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Introduction: Ripretinib (Rip) is a kinase inhibitor with broad preclinical activity against mutant KIT. Based on the INVICTUS trial, Rip was approved for patients with Gastrointestinal Stromal Tumors (GIST) after treatment with 3 or more kinase inhibitors. Most patients in this ≥4th-line setting progress on Rip within one year. Here, we characterized Rip-progressing GIST samples to identify resistance mechanisms. Methods: Progressing lesions in 25 patients were analyzed by NGS after Rip failure. KIT mutations (muts) were recapitulated by gene editing. Activities of Rip, sunitinib (SU), and novel TKIs were characterized by cell viability assays and immunoblot. Mutagenesis experiments were performed using ENU. SU- and Rip-resistant cell lines were pooled and treated with various regimens, with clonal composition deconvoluted by cDNA-based amplicon sequencing. Results: 19/25 Rip-progressing GISTs displayed muts in the ATP-binding pocket (ATP-BP; e13/14). Four of these had pre-existing muts in the activation loop (AL; e17/18), which were confirmed to be in-cis with the primary and ATP-BP muts (triple in cis; TIC). Mutagenesis screens using a double KIT-mutant GIST line (e11 + AL) as a starting point confirmed that TIC-muts are a predominant escape mechanism when treated with Rip. A GIST subline (T1-triple) with TIC-muts in e11, e18 (A829P), and e13 (V654A) was highly resistant to Rip (GR50 > 2µM). Structural analyses of ATP-BP muts suggest steric interference and loss of van der Waals interactions that impede Rip binding and thereby confer Rip resistance. Another 3/25 Rip-progressing GISTs harbored pathogenic KIT muts in e9 only. A novel GIST cell line with primary KIT e9 mut (T1-e9) was 14-fold less sensitive to Rip than isogenic e11-driven cells (GR50 = 115 vs 8nM). Notably, adding a typical AL mut to T1-e9 (T1-e9-N822K) sensitized the cells to Rip (GR50 = 20nM). Immunoblots showed >95% inhibition of phospho-KIT in AL-mutant cell lines at Rip 100nM, whereas inhibition was weaker in T1-e9 (77%) and T1-V654A (46%), and absent in T1-triple. A compound screen of FDA-approved kinase inhibitors identified Nintedanib (NIN) as the most active compound against T1-triple. Clonal outgrowth assays of mixed cultures revealed SU (93% inhibition), and a weekly switch between Rip and either SU (96%) or NIN (79%) as the most effective inhibitors of pooled cell growth. Conclusions: KIT e9 primary muts and e13/14 (ATP-BP) secondary muts are enriched in post-progression biopsies following Rip treatment. KIT TIC-muts are novel frequent events driving GIST clinical progression and confer a high degree of resistance. Strategies to overcome resistance may include combinations or sequences of approved drugs, and novel drugs that more efficiently inhibit TIC-mutant KIT. Citation Format: Thomas Mühlenberg, Johanna Falkenhorst, Tom Schulz, Benjamin S. Fletcher, Alina Teuber, Dawid Krzeciesa, Isabella Klooster, Jonas Lategahn, Wen-Bin Ou, Meijun Lundberg, Margaret von Mehren, Susanne Grunewald, Alicia I. Tüns, Mehdi Brahmi, Michael C. Heinrich, Cesar Serrano, Hans-Ulrich Schildhaus, Sonja Sievers, Jürgen Treckmann, Lydia Wilson, Chandrajit P. Raut, Adrian Marino-Enriquez, Suzanne George, Daniel Rauh, Jonathan A. Fletcher, Sebastian Bauer. ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3887.

Gastrointestinal stromal tumor (GIST) is the most common GI sarcoma and is generally initiated by KIT or PDGFRA mutations which are compelling therapeutic targets for tyrosine kinase inhibitors (TKI). However, the emergence of secondary mutations results in clinical resistance to available TKIs. GIST progression is driven by genomic events which incrementally target the p16-CDK4/6-RB1 and p14-TP53-RB1 pathways to create CDK4/6 and CDK2 oncogenic co-dependency. Based on limited efficacy of single-agent CDK4/6-inhibitor (CDK4/6i) therapy in GIST, we evaluated strategies of co-targeting CDK2 and CDK4/6. Multiplexed protein imaging (via Immuno-SABER) was validated for the detection of cell cycle regulator aberrations in GIST clinical samples (N=18), 7 of which were TKI-resistant, and including 3 patients in whom multiple metastases were analyzed. The impact of various CDK perturbants using CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in GIST cell lines and murine xenografts. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. Abnormal expression/biallelic inactivation of CDKN2A/p16, RB1, and TP53 were identified in 7 (39%), 2 (11%), and 2 (11%) of 18 GISTs, respectively. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. Since 5 of 7 RB1-intact advanced GISTs had co-dysregulation of the CDK2 and CDK4/6 pathways, we evaluated co-inhibition of CDK2 and CDK4/6 in vitro and in vivo which inhibited cell proliferation (P Citation Format: Inga-Marie Schaefer, Meijun Z. Lundberg, Matthew L. Hemming, Sinem K. Saka, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven Gygi, George D. Demetri, Ewa Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, Wen-Bin Ou, Jonathan A. Fletcher. Response and resistance to CDK2 and CDK4/6 inhibition in GIST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5648.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107–17. ©2017 AACR.

The MDM2-p53 pathway plays a prominent role in various cancer pathogenesis. Nutlin-3, a small-molecule antagonist of MDM2, inhibits proliferation in cancer cells with wild-type p53 by blocking the interaction of MDM2 and p53. However, different cancer cells differ greatly sensitivity of Nutlin-3 because of MDM2 amplification and/or p53 mutation status. Herein, we evaluate the sensitivity of Nutlin-3 in different cancer cell lines including four mesothelioma cell lines, one breast cancer line, and one leiomyosarcoma cell line with nonmutant p53 expression, two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one control mesothelioma cell line JMN1B harboring p53 point mutation. Unexpectedly, Nutlin-3 treatment induced cyclin D1 expression accompanied with inhibition of MDM2-p53 interaction in nonmutant cancer cell lines. Additive effects were obtained through a coordinated attack on MDM2-p53 interaction and cyclin D1, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that lentiviral mediated cyclin D1 knockdown or drug inhibition, and block of MDM2-p53 interaction, in cancer cell lines containing wild-type p53, induced greater anti-proliferative effects, compared to either intervention alone. Further results demonstrated that upregulation of cyclin D1 after Nutlin-3 treatment to some extent was dependent on expression and ubiquitin E3-ligase activity of MDM2. These compelling anti-proliferative responses indicate that combination inhibition of cyclin D1 and MDM2-p53 interaction warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53. Citation Format: Wenbin Ou, Weicai Chen, Jiaqing Zhu, Grant Eilers, Xuhui Li, Peipei Yang, Hailong Li, Fanguo Meng, Jonathan Fletcher. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. [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 2707. doi:10.1158/1538-7445.AM2015-2707

Liposarcoma (LPS) is the most common sarcoma of humans. There are no systemic therapeutic regimens known to improve survival when complete surgical resection is not feasible, underscoring the need for an improved molecular understanding of LPS to stimulate the development of effective targeted therapies. The MDM2-p53 pathway plays a prominent role in WDLPS pathogenesis, with the vast majority of human tumors harboring either MDM2 amplifications or p53 mutations. Nutlin-3, a small-molecular antagonist of MDM2, inhibits cell proliferation via blocking the interaction of MDM2-p53 in LPS with wild-type p53. We wonder whether MDM2 overexpression due to amplification or/and mutant p53 still plays a crucial oncogenic role in LPS containing p53 mutation. Herein, we developed isogenic liposarcoma lines in which the parental forms were MDM2 amplification and p53 wild-type, whereas sublines had mutation p53 expression, showed Nutlin-3 resistance. mRNA sequencing and immunoblotting showed that MDM2 and CDK4 were overexpression in the parental lines and sublines, whereas MDM2 and CDK4 expression was low in mesothelioma and GIST cell lines. HDAC inhibitor (HDACi, SAHA and LBH589) treatment resulted in the dephosphorylation and degradation of MDM2 and p53, but little affected CDK4 and JUN expression, irrespective of p53 mutational status in LPS with MDM2 amplification, and in a mesothelioma cell line JMN1B with p53 mutation, but not two mesothelioma lines containing normal MDM2 level and wild-type p53. Our findings indicate that regulation of wild-type 53 degradation by HDACi is MDM2 amplification-dependent. HDAC inhibition by SAHA and LBH589 had a substantially effect on LPS and mesothelioma proliferation and survival associated with upregulation of the PTEN and p21, inhibition of cell proliferation marker cyclin A and PCNA expression, induction of G1 or G2 phase arrest; induction of apoptosis showing an increase of caspase 3/7 activity and expression, PARP cleavage, and the sub-G1 apoptotic population. Moreover, we characterize biological functions of MDM2-p53 axis in nutlin-3-sensitive and nutlin-3-resistant LPS, showing MDM2 knockdown in the four LPS lines and p53 knockdown in the three mutant-p53 LPS lines resulted in anti-proliferative effects. Additive effects were obtained through a coordinated attack on MDM2 and p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that MDM2 and p53 knockdown, in LPS cell lines containing the p53 mutation, induced greater cell apoptosis and anti-proliferative effects, compared to either intervention alone, which is comparable to the effects seen after HDAC inhibition by SAHA and LBH589. These compelling pro-apoptotic and anti-proliferative responses indicate that HDAC inhibition warrants clinical evaluation as a novel therapeutic strategy in LPS, including nutlin-3 resistant sublines with the p53 mutation. Note: This abstract was not presented at the meeting. Citation Format: Wen-bin Ou, Hailong Li, Li Liu, Shengmei Zhou, Ye Kuang, Ziqin Yan, Zhe Jiang, Si Shi, Fanguo Meng, Qing Sheng, Haimeng Zhou, Jonathan A. Fletcher. HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status. [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 5136. doi:10.1158/1538-7445.AM2014-5136

Lung cancer is the leading cause of cancer deaths in the United States with an estimated >160,000 deaths and >200,000 newly diagnosed cases each year. Over the past 20 years, the survival rate for patients remains extremely poor (∼15% over 5 years). Discovery of novel pathway involved in the pathogenesis of the lung cancer can provide new therapeutic opportunities. Stem cell factor SALL4 has recently merged to be a diagnostic tool and therapeutic target for acute myeloid leukemia and liver cancer. However, very little is known on its role in lung cancer. In order to investigate the functional role of SALL4 in lung cancer tumorigenesis and progression, we first performed gene expression profile analysis and revealed that SALL4 expression level was significant higher in all types of non-small cell lung cancer samples compared to normal lung tissue control, which was validated by IHC. Furthermore, we performed loss of function study using multiple shRNAs. Our data showed that knockdown of SALL4 could induce dramatic cell growth arrest in multiple lung cell lines including large cell carcinoma H661 and adenocarcinoma H522, H292 and PC9, but not in HCC827 cells with none/low SALL4 level. In order to elucidate the downstream pathway of SALL4 in lung cancer, we first conducted gene expression microarray using SALL4 knockdown H661 cells and cells treated with scramble control vectors. Combination of one-way ANOVA analysis and pathway analysis using Pathway Interaction Database (PID) showed that both the EGFR and IGF1R signaling pathways were affected by SALL4 knockdown in H661 lung cancer cells. Using western blot, we further validated that SALL4 depletion could lead to a decrease in both EGFR and IGF1R protein expressions in lung cancer cells. However, we did not observe any changes at the mRNA level for these two receptors upon SALL4 depletion. This leads us to wonder whether SALL4 could affect these two molecules indirectly. It has been well established that receptor tyrosine kinase (RTK) including EGFR and IGF1R can be negatively regulated by Cbl RING ubiquitin ligases. Of interest, our microarray data also showed that loss of SALL4 led to a dramatic upregulation of Cbl-b, a member of Cbl RING ligases, which was further validated by using real-time RT-PCR. In addition, SALL4 downregulation also dramatically increased Cbl-b protein expression in these lung cancer cells. Therefore, we propose that SALL4 can indirectly positively affect the expression of EGFR and IGF1R proteins through Cbl-b. In summary, we have demonstrated that SALL4 expression is aberrantly elevated in non-small cell lung cancer cells. Furthermore our data suggested that SALL4 plays an essential role in lung cancer tumor growth and maintenance by affecting both EGFR and IGFR signaling pathways via regulating Cbl-b expression. Citation Format: Ailing Li, Wenbin Ou, Jonathan Fletcher, Kol Jia Yong, Henry Yang, Ross Soo, Daniel Tenen, Li Chai. Targeting SALL4 in lung cancer through a novel Cbl-b/IGF1R&EGFR axis. [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 3389. doi:10.1158/1538-7445.AM2014-3389

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with this asbestos-associated disease. The receptor tyrosine kinases EGFR and MET are activated in subsets of mesothelioma, suggesting these kinases might represent novel therapeutic targets. However, clinical trials have not shown activity for EGFR inhibitors in mesothelioma. We had previously demonstrated activation of multiple receptor tyrosine kinases, including EGFR, MET, and AXL, in individual mesothelioma cell lines, targeting HSP90 suppressed the multiple activated RTKs in mesothelioma cell lines, resulting in apo-apoptotic and anti-proliferative effects. Thus, we hypothesized that a coordinated network of multi-RTK activation contributes collectively to activate PI3-K/AKT/mTOR and/or RAF/MAPK signalings for the tumorigenesis of mesothelioma. Herein, we demonstrate abnormal activation of the PI3-K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, as compared with normal mesothelial cells. Multiple receptor tyrosine kinases, including EGFR, MET, and AXL, in individual mesothelioma cell lines, collectively activated survival signaling intermediate AKT, but not MAPK. The anti-proliferative responses of the coordinate inhibition of these multi-kinase signaling were comparable with suppression of PI3-K/AKT/mTOR pathway. Dual targeting AKT/mTOR signaling has substantially greater effect on mesothelioma proliferation and survival, compared to inhibition of individual activated kinases and downstream signaling alone, suggesting that no one activated RTK is the predominant oncogenic control mechanism in mesothelioma and PI3-K/AKT/mTOR is a crucial survival pathway. The AKT/mTOR signaling inhibition by BEZ235 resulting in pro-apoptotic and anti-proliferative effects in mesothelioma, was associated with the blockage of MDM2-p53 interaction. Citation Format: Shengmei Zhou, Li Liu, Weijiang Hu, Hailong Li, Fanguo Meng, Haimeng Zhou, Jonathan A. Fletcher, Wenbin Ou. Dual targeting of the AKT/mTOR signaling pathwayinhibits mesothelioma: Targeted therapies against multiple activated receptor tyrosine kinases. [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 2168. doi:10.1158/1538-7445.AM2013-2168

Ovarian cancer is a leading cause of cancer death among women in Western Europe and the United States, which has the highest mortality rate of all gynecologic malignancy. Although more than 70% patients have increased 5-year survival rates after surgery followed by chemotherapy and second-line therapies, the low overall cure rates and the intolerable side effects of systemic chemotherapy asks for the development of novel and more effective pharmacological interventions. The increasing evidences suggest that receptor tyrosine kinase (RTK) activation participates in the oncogenic progression from nonneoplastic mesothelial lining of the ovaries or the fallopian tube epithelium to epithelial ovarian cancer. The RTKs, including EGFR, ERBB2, PDGFR, VEGFR and MET, are activated in subsets of ovarian cancer, suggesting that these kinases might represent novel therapeutic targets. However, clinical trials have not or just partially shown benefit to ovarian cancers treated with EGFR, ERBB2, or PDGFR inhibitors. Despite multiple RTK activation in ovarian cancer pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. We hypothesized that a coordinated network of multi-RTK activation is important for the tumorigenesis of ovarian cancers. Herein, we demonstrate co-activation of multiple RTKs (EGFR, ERBB2, ERBB4, MET and/or AXL) in individual ovarian cancer cell lines and primary tumors. We also show that coordinate inhibition of this multi-kinase signaling has substantially greater effect on ovarian cancer proliferation and survival, compared to inhibition of individual activated kinases. The inhibition of this multi-RTK signaling by HSP90 suppression results in profound pro-apoptotic and anti-proliferative effects, and is associated with the inactivation of RTK downstream PI3-K/AKT/mTOR and RAF/MAPK signaling. These studies suggest that anti-multiple RTK strategy could be useful in the treatment of ovarian cancer. 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 1092. doi:1538-7445.AM2012-1092

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors due to acquisition of secondary mutations in the KIT kinase domain, genomic amplification of KIT, or activation of alternate oncoproteins. Approximately 10% of KIT-dependent GIST metastases lose KIT expression at time of clinical progression during imatinib therapy. We performed whole transcriptome sequencing in KIT-expressing GIST vs. KIT-negative GIST, and thereby identified strong cyclin D1 expression only in KIT-negative GISTs. KIT-negative GISTs showed complete loss of the GIST biomarker, PKCθ, and restoration of PKCθ expression in these cells resulted in loss of Cyclin D1 expression. Lentivirus-mediated CCND1 (Cyclin D1) short hairpin RNA (shRNA) knockdown resulted in profound anti-proliferative and pro-apoptotic effects in KIT-negative GIST cell lines, and was associated with activation (dephosphorylation) of the tumor suppressor Rb and upregulation of the p27 CDK inhibitor. c-Jun expression levels paralleled the Cyclin D1 expression in the GIST lines, and c-Jun shRNA knockdown resulted in downregulation of cyclin D1 in the KIT-negative GIST lines, and was accompanied by profound anti-proliferative effects, including p27 upregulation, and pro-apoptotic effects as evidenced by PARP cleavage. By contrast, cyclin D1 and c-Jun silencing showed little anti-proliferative and pro-apoptotic effects in KIT-positive, KIT-dependent GIST lines. These findings show that cyclin D1 and c-Jun overexpression serve a crucial oncogenic role in KIT-independent GISTs, including those enabling clinical resistance to imatinib or sunitinib targeted kinase inhibitor therapies. Our findings also show that PKCθ downregulation and c-Jun upregulation coordinately enable cyclin D1 overexpression in KIT-independent GISTs. These novel findings highlight the relevance of c-Jun/cyclin D1 as novel therapeutic targets in GISTs that have lost KIT oncoprotein expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 952. doi:10.1158/1538-7445.AM2011-952

Endometrial carcinoma is the most common gynecologic malignancy in the world. While most patients (85%) are surgically cured with a hysterectomy, unfortunately, the remaining 15% cases tend to have persistent or recurrent cancer that are resistant to current radiation and chemotherapy. Discovery of novel pathway(s) involved in the pathogenesis of the endometrial cancer can provide new therapeutic opportunities. The transcription factor SALL4 is required for early embryonic development and for the maintenance of human and murine embryonic stem cell (ESC) pluripotency. Our previous studies have shown that SALL4 also plays an essential role in leukemogenesis. We hypothesize that SALL4 may play a similar role in the pathogenesis of sold tumors. We initiated our study by surveying the expression of SALL4 in various solid tumors. We found that 50% of endometrial carcinoma patients had aberrant expression of SALL4 compared to that of normal tissue by immunohistochemistry staining (n=30). This finding was further validated by quantitative real-time PCR (N=8). In addition, we evaluated SALL4 expression in several endometrial cancer cell lines, such as poorly-differentiated cell line AN3CA, KLE, and moderately differentiated cell line, HEC-1A. Western blot showed that SALL4 was highly expressed in AN3CA and KLE, but not detectable in HEC-1A cells. To test whether SALL4 plays a functional role in this disease, we chose to use loss-of-function approach. Knock-down of SALL4 in AN3CA and KLE was achieved by SALL4 specific shRNA. On day 5 after infection, down-regulation of SALL4 induced a high degree of cell death (50%). MTT proliferation assay also confirmed cell growth rate was significantly decreased compared to that from scramble control. We further investigated whether this decreased viable cells was due to apoptosis or cell cycle arrest or both. Western blot showed that knock-down of SALL4 could induce caspase-dependent apoptosis, as evidenced by detection of more caspase 3 cleavage products compared to that of scramble control. In addition, BrdU cell cycle assay showed that knock-down of SALL4 led to decreased S phase along with G2 arrest. Using ChIP-on-chip assay, we have found that SALL4 could bind to c-MYC promoter region. Consistently, our western blot show c-MYC was dramatically decreased after SALL4 knocking down in AN3CA cells compared to scramble control. In summary, for the first time our data show that SALL4 is aberrantly expressed both in primary endometrial carcinoma and poorly differentiated endometrial cell lines. Loss-of -function study demonstrated that SALL4 play an essential role in cell survival in endometrial carcinoma by activating c-MYC pathway. Further study will reveal whether SALL4 can be used as a novel therapeutic target for those advanced, progressive or recurrent endometrial carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2011-3047

Mesothelioma is a chemotherapy-resistant neoplasm, as is evident in the dismal survival for patients with this asbestos-associated disease. Conventional chemotherapies and radiation therapy have only limited efficacy, and improved survival will require development of novel and more effective pharmacological interventions. Although p53 genomic mutations are uncommon in mesothelioma, we hypothesized that functional p53 inactivation (resulting from NF2-dysregulated FAK activation) might be a frequent event. Using proteomic methods, with tandem mass spectrometry analyses of mesotheliomas after double immuno-affinity purifications for p53 and phosphotyrosine, we find that FAK is consitutively activated in 10 of 10 mesothelioma cell lines and 9 of 9 mesothelioma surgical specimens, and associated with p53 in 6 of 6 mesothelioma cell lines. In four mesotheliomas with non-mutant p53, FAK silencing by lentiviral short hairpin RNA (shRNA) induced expression and phosphorylation of p53 at Ser15. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared to MESO924, MESO257, MESO296, and MESO428 cells, which have nonmutant p53. Additive effects were obtained through a coordinated attack on p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that FAK knockdown and MDM2 inhibition (nutlin-3) induced greater p53 expression, cell apoptosis, anti-proliferative effects and cell cycle arrest, compared to either intervention alone. These findings highlight novel therapeutic opportunities in mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5031.