasis, we undertook two non-candidate analyses of Ezrin function including a microarray subtraction of high and low Ezrin expressing cells and a proteomic approach to identify proteins that bind the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis we found Ezrin to be part of a ribonucleoprotein complex and to bind with poly A binding protein 1 (PABA1; PABPC1). Using luciferase reporter-based assays, we have shown that OS cells expressing high levels of Ezrin are able to translate mRNAs containing a stem loop 5′ UTR structure, so called “weakly translated mRNAs,” more efficiently than low Ezrin OS cells. This finding suggests that Ezrin's contribution to the metastatic phenotype may be due in part to enhanced translation of specific mRNAs during metastasis that are normally expressed at low levels outside of the metastatic context. Ongoing studies will now assess the ability of Ezrin to enhance the expression of specific mRNAs in 3-dimensional contexts more relevant to cancer cell growth in vivo. We have developed stable high and low Ezrin osteosarcoma cell lines that express a GFP reporter with or without a complex 5′ UTR stem loop structure in the mRNA transcript and with and without protein destabilizing elements. Initial studies have shown that the addition of the 5′ UTR structure and destabilizing elements progressively limits GFP reporter expression. We plan to confirm that this reduction in expression is due to reduced translation and to assess whether 3-D culture conditions or the in vivo environment enhance expression of these tunable reporters of translation in an Ezrin-dependant manner. We expect these results to provide a novel mechanistic basis to consider how Ezrin may contribute to metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4190. doi:1538-7445.AM2012-4190

Though advancements in chemotherapy and surgical techniques have ameliorated treatment of primary osteosarcoma (OS), the metastatic phenotype remains a clinical challenge. Overall five-year survival rates for patients with localized OS have improved to 60-70%, yet survival rates of patients with metastasis remains at 20-30% with mortality linked to metastatis-induced respiratory failure. Therefore, targeting fundamental molecular events that lead to metastasis may yield significant benefit to patients with OS. Accumulating evidence from clinical samples and pre-clinical animal models suggests that ezrin is a key regulator in the metastasis of OS cells. Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins and a critical component for cell motility, adhesion and shape. We have recently identified that a small molecule, NSC 668394 acts as a potent and selective inhibitor of ezrin function and inhibits migration in both in-vitro and in-vivo models. Moreover, suppression of ezrin phosphorylation by NSC 668394 significantly reduced the metastatic behavior in cellular and animal models and has thus emerged as an important lead inhibitor. Consequently, we conducted a series of structure-activity-relationship (SAR) studies that monitored direct binding; OS cell migration and HUVEC monolayer invasion in ‘real-time’ with surface-plasmon resonance and electrical impedance technology. From our 2nd generation library, we have designed novel candidate inhibitors which feature enhanced ezrin-binding affinity, and improved anti-migration and anti-invasion activities. In addition to their potency against the different stages of OS metastasis, these compounds possess other desirable attributes for development including good druggable physical-chemical characteristics and are currently undergoing further preclinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3906. doi:1538-7445.AM2012-3906

Chimeric transcription factors have long been considered ideal anti-cancer targets since they are only present in tumor cells, however, their lack of enzymatic activity has placed them in a category of ‘undruggable’ proteins. The EWS-FLI1 fusion protein of Ewing Sarcoma (ES) has been validated as an anticancer target both alone and as a partner of RNA Helicase A (RHA). Our prior work identified the small molecule YK-4-279 as a specific inhibitor of EWS-FLI1 by blocking the interaction with RHA. Blocking this interaction leads to apoptosis. Data suggests that relatively long exposures of drug are necessary to keep EWS-FLI1 and RHA apart. We have modeled YK-4-279 treatment using cell lines (TC71, TC32, A4573, SK-ES, and RD-ES) to establish the duration of YK-4-279 exposure that leads to apoptosis. Apoptosis was measured by caspase-3 cleavage. A validated plasma detection method allows for HPLC measurement of YK-4-279. Pharmacokinetic (PK) models of YK-4-279 for both intraperitoneal (IP) and intravenous (IV) administration were developed in SD rats and BL6 mice. ES cell lines were exposed to YK-4-279 over a time-course, followed by a caspase-3 activity assay that demonstrated a minimum of 16 hours exposure was necessary to achieve maximal apoptosis using either 3 or 10 microM compound. A dose-dependency assay demonstrated that while apoptosis could be achieved with 30 - 80 microM YK-4-279 after 4 hours of treatment, caspase-3 activity was less than or equal to 3 microM for 16 hours. Rat PK modeling demonstrated a t1/2 of 30 minutes following IV administration. BL6 mice demonstrated similar kinetics to the rat. SCID/bg mice, which are necessary for tumor efficacy studies, demonstrated approximately 50% faster clearance than either rat or BL6 mice. Absolute bioavailability for IP administration was 41%. Models that use cell culture based targets for plasma levels and duration of exposure will be created to optimize IP dosing regimen. The optimized IP dosage and dosing intervals will be evaluated in tumor bearing animals in order to advance development of YK-4-279. The results of these studies will be used to guide toxicology studies in animals. In addition, pharmacodynamics models are being developed to compare YK-4-279 levels with functional activity. The combined results of these investigations will lead to human clinical trials for this first-in-chemical class, first-in-mechanism drug candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-316. doi:1538-7445.AM2012-LB-316

Osteosarcoma is the predominant primary bone cancer in children with a low survival rate due to pulmonary metastasis. Research on the molecular mechanism driving pulmonary metastasis implicates the cytoskeletal protein ezrin as the critical component of the disease pathology. Ezrin links the actin cytoskeleton to the plasma membrane proteins, and its overexpression is associated with poor survival in patients and increased invasion in osteosarcoma cell lines. We used surface plasmon resonance technology to screen small molecule libraries for compounds directly binding to ezrin. Two molecules, NSC305787 and NSC668394, were chosen based on their ezrin binding affinities and ability to inhibit ezrin in multiple functional, biochemical, cellular, and in vivo assays. To test the anti-metastatic activity of these molecules in vivo, we utilized a tissue-specific transgenic mouse model expressing Cre recombinase to excise the Retinoblastoma (Rb) and p53 tumor suppressor genes in osteoblast progenitor cells. These animals develop spontaneous osteosarcoma with 100% penetrance and high frequency of liver and lung metastases, mimicking human disease. We tested the anti-metastatic potential of ezrin inhibitors that we discovered, NSC305787 and NSC668394, in this clinically relevant mouse model. We started the treatment on one group of animals at 2 months of age before any detectable tumor is present and on a second group of animals only after detection of smallest palpable tumor. Animals in each group were randomly assigned to one of three treatment options: DMSO (Control), NSC305787, or NSC668394. Animals were observed for primary tumor growth and metastasis formation. All animals were euthanized when primary tumor volume reached 2.0cm3, when animals had severe cachexia, or when animals showed signs of pulmonary insufficiency. Mice treated with NSC305787 and NSC668394 did not show any changes in the growth of their primary tumors and experienced similar survival times. However, our results suggest that NSC305787 inhibits osteosarcoma metastasis to lungs as confirmed by histopathological analysis. Therefore, when combined with adjuvant chemotherapy and surgical resection of the primary tumor, targeting ezrin with small molecules may yield a positive outcome and greater survival in a clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4874. doi:1538-7445.AM2012-4874