Thanasak Sueblinvong

Water-fat MRI for assessing changes in bone marrow composition due to radiation and chemotherapy in gynecologic cancer patients.

To assess the feasibility of using fat‐fraction imaging for measuring marrow composition changes over large regions in patients undergoing cancer therapy.

Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

Predicting response to the anti-estrogen fulvestrant in recurrent ovarian cancer

BACKGROUND Anti-estrogen therapy appears to have efficacy in a subset of ovarian cancers, as demonstrated in multiple phase II studies. Identifying sensitive patients early in treatment may allow for targeted, low-toxicity primary therapy or prevention of recurrence. We have previously demonstrated that the likelihood of response to letrozole could be improved by patient selection based on estrogen-pathway marker expression. We sought to identify ovarian cancer biomarkers that might indicate sensitivity to fulvestrant, an estrogen receptor antagonist. METHODS Tissue samples from the primary tumors of patients enrolled in a phase II study of fulvestrant for the treatment of multiply-recurrent ovarian cancer were embedded randomly in a tissue microarray (TMA). Estrogen receptor alpha (ERα) expression was assessed by both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (IF) (AQUA) while expression of 14 other estrogen-regulated markers was assessed by quantitative IF and correlated with clinical outcomes. RESULTS Almost half of patients experienced clinical benefit (CR+PR+SD) at 90 days despite a median of 5 previous treatment regimens. 24 of 26 patient samples were available and included in the TMA. ERα expression, measured either by conventional IHC or by AQUA analysis, was associated with clinical benefit, while TFF1 and vimentin expression (measured by IF AQUA score) was predictive of progression-free survival. CONCLUSIONS These results confirm our previous observation that clinical ovarian cancer includes a subset of tumors with sensitivity to estrogen pathway blockade. Expression profile of sensitive tumors appears to be detectably different from insensitive tumors, suggesting that further improvements in treatment efficacy can be obtained through appropriate patient selection.

Hyperthermic intraperitoneal chemotherapy with carboplatin for optimally-cytoreduced, recurrent, platinum-sensitive ovarian carcinoma: A pilot study

OBJECTIVE We aimed to evaluate the feasibility and tolerability of hyperthermic intraperitoneal carboplatin (HIPEC-carboplatin) following secondary cytoreduction for recurrent, platinum-sensitive ovarian cancer. METHODS In a single institution prospective, pilot study, ten patients underwent secondary cytoreductive surgery followed by HIPEC-carboplatin at 1000 mg/m(2). Consolidation (6 cycles) was with platinum-based regimens. Adverse and quality of life were measured throughout treatment. RESULTS Twelve patients were enrolled of which 2 were excluded (one each for extra-abdominal disease indentified before surgery and suboptimal cytoreduction). All 10 remaining patients received prescribed HIPEC-carboplatin. There were no intra-operative complications or AEs attributable to HIPEC-therapy. Grade 1/2 nausea was the most common post-operative toxicity (6/10 patients). Two patients had grade 4 post-operative neutropenia and thrombocytopenia but only one experienced transient treatment delay. The median hospital stay was 5.5 days. 69/70 (98%) of planned chemotherapy doses were ultimately delivered with 1 patient electively forgoing her final treatment. At a median (range) follow-up of 16 (6-23) months, three patients have recurred at 8, 14, and 16 months from surgery. The median disease-free and overall survivals have not been reached. Fact-O scores were significantly lower following surgery (126 vs. 108, p<.01), but improved by completion of therapy (108 vs. 113, p=0.27). CONCLUSIONS HIPEC-carboplatin at 1000 mg/m(2) following optimal cytoreduction for ovarian cancer is feasible. Surgical complications were not observed, and post-operative AEs were largely within expected ranges. Consolidation using standard platinum-based regimens was feasible following HIPEC-carboplatin, and preliminary survival data suggests efficacy. Further investigation of HIPEC-carboplatin in the setting of debulkable cancer recurrence is warranted.

A phase I feasibility study of multi-modality imaging assessing rapid expansion of marrow fat and decreased bone mineral density in cancer patients ☆

PURPOSE Cancer survivors are at an increased risk for fractures, but lack of effective and economical biomarkers limits quantitative assessments of marrow fat (MF), bone mineral density (BMD) and their relation in response to cytotoxic cancer treatment. We report dual energy CT (DECT) imaging, commonly used for cancer diagnosis, treatment and surveillance, as a novel biomarker of MF and BMD. METHODS We validated DECT in pre-clinical and phase I clinical trials and verified with water-fat MRI (WF-MRI), quantitative CT (QCT) and dual-energy X-ray absorptiometry (DXA). Basis material composition framework was validated using water and small-chain alcohols simulating different components of bone marrow. Histologic validation was achieved by measuring percent adipocyte in the cadaver vertebrae and compared with DECT and WF-MRI. For a phase I trial, sixteen patients with gynecologic malignancies (treated with oophorectomy, radiotherapy or chemotherapy) underwent DECT, QCT, WF-MRI and DXA before and 12months after treatment. BMD and MF percent and distribution were quantified in the lumbar vertebrae and the right femoral neck. RESULTS Measured precision (3mg/cm(3)) was sufficient to distinguish test solutions. Adiposity in cadaver bone histology was highly correlated with MF measured using DECT and WF-MRI (r=0.80 and 0.77, respectively). In the clinical trial, DECT showed high overall correlation (r=0.77, 95% CI: 0.69, 0.83) with WF-MRI. MF increased significantly after treatment (p<0.002). Chemotherapy and radiation caused greater increases in MF than oophorectomy (p<0.032). L4 BMD decreased 14% by DECT, 20% by QCT, but only 5% by DXA (p<0.002 for all). At baseline, we observed a statistically significant inverse association between MF and BMD which was dramatically attenuated after treatment. CONCLUSION Our study demonstrated that DECT, similar to WF-MRI, can accurately measure marrow adiposity. Both imaging modalities show rapid increase in MF following cancer treatment. Our results suggest that MF and BMD cannot be used interchangeably to monitor skeletal health following cancer therapy.

Establishment, Characterization and Downstream Application of Primary Ovarian Cancer Cells Derived from Solid Tumors

Ovarian cancer is the deadliest of the gynecological diseases and the fifth cause of cancer death among American women. This is mainly due to the lack of prognostic tools capable of detecting early stages of ovarian cancer and to the high rate of resistance to the current chemotherapeutic regimens. In this scenario the overall 5-year survival rate for ovarian cancer patients diagnosed at late stage is less than 25%. Abnormalities associated with the malignant phenotype and the mechanisms of tumor progression are not clearly understood. In vitro studies are necessary, yet have been hampered due to the limitations accompanied with the use of ovarian cancer cell lines and the heterogeneity of the ovarian cancer cell population derived from ascites fluids. In this study we present a simple, rapid and reproducible method for the isolation and characterization of ovarian cancer cells from solid tumor tissue and show that enzymatic digestion for 30 minutes with dispase II results in the most effective recovery of viable epithelial ovarian cancer (EOC) cells. The resulting cancer (EOC) cell preparations demonstrate a significant yield, high levels of viability and are fibroblast-free. They grow for up to six passages and retain the capacity of forming spheroids-like structures in agarose. In addition, they can be genetically manipulated and used for drug screening, thus rendering them highly suitable for downstream applications. Notably, isolation of ovarian cancer cells from solid specimens using this method has the advantage of allowing for isolation of cancer cells from early stages of ovarian cancer as well as obtaining cells from defined either primary and/or metastatic ovarian cancer sites. Thus, these cells are highly suitable for investigations aimed at understanding ovarian cancer.

Solitary fibrous tumors arising from the female pelvis

BACKGROUND: Solitary fibrous tumor is a rare mesenchymal tumor reported initially in the pleura but that is now reported in widely ranging anatomic sites with a variable clinical course. Solitary fibrous tumor arising from the female genital tract is extremely rare and the management of this condition is controversial. CASES: We report three cases of female genital tract solitary fibrous tumors displaying different clinical behaviors and review literature with regard to diagnosis, possible prognostic factors, and management of this tumor. CONCLUSION: The primary treatment of this disease should be surgical. The rarity and disparate clinical manifestations of this disease preclude a definitive statement on use and optimization of adjuvant therapy. Nevertheless, both pathologic and clinical findings may be useful in gauging risk and assessing the merits of individualized adjuvant therapy.

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

Abstract POSTER-THER-1405: Targeting proteasome-associated deubiquitinating enzymes for ovarian cancer treatment

Purpose of the study: Recent studies have shown that genetic events in cancer activate signaling pathways that ultimately alter cell metabolism, thereby rendering cancer cells more dependent upon “metabolic pathways” as compared to normal cells. These findings have resulted in refocused efforts in the strategy for the selective design of anticancer therapies to target the metabolic dependencies of cancer cells. Deubiquitinating enzymes (DUBs) are an essential component of the ubiquitin-dependent protein degradation system and, owing to their recently described role during cancer progression and chemoresistance, DUBs are an attractive new target for cancer treatment. The purpose of our study is to develop novel small molecule inhibitors of proteasome-associated DUBs as an alternative therapeutic approach for treatment of resistant forms of ovarian cancer. The rationale driving this study is that up-regulation of DUBs (at protein and enzymatic activity levels) is a feature of cancer and of chemoresistant versus chemosensitive ovarian cancer. Experimental Procedure: We conducted a high-throughput screening for small molecule inhibitors of DUBs, resulting in the identification of a lead compound, RA-9. Potency, selectivity, cell permeability and potential off-target effects of RA-9 were evaluated in vitro in primary ovarian cancer cells derived from clinical specimens and in vivo in a preclinical mouse xenograft model of human ovarian cancer. Preclinical efficacy and drug safety were also evaluated in a mouse model of human ovarian cancer. Summary of the data: RA-9 was shown to be a potent, selective and cell permeable inhibitor of a specific subset of DUBs associated with the 19S subunit of proteasomes. RA-9 selectively killed primary ovarian cancer cells derived from patients resistant to chemotherapy at clinically achievable concentrations. Mechanistically, RA-9 caused the onset of apoptosis in ovarian cancer cells via a mechanism involving exacerbation of endoplasmic-reticulum stress responses. In vivo, RA-9 was well tolerated, significantly decreased tumor burden, and increases overall survival in a preclinical model of human ovarian cancer. Conclusion: Our findings indicate that RA-9 has significant in vitro and in vivo anti-cancer activity at doses well tolerated in a mouse xenograft model. Based on these promising data, our laboratory is committed to the further development of this class of inhibitors. This includes medicinal chemistry study to optimize selectivity and potency of our current lead compound and pharmacology efforts to optimize pharmacokinetic and pharmacological properties of RA-9 and its most promising derivatives. Our immediate goal is to rapidly translate our findings from the benchside by performing a phase 0 clinical trial with our new investigational drugs. Importantly, Phase 0 studies are highly feasible in that they require: GLP grade drugs, single doses of drugs that are about 1% of the amount designed for clinical trials and a relatively low number of subjects. This work was supported by the Department of Defense Ovarian Cancer Research Program (OCRP) OC093424 and Minnesota Ovarian Cancer Alliance to MB. Citation Format: Kathleen Coughlin, Thanasak Sueblinvong, Rachel Isaksson Vogel, Ravi Anchoori, Martina Bazzaro. Targeting proteasome-associated deubiquitinating enzymes for ovarian cancer treatment [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1405.

Abstract LB-258: Small-molecule inhibitors of de-ubiquitinating enzymes for cervical cancer treatment

Title. Small-Molecule Inhibitors of de-Ubiquitinating Enzymes for Cervical Cancer Treatment Abstract: Human Papillomavirus (HPV) is the primary cause of cervical cancer and responsible for 5% of all cancers worldwide. While HPV vaccines can be an effective preventive measure against cervical cancer, there are currently no virus-specific therapies for it, and the efficacy of standard surgical and chemo/radiotherapies is limited for advanced disease. The E6 oncoprotein of HPV exerts its oncogenic activity by binding to the E3 ubiquitin ligase E6-AP and redirects its activity towards p53 and other tumor suppressor proteins for rapid ubiquitin-mediated proteasomal degradation. Therefore, stabilization of p53 via preventing its ubiquitin-mediated degradation represents a valid therapeutic approach for cervical cancer treatment. De-ubiquitinating enzymes are an attractive novel “druggable” target for cervical cancer as they are responsible for controlling the steady state levels of proteins (i.e. p53 and E6-AP) crucial for maintaining the transformed status of cervical cancer cells, upstream proteasome. Thus, their inhibition is potentially associated with less toxic effects as compared to inhibition of proteasomes. During a structure-based screening of chalcone-based small-molecule inhibitors of ubiquitin-mediated protein degradation we have recently identified a novel class of molecules capable of inhibiting the ubiquitin-mediated protein degradation upstream proteasomes. The lead compound of the series, contains the α-β unsaturated carbonyl system, as the molecular determinant for inhibiting de-ubiquitinating enzyme activities and: i) is capable of inducing acute perturbation of the ubiquitin-mediated protein degradation pathways accompanied by accumulation of poly-ubiquitinated species and reduction of mono-, di-, and tri-ubiquitin species, ii) has no effect on the catalytic activities of purified proteasomes or proteasome in living cells, iii) is capable of induce aggresome formation, iv) is capable of rescuing p53 levels/functions in cervical cancer cells which are accompanied by decrease in the levels of Cyclin D1 and failure for the cells to enter the S-phase of the cell cycle, v) is capable of preventing tumor-colony formation and induces onset of apoptosis via caspase-3 activation in cervical cancer cells without affecting the viability of normal cells. Therefore, we believe in the feasibility of using our lead compound in preclinical model of cervical cancer to test its efficacy as antineoplastic agent. 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 LB-258. doi:10.1158/1538-7445.AM2011-LB-258