Search

PDF file - 548K, Knockdown of HRK mRNA by siRNA to the human HRK gene (siHRK)

PDF file - 122K

PDF file - 81K, Alterations of the apoptosis regulating genes in SUZ12 knockdown EOC cells

Pathway and network analysis.

PDF file - 857K, SUZ12 knockdown inhibits the growth of human EOC cells

Flowchart depicting the number of patients in each step of the study.

PDF file - 2MB, SUZ12 is expressed at higher levels in human EOC cells

Table 1. Tumor Histology, Cellularity, and Chemotherapy Respons. Table 2. Significance and Fold Change of 770 genes and corresponding pathways in pre- and post-NACT. Table 3. List of top canonical pathways, molecular and cellular function, toxicity list, and networks. Table 4. Classification of all variants identified in DNA and cell-free DNA of pre- and post-NACT. Protein Change. Table 5. Number of mutant genes and specific variants in pre- and post-NAC. Table 6. All variants in pre- and post-NACT and recurrent plasma samples.Table 7. Potential FDA-approved targeted therapies and clinical trials based on gene level alterations.

Heatmap. Pathway changes pre- and post-NACT.

R-plot PCA by cellularity.

Plasma cfDNA vs. Tumor DNA variant overlap.

PDF file - 55K, Upregulation of HRK contributes to apoptosis induced by SUZ12 knockdown

PDF file - 314K, HRK contributes to apoptosis induced by SUZ12 knockdown

Supplementary Table and Figure Legends

While high-grade serous ovarian carcinoma (HGSOC) is the most common histologic subtype of ovarian cancer, significant tumor heterogeneity exists. In addition, chemotherapy induces changes in gene expression and alters the mutational profile. To evaluate the notion that patients with HGSOC could be better classified for optimal treatment based on gene expression, we compared genetic variants [by DNA next-generation sequencing (NGS) using a 50 gene Ion Torrent panel] and gene expression (using the NanoString PanCancer 770 gene Panel) in the tumor from 20 patients with HGSOC before and after neoadjuvant chemotherapy (NACT). NGS was performed on plasma cell free DNA (cfDNA) on a select group of patients (n = 14) to assess the utility of using cfDNA to monitor these changes. A total of 86 genes had significant changes in RNA expression after NACT. Thirty-eight genetic variants (including SNPs) from 6 genes were identified in tumors pre-NACT, while 59 variants from 19 genes were detected in the cfDNA. The number of DNA variants were similar after NACT. Of the 59 variants in the plasma pre-NACT, only 6 persisted, whereas 33 of 38 specific variants in the tumor DNA remained unchanged. Pathway analysis showed the most significant alterations in the cell cycle and DNA damage pathways.Implications: Gene expression profiles at the time of interval debulking provide additional genetic information that could help impact treatment decisions after NACT; although, continued collection and analysis of matched tumor and cfDNA from multiple time points are needed to determine the role of cfDNA in the management of HGSOC. Mol Cancer Res; 16(5); 813–24. ©2018 AACR.

PDF file - 41K, 5-Azacytidine (5-AzaC) does not upregulate HRK expression in EOC cells

XLS file - 49K, List of genes differentially expressed in SKOV3 EOC cells expressing shSUZ12 compared with controls using an apoptosis-regulating genes real-time PCR array from SA-Bioscience

PDF file - 45K

PDF file - 189K

PDF file - 58K

PDF file - 72K, QPCR array for DNA Damage genes after endoglin downregulation

Explanation and example analysis pipeline used to quantitate changes in cell size and other parameters. Supplemental Figure 1: (A) Sample images of immunofluorescence of F-actin (red) and DAPI (blue) before and after IC50treatment with CX-5461. Images were taken at random at 200x magnification. Thresholding was performed to identify DAPI stained nuclei using the Mixture of Gaussian (MoG) technique to bin pixels into foreground and background classes. Cytoplasm, marked by F-actin, was segmented using the watershed technique after Sobel transformation followed by Otsu thresholding. All image segmentation and quantitation was performed by CellProfiler. (B) Summary quantitation in representative images of changes to segmented cells' parameters before and after treatment.

Purpose: Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer.Experimental Design: Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin.Results: Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%–41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%–62% reduction, P < 0.001).Conclusions: Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer. Clin Cancer Res; 19(1); 170–82. ©2012 AACR.

Purpose: A hallmark of neoplasia is increased ribosome biogenesis, and targeting this process with RNA polymerase I (Pol I) inhibitors has shown some efficacy. We examined the contribution and potential targeting of ribosomal machinery in chemotherapy-resistant and -sensitive models of ovarian cancer.Experimental Design: Pol I machinery expression was examined, and subsequently targeted with the Pol I inhibitor CX-5461, in ovarian cancer cell lines, an immortalized surface epithelial line, and patient-derived xenograft (PDX) models with and without chemotherapy. Effects on viability, Pol I occupancy of rDNA, ribosomal content, and chemosensitivity were examined.Results: In PDX models, ribosomal machinery components were increased in chemotherapy-treated tumors compared with controls. Thirteen cell lines were sensitive to CX-5461, with IC50s 25 nmol/L–2 μmol/L. Interestingly, two chemoresistant lines were 10.5- and 5.5-fold more sensitive than parental lines. CX-5461 induced DNA damage checkpoint activation and G2–M arrest with increased γH2AX staining. Chemoresistant cells had 2- to 4-fold increased rDNA Pol I occupancy and increased rRNA synthesis, despite having slower proliferation rates, whereas ribosome abundance and translational efficiency were not impaired. In five PDX models treated with CX-5461, one showed a complete response, one a 55% reduction in tumor volume, and one maintained stable disease for 45 days.Conclusions: Pol I inhibition with CX-5461 shows high activity in ovarian cancer cell lines and PDX models, with an enhanced effect on chemoresistant cells. Effects occur independent of proliferation rates or dormancy. This represents a novel therapeutic approach that may have preferential activity in chemoresistant populations. Clin Cancer Res; 23(21); 6529–40. ©2017 AACR.

Purpose: Within heterogeneous tumors, subpopulations often labeled cancer stem cells (CSC) have been identified that have enhanced tumorigenicity and chemoresistance in ex vivo models. However, whether these populations are more capable of surviving chemotherapy in de novo tumors is unknown.Experimental Design: We examined 45 matched primary/recurrent tumor pairs of high-grade ovarian adenocarcinomas for expression of CSC markers ALDH1A1, CD44, and CD133 using immunohistochemistry. Tumors collected immediately after completion of primary therapy were then laser capture microdissected and subjected to a quantitative PCR array examining stem cell biology pathways (Hedgehog, Notch, TGF-β, and Wnt). Select genes of interest were validated as important targets using siRNA-mediated downregulation.Results: Primary samples were composed of low densities of ALDH1A1, CD44, and CD133. Tumors collected immediately after primary therapy were more densely composed of each marker, whereas samples collected at first recurrence, before initiating secondary therapy, were composed of similar percentages of each marker as their primary tumor. In tumors collected from recurrent platinum-resistant patients, only CD133 was significantly increased. Of stem cell pathway members examined, 14% were significantly overexpressed in recurrent compared with matched primary tumors. Knockdown of genes of interest, including endoglin/CD105 and the hedgehog mediators Gli1 and Gli2, led to decreased ovarian cancer cell viability, with Gli2 showing a novel contribution to cisplatin resistance.Conclusions: These data indicate that ovarian tumors are enriched with CSCs and stem cell pathway mediators, especially at the completion of primary therapy. This suggests that stem cell subpopulations contribute to tumor chemoresistance and ultimately recurrent disease. Clin Cancer Res; 18(3); 869–81. ©2011 AACR.

While high-grade serous ovarian carcinoma (HGSOC) is the most common histologic subtype of ovarian cancer, significant tumor heterogeneity exists. In addition, chemotherapy induces changes in gene expression and alters the mutational profile. To evaluate the notion that patients with HGSOC could be better classified for optimal treatment based on gene expression, we compared genetic variants [by DNA next-generation sequencing (NGS) using a 50 gene Ion Torrent panel] and gene expression (using the NanoString PanCancer 770 gene Panel) in the tumor from 20 patients with HGSOC before and after neoadjuvant chemotherapy (NACT). NGS was performed on plasma cell free DNA (cfDNA) on a select group of patients (n = 14) to assess the utility of using cfDNA to monitor these changes. A total of 86 genes had significant changes in RNA expression after NACT. Thirty-eight genetic variants (including SNPs) from 6 genes were identified in tumors pre-NACT, while 59 variants from 19 genes were detected in the cfDNA. The number of DNA variants were similar after NACT. Of the 59 variants in the plasma pre-NACT, only 6 persisted, whereas 33 of 38 specific variants in the tumor DNA remained unchanged. Pathway analysis showed the most significant alterations in the cell cycle and DNA damage pathways. Implications: Gene expression profiles at the time of interval debulking provide additional genetic information that could help impact treatment decisions after NACT; although, continued collection and analysis of matched tumor and cfDNA from multiple time points are needed to determine the role of cfDNA in the management of HGSOC. Mol Cancer Res; 16(5); 813–24. ©2018 AACR.

Purpose: A hallmark of neoplasia is increased ribosome biogenesis, and targeting this process with RNA polymerase I (Pol I) inhibitors has shown some efficacy. We examined the contribution and potential targeting of ribosomal machinery in chemotherapy-resistant and -sensitive models of ovarian cancer. Experimental Design: Pol I machinery expression was examined, and subsequently targeted with the Pol I inhibitor CX-5461, in ovarian cancer cell lines, an immortalized surface epithelial line, and patient-derived xenograft (PDX) models with and without chemotherapy. Effects on viability, Pol I occupancy of rDNA, ribosomal content, and chemosensitivity were examined. Results: In PDX models, ribosomal machinery components were increased in chemotherapy-treated tumors compared with controls. Thirteen cell lines were sensitive to CX-5461, with IC50s 25 nmol/L–2 μmol/L. Interestingly, two chemoresistant lines were 10.5- and 5.5-fold more sensitive than parental lines. CX-5461 induced DNA damage checkpoint activation and G2–M arrest with increased γH2AX staining. Chemoresistant cells had 2- to 4-fold increased rDNA Pol I occupancy and increased rRNA synthesis, despite having slower proliferation rates, whereas ribosome abundance and translational efficiency were not impaired. In five PDX models treated with CX-5461, one showed a complete response, one a 55% reduction in tumor volume, and one maintained stable disease for 45 days. Conclusions: Pol I inhibition with CX-5461 shows high activity in ovarian cancer cell lines and PDX models, with an enhanced effect on chemoresistant cells. Effects occur independent of proliferation rates or dormancy. This represents a novel therapeutic approach that may have preferential activity in chemoresistant populations. Clin Cancer Res; 23(21); 6529–40. ©2017 AACR.

To investigate whether tumor cells could be detected in the vagina of women with serous ovarian cancer through TP53 analysis of DNA samples collected by placement of a vaginal tampon.Women undergoing surgery for a pelvic mass were identified in the gynecologic oncology clinic. They placed a vaginal tampon before surgery, which was removed in the operating room. Cells were isolated and DNA was extracted from both the cells trapped within the tampon and the primary tumor. In patients with serous carcinoma, the DNA was interrogated for the presence of TP53 mutations using a method capable of detecting rare mutant alleles in a mixture of mutant and wild-type DNA.Thirty-three patients were enrolled. Eight patients with advanced serous ovarian cancer were included for analysis. Three had a prior tubal ligation. TP53 mutations were identified in all eight tumor samples. Analysis of the DNA from the tampons revealed mutations in three of the five patients with intact tubes (sensitivity 60%) and in none of the three patients with tubal ligation. In all three participants with mutation detected in the tampon specimen, the tumor and the vaginal DNA harbored the exact same TP53 mutation. The fraction of DNA derived from exfoliated tumor cells ranged from 0.01% to 0.07%.In this pilot study, DNA derived from tumor was detected in the vaginas of 60% of patients with ovarian cancer with intact fallopian tubes. With further development, this approach may hold promise for the early detection of this deadly disease.

// Zachary C. Dobbin 1,2 , Ashwini A. Katre 1 , Adam D. Steg 1 , Britt K. Erickson 1 , Monjri M. Shah 1 , Ronald D. Alvarez 1 , Michael G. Conner 3 , David Schneider 4 , Dongquan Chen 5 and Charles N. Landen 6 1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Alabama at Birmingham 2 NIH Medical Scientist Training Program, University of Alabama at Birmingham 3 Department of Pathology, University of Alabama at Birmingham 4 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 5 Division of Preventative Medicine, Department of Medicine, University of Alabama at Birmingham 6 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of Virginia, Charlottesville, VA Correspondence: Charles N. Landen Jr, email: // Keywords : Ovarian Cancer, Patient-derived xenograft, cancer stem cells, chemoresistance, animal models of cancer Received : August 08, 2014 Accepted : August 18, 2014 Published : August 19, 2014 Abstract A cornerstone of preclinical cancer research has been the use of clonal cell lines. However, this resource has underperformed in its ability to effectively identify novel therapeutics and evaluate the heterogeneity in a patient’s tumor. The patient-derived xenograft (PDX) model retains the heterogeneity of patient tumors, allowing a means to not only examine efficacy of a therapy, but also basic tenets of cancer biology in response to treatment. Herein we describe the development and characterization of an ovarian-PDX model in order to study the development of chemoresistance. We demonstrate that PDX tumors are not simply composed of tumor-initiating cells, but recapitulate the original tumor’s heterogeneity, oncogene expression profiles, and clinical response to chemotherapy. Combined carboplatin/paclitaxel treatment of PDX tumors enriches the cancer stem cell populations, but persistent tumors are not entirely composed of these populations. RNA-Seq analysis of six pair of treated PDX tumors compared to untreated tumors demonstrates a consistently contrasting genetic profile after therapy, suggesting similar, but few, pathways are mediating chemoresistance. Pathways and genes identified by this methodology represent novel approaches to targeting the chemoresistant population in ovarian cancer

Ovarian cancer is the fifth most common cause of death due to cancer in women despite being the tenth in incidence. Unfortunately, the five-year survival rate is only 45%, which has not improved much in the past 30 years. Even though the majority of women have successful initial therapy, the low rate of survival is due to the eventual recurrence and succumbing to their disease. With the recent release of the Cancer Genome Atlas for ovarian cancer, it was shown that the PI3K/AKT/mTOR pathway was one of the most frequently mutated or altered pathways in patients' tumors. Researching how the PI3K/AKT/mTOR pathway affects the progression and tumorigensis of ovarian cancer will hopefully lead to new therapies that will increase survival for women. This review focuses on recent research on the PI3K/AKT/mTOR pathway and its role in the progression and tumorigensis of ovarian cancer.

Preclinical research in gynecologic malignancies has largely relied upon cloned cancer-derived cell lines and tumor xenografts derived from these cell lines. Unfortunately, the use of cell lines for translational research has disadvantages because genetic and phenotypic alterations from serial passaging have resulted in expression profiles that are different from the original patient tumors. The patient-derived xenograft (PDX) model derived from human tumor not previously cultured has shown better representation of the heterogeneity of gynecologic malignancies and the human tumor microenvironment with preservation of cytogenetics, cellular complexity, and vascular and stromal tumor architecture. Studies have shown promise with these models to analyze tumor development and adaptation, test drug efficacy, and predict clinical outcomes. Their ultimate value may be seen with preclinical drug screening including novel targeted therapies, biomarker identification, and the development of individualized treatment plans. This article reviews PDX model development, current studies testing chemotherapeutics and targeted therapies, and limitations of the PDX model in gynecologic malignancies.

We introduce a novel statistical approach that quantifies, for the first time, the amount of colocalization of two fluorescent-labeled proteins in an image automatically, removing the bias of visual interpretation. This is done by estimating simultaneously the maximum threshold of intensity for each color below which pixels do not show any statistical correlation. The sensitivity of the method was illustrated on simulated data by statistically confirming the existence of true colocalization in images with as little as 3% colocalization. This method was then tested on a large three-dimensional set of fixed cells cotransfected with CFP/YFP pairs of proteins that either co-compartmentalized, interacted, or were just randomly localized in the nucleolus. In this test, the algorithm successfully distinguished random color overlap from colocalization due to either co- compartmentalization or interaction, and results were verified by fluorescence resonance energy transfer. The accuracy and consistency of our algorithm was further illustrated by measuring, for the first time in live cells, the dissociation rate (kd) of the HIV-1 Rev/CRM1 export complex induced by the cytotoxin leptomycin B. Rev/CRM1 colocalization in nucleoli dropped exponentially after addition of leptomycin B at a rate of 1.25 3 10 � 3 s � 1 . More generally, this algorithm can be used to answer a variety of biological questions involving protein-protein interactions or co-compartmentalization and can be generalized to colocalization of more than two colors.

Epithelial ovarian cancer remains a deadly diagnosis with poor prognosis and patients succumbing to chemoresistant disease in 80% of cases. Mechanisms of chemoresistance are numerous, but we have identified ribosomal biogenesis basal machinery as being a common upregulated signaling network in response to chemotherapeutic insult. Tumors treated with chemotherapy showed dramatic increases in size and morphology of nucleoli, suggesting global changes in ribosomal synthesis with stress. We next sought to use the RNA Polymerase I inhibitor CX-5461 to identify biologic changes in targeting ribosomal machinery that may allow resensitization to chemotherapy. CX-5461 induced autophagy, senescence and mitotic catastrophe in ovarian cancer cell lines Overall, 13 ovarian cancer lines examined were highly sensitive to Pol I inhibition by MTT, with IC50s ranging from 25nM to 2μM, and chemoresistant lines were generally more sensitive to CX-5461. In p53 mutant cells CX-5461 appears to be primarily cytostatic with mitotic catastrophe being a relatively rare event accompanying cytokinesis failure. This could be indicative of the cells successfully using autophagy to decrease ribosome requirements and evade treatment. When treated in combination with chloroquine as an autophagy inhibitor IC50s decreased to 550nM in HeyA8 (from 2μM), 70nM for HeyA8MDR (from 191nM), 110nM in SKOV3 (from 595nM) and 60nM for SKOV3TR-ip2 (from 109nM) with abundant multinucleated cells, suggesting synergy between ribosomal synthesis and autophagy inhibitors in vitro. Chloroquine administration appears to push cell to mitotic catastrophe after co-administration with CX-5461, thereby enhancing cytotoxicity over cytostatic endpoints after treatment, even in multi-drug resistant cell populations. Inhibition of autophagy appears to enhance efficacy of RNA Polymerase I inhibition in both sensitive and chemoresistant populations, prompting a potential new approach to chemoresistant disease. Citation Format: Robert Cornelison, Danielle C. Llaneza, Yulia Petrova, Zachary C. Dobbin, David A. Schneider, Charles N. Landen. Inhibition of autophagy potentiates cytotoxicity of CX-5461 treatment in chemoresistant epithelial ovarian cancer [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 3211. doi:10.1158/1538-7445.AM2017-3211

The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance.

BRCA-positive ovarian cancer patients derive benefit PARP inhibitors. Approximately 50% of ovarian cancer tumors have homologous recombination (HR) deficiencies and are therefore "BRCA-like," possibly rendering them sensitive to PARP inhibition. However, no predictive assay exists to identify these patients. We sought to determine if irradiation-induced Rad51 foci formation, a known marker of HR, correlated to PARP inhibitor response in an ovarian cancer model.Ovarian cancer cell lines were exposed to PARP-inhibitor ABT-888 to determine effect on growth. Rad51 protein expression prior to irradiation was determined via Western blot. Cultured cells and patient-derived xenograft tumors (PDX) were irradiated and probed for Rad51 foci. In vivo PDX tumors were treated with ABT-888 and carboplatin; these results were correlated with the ex vivo ionizing radiation assay.Three of seven cell lines were sensitive to ABT-888. Sensitive lines had the lowest Rad51 foci formation rate after irradiation, indicating functional HR deficiency. Approximately 50% of the PDX samples had decreased Rad51 foci formation. Total Rad51 protein levels were consistently low, suggesting that DNA damage induction is required to characterize HR status. The ex vivo IR assay accurately predicted which PDX models were sensitive to PARP inhibition in vitro and in vivo. ABT-888 alone reduced orthotopic tumor growth by 51% in A2780ip2 cell line, predicted to respond by the ex vivo assay. Three PDX models' response also correlated with the assay.The ex vivo IR assay correlates with response to PARP inhibition. Analysis of total Rad51 protein is not a reliable substitute.

Objectives: The objectives of this study were to use the heterogeneity of the patient-derived xenograft (PDX) model to characterize the surviving population for de novo mediators of chemotherapy resistance, and subsequently target pathways contributing to that resistance. Methods: Tumors removed during primary tumor-reductive surgery were implanted directly into SCID mice, either subcutaneously and intraperitoneally. Mice with PDX tumors were treated with combination carboplatin/paclitaxel for 4 weeks. In addition, chemoresistant models were developed by treatment until complete response was achieved, and recurrent tumors retreated until resistant. Patient tumors, developing PDX tumors, and post-chemo surviving residual tumors were subjected to qPCR arrays, RNA-Seq, Methyl-Seq, and proteomic analysis; and IHC for Ki-67 and cancer stem cell markers CD133, ALDH1, and CD44. The Pol-I inhibitor CX-5461 was used to treat five PDX models. Results: At the mRNA level, the original patient tumors were similar to PDX tumors in mice as assessed by an 84-oncogene expression panel. Subcutaneous and intraperitoneal tumors were also remarkably similar. Patient responses correlated well with responses in mice. Proteomic analysis identified 14,023 unique proteins, of which approximately 30% were murine, and 70% were human in each sample. A total of 1,202 of human proteins were found to be differently expressed (p-value < 0.01) between patient and PDX tumors. Of these, 996 (82.9%) were reduced in the PDX model, and predominantly participants in the immune system. PDX tumors collected after chemotherapy showed that the surviving population was significantly more dormant than the original tumor (Ki-67 67% versus 31%, P Conclusion: The ovarian-PDX model exhibits consistent molecular and biologic similarity to patient tumors. Post-chemo tumors reveal multiple pathways to be consistently altered. Taking this information back to the PDX model demonstrated that inhibition of ribosomal RNA synthesis was highly effective, offering a novel opportunity to preferentially target the chemoresistant population in ovarian cancer. Citation Format: Zachary Dobbin, Robert Cornelison, Ashwini K. Katre, David A. Schneider, Karen Watters, Fabian Coscia, Ernst Lengyel, Aaron Buechlein, Fang Fang, Kenneth Nephew, Charles N. Landen, Jr.. Identification and targeting mediators of chemoresistance using the patient-derived xenograft model of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B39.

BACKGROUND: One of the first identified hallmarks of neoplastic transformation was an increased rate of ribosome biogenesis, and increased activity of RNA polymerase I (RNA pol I) is observed in most human cancers. Ribosome biogenesis is an energy intensive and tightly regulated process, and recent studies have shown that CX-5461, a potent and specific RNA pol I inhibitor, has selective toxicity to cancer cells. Here we demonstrate that CX-5461 is an effective anti-proliferative agent in epithelial ovarian carcinoma cells with an increased efficacy in chemoresistant ovarian cancer cells compared to their sensitive parental lines. METHODS AND RESULTS: Ovarian cancer cells were demonstrated to be highly sensitive to RNA pol I inhibition by MTT assay, with an IC50 range from 25nM to 2uM in 13 different ovarian cancer cell lines, and an IC50 of 3uM in the nontransformed immortalized normal surface epithelial line HIO-180. Notably, chemoresistant cell populations were generally more sensitive to CX-5461, compared to matched chemosensitive lines. IC50 in HeyA8MDR was 80nM compared to 2000nM in parental HeyA8, and was 100nM in SKOV3TRip2 compared to 400nM in SKOV3ip1. CX-5461 induces G2/M arrest (averaging 52.7% in G2 compared to 32.5% in controls), primarily through mitotic catastrophe, inducing an accumulation of multinucleated senescent cells. DNA damage pathways are activated, operating through ATM/ATR, as demonstrated by rescue with caffeine and ATR specific inhibitors. In an effort to identify why chemoresistant cells might be more susceptible, ribosomal synthesis processes were examined. Chemoresistant cells were found to have 2-4-fold increased Pol I occupancy on rDNA by ChIP, and increased rRNA synthesis by isotopic incorporation, while ribosome abundance and overall translation efficiency, measured by sucrose gradient fractionation, were unchanged. Treatment of five separate patient-derived xenograft models (PDX) with CX-5461 showed a complete response in one model, partial 55% reduction in tumor size in a second, stable disease in a 3rd, and progression in two models. CONCLUSIONS: CX-5461 shows high activity in ovarian cancer cell lines and patient-derived xenograft models, with a preferential effect on chemoresistant cells. Enhanced sensitivity to the compound is due at least in part to cancer cells’ increased demand for rRNA synthesis. RNA pol I inhibitors represent an exciting new treatment option that may have preferential effect on the chemoresistant population. Citation Format: Robert Cornelison, Zachary C. Dobbin, Ashwini A. Katre, Dae Hoon Jeong, Yulia Petrova, Danielle C. Llaneza, Adam D. Steg, Yinfeng Zhang, David A. Schneider, Charles N. Landen. Targeting RNA-polymerase I using CX-5461 as a mechanism for treating chemotherapy resistant epithelial ovarian cancer. [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 324.

e17103Background: While the majority of patients diagnosed with endometrial cancer (EMCA) have low-risk disease at the time of diagnosis, a subset of patients with Stage I disease are classified as...

Purpose: Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer. Experimental Design: Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin. Results: Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%–41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%–62% reduction, P < 0.001). Conclusions: Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer. Clin Cancer Res; 19(1); 170–82. ©2012 AACR.

Epithelial ovarian cancer (EOC) ranks first as the cause of death for gynecological cancers in the United States. SUZ12 is a component of the polycomb repressive complex 2 (PRC2) and is essential for PRC2-mediated gene silencing by generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3). The role of SUZ12 in EOC has never been investigated. Here, we show that SUZ12 is expressed at significantly higher levels in human EOC (n = 117) compared with either normal human ovarian surface epithelium (n = 35, P < 0.001) or fallopian tube epithelium (n = 15, P < 0.001). There is a positive correlation between expression of SUZ12 and EZH2 in human EOC (P < 0.001). In addition, expression of SUZ12 positively correlates with Ki67, a marker of cell proliferation (P < 0.001), and predicts shorter overall survival (P = 0.0078). Notably, knockdown of SUZ12 suppresses the growth of human EOC cells in vitro and in vivo in both orthotopic and subcutaneous xenograft EOC models. In addition, SUZ12 knockdown decreases the levels of H3K27Me3 and triggers apoptosis of human EOC cells. Mechanistically, we identified Harakiri (HRK), a proapoptotic gene, as a novel SUZ12 target gene, and showed that HRK upregulation mediates apoptosis induced by SUZ12 knockdown in human EOC cells. In summary, we show that SUZ12 promotes the proliferation of human EOC cells by inhibiting apoptosis and HRK is a novel SUZ12 target gene whose upregulation contributes to apoptosis induced by SUZ12 knockdown. Mol Cancer Res; 10(11); 1462–72. ©2012 AACR.

Paclitaxel, an anti-microtubule agent, is an effective chemotherapeutic drug in breast cancer. Nonetheless, resistance to paclitaxel remains a major clinical challenge. The need to better understand the resistant phenotype and to find biomarkers that could predict tumor response to paclitaxel is evident. In estrogen receptor α-positive (ER(+)) breast cancer cells, phosphorylation of caveolin-1 (CAV1) on Tyr-14 facilitates mitochondrial apoptosis by increasing BCL2 phosphorylation in response to low dose paclitaxel (10 nM). However, two variants of CAV1 exist: the full-length form, CAV1α (wild-type CAV1 or wtCAV1), and a truncated form, CAV1β. Only wtCAV1 has the Tyr-14 region at the N terminus. The precise cellular functions of CAV1 variants are unknown. We now show that CAV1 variants play distinct roles in paclitaxel-mediated cell death/survival. CAV1β expression is increased in paclitaxel-resistant cells when compared with sensitive cells. Expression of CAV1β in sensitive cells significantly reduces their responsiveness to paclitaxel. These activities reflect an essential role for Tyr-14 phosphorylation because wtCAV1 expression, but not a phosphorylation-deficient mutant (Y14F), inactivates BCL2 and BCLxL through activation of c-Jun N-terminal kinase (JNK). MCF-7 cells that express Y14F are resistant to paclitaxel and are resensitized by co-treatment with ABT-737, a BH3-mimetic small molecule inhibitor. Using structural homology modeling, we propose that phosphorylation on Tyr-14 enables a favorable conformation for proteins to bind to the CAV1 scaffolding domain. Thus, we highlight novel roles for CAV1 variants in cell death; wtCAV1 promotes cell death, whereas CAV1β promotes cell survival by preventing inactivation of BCL2 and BCLxL via JNK in paclitaxel-mediated apoptosis.

Purpose: Within heterogeneous tumors, subpopulations often labeled cancer stem cells (CSC) have been identified that have enhanced tumorigenicity and chemoresistance in ex vivo models. However, whether these populations are more capable of surviving chemotherapy in de novo tumors is unknown. Experimental Design: We examined 45 matched primary/recurrent tumor pairs of high-grade ovarian adenocarcinomas for expression of CSC markers ALDH1A1, CD44, and CD133 using immunohistochemistry. Tumors collected immediately after completion of primary therapy were then laser capture microdissected and subjected to a quantitative PCR array examining stem cell biology pathways (Hedgehog, Notch, TGF-β, and Wnt). Select genes of interest were validated as important targets using siRNA-mediated downregulation. Results: Primary samples were composed of low densities of ALDH1A1, CD44, and CD133. Tumors collected immediately after primary therapy were more densely composed of each marker, whereas samples collected at first recurrence, before initiating secondary therapy, were composed of similar percentages of each marker as their primary tumor. In tumors collected from recurrent platinum-resistant patients, only CD133 was significantly increased. Of stem cell pathway members examined, 14% were significantly overexpressed in recurrent compared with matched primary tumors. Knockdown of genes of interest, including endoglin/CD105 and the hedgehog mediators Gli1 and Gli2, led to decreased ovarian cancer cell viability, with Gli2 showing a novel contribution to cisplatin resistance. Conclusions: These data indicate that ovarian tumors are enriched with CSCs and stem cell pathway mediators, especially at the completion of primary therapy. This suggests that stem cell subpopulations contribute to tumor chemoresistance and ultimately recurrent disease. Clin Cancer Res; 18(3); 869–81. ©2011 AACR.

Introduction: A cornerstone of preclinical ovarian cancer research over the last 30 years has been the use of cell lines both in vitro and in vivo. This resource has underperformed in its ability to identify effective novel therapeutics and evaluate the heterogeneity of an ovarian neoplasm. Developing a patient-derived xenograft (PDX) allows for a comprehensive study of the heterogeneous tumor and potentially the identification of cellular populations responsible for chemoresistance and recurrence, but has not been fully characterized for ovarian cancer. We have developed an ovarian PDX model to demonstrate many important similarities and differences between these and primary patient tumors, and begun to explore its utility in identifying mediators of chemoresistance and personalizing therapy. Methods: Tumors removed from patients undergoing primary tumor reductive surgery were implanted into the subcutaneous flanks of SCID mice. Once a tumor developed, it was expanded into additional mice to propagate a PDX line. Tumors were analyzed for heterogeneity by examination of the tumor initiating cell populations, its stromal content with HLA immunohistochemistry, and proliferation with Ki-67. Cohorts of PDX lines were treated with maximum-tolerated dose of combined carboplatin and paclitaxel or vehicle and response was compared between the PDX and the patient. Chemoresistant PDX lines were generated by treatment with MTD carboplatin and paclitaxel until no evidence of disease (NED), observation until recurrence, and retreatment until fully resistant. RNAseq was conducted comparing treated PDX lines to the untreated PDX lines (n=6 pair) to identify important mediators of chemotherapy resistance. Finally, PDX lines were characterized for defects in homologous recombination (HR) repair with an ex vivo assay, correlating HR status with response to PARP inhibition in vivo. Results: Refinement of xenografting procedures yields an 85% success rate in establishing a PDX. PDX tumors maintain the patient's histology, but the stromal component is replaced by murine cells in the first generation. The xenografts retain primary tumor heterogeneity, at least in expression of putative tumor initiating cells (TIC) (ALDH: 17% vs 19%, CD44: 2.4% vs 5%, CD133 10% vs 3%). However, treatment with chemotherapy significantly increases these populations (ALDH1 increased to 36.1% from 16.2% (p=0.002); CD133 increased to 33.8% from 16.2% (p=0.026), suggesting a role in chemotherapy resistance. The treated PDX tumors showed a decrease in Ki67 staining (from 64% to 34%, p=0.001), suggesting dormancy is induced in the surviving population. The PDX tumors show similar response to chemotherapy as patient tumors, in that PDX tumors from patients with a partial response respond more slowly (if at all) than those from patients achieving a complete response (mean change in tumor volume -5.11% vs -63.73%, p=0.029). RNAseq comparing treated and untreated PDX lines indicate only 54 common genes with a significant change in mRNA (p Conclusions: The ovarian PDX model recapitulates patient tumor heterogeneity and mirrors response to chemotherapy. This model may prove superior in predicting response to therapy in patients, and allows a better model to study complex tumor biology that is impacted by tumor heterogeneity, such as survival of small populations with chemotherapy. Citation Format: Zachary C. Dobbin, Ashwini K. Katre, Monjri M. Shah, Britt K. Erickson, Hauping Chen, Ronald D. Alvarez, Michael G. Conner, Dongquan Chen, Charles N. Landen. An ovarian patient-derived xenograft model to identify the chemoresistant population. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A58.

Background: Endoglin (ENG, CD105) is a protein markedly overexpressed in tumor-associated endothelial cells, and a target for anti-angiogenic therapy. Recently we have noted it to be overexpressed in chemoresistant ovarian cancer cells as well. Our objective was to evaluate the effects and mechanisms of targeting endoglin specifically in ovarian cancer cells. Methods: Endoglin expression was assessed in multiple ovarian cancer lines by Western blot, immunohistochemistry, and flow cytometry. Anti-endoglin siRNAs were used to downregulate expression in ES2 and HeyA8MDR. In vitro, the effects of endoglin-knockdown individually and with chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation and qPCR array for mediators of DNA damage and repair. In an orthotopic murine model, mice inoculated with ES2 or HeyA8MDR cell lines were administered chitosan-encapsulated anti-ENG siRNA or control siRNA with and without carboplatin. PCNA, γH2AX, and 53BP1 IHC and the TUNEL assay were performed to examine biologic effects of endoglin knockdown. Results: Endoglin is expressed to varying degrees by multiple ovarian cancer cell lines. Expression was on the cell surface, consistent with its recognized role as a cofactor with TGF-beta receptor, in only 5% of cells, with most other cells having cytoplasmic expression. In ES2 and HeyA8MDR cell lines, endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Endoglin downregulation led to a decrease in expression of several DNA repair genes, including NBN, NTHL1, BARD1, H2AFX and SIRT1, and an increase in expression of DDIT3 and PPP1R15A. BARD1-specific downregulation, in turn, led to a significant decrease in BRCA1 expression, likely through ubiqutinylation. In vivo, anti-endoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared to control (35-41% reduction, p Citation Format: Angela J. Ziebarth, Somaria Nowsheen, Adam D. Steg, Monjri M. Shah, Ashwini A. Katre, Zachary C. Dobbin, Anil K. Sood, Michael G. Conner, Eddy S. Yang, Charles N. Landen. Endoglin (CD105) is a target for ovarian cancer cell-specific therapy through induction of DNA damage. [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 5475. doi:10.1158/1538-7445.AM2013-5475 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Background: Traditionally, epithelial ovarian cancer (EOC), and its various histologic subtypes, was considered one disease process. With an improved understanding of underlying carcinogenesis, it is now hypothesized that ovarian cancers of different histologies may originate in different sites - serous from the fallopian tube, endometrioid and clear cell from endometriotic implants, and mucinous from the GI tract. However, five percent of EOC are classified as “mixed” tumors, with two or more histologic subtypes present in the same tumor. Whether these cancers represent phenotypic diversions of the same cancer, or synchronous tumors from different sites is not known, and if discovered, may shed light on the etiology of single-histology malignancies. Objective: By comparing the TP53 mutational signatures of both histologic subtypes within the same tumor, we sought to determine if mixed histology ovarian cancers arise from the same tumor initiating cells. Methods: After IRB approval, formalin fixed paraffin embedded samples of mixed histology tumors were identified. After review by a gynecologic pathologist, the specific area of each histologic subtype was identified and isolated using Laser Capture Microdissection (LCM). DNA was extracted separately from each histologic subtype. PCR reactions were performed to amplify TP53 exons 4-8. Gel electrophoresis was performed to specifically isolate the desired exon, which was extracted and purified for sequencing. Sanger sequencing was performed in forward and reverse directions, and mutations were identified by comparison to the TP53 reference sequence (NC_000017). TP53 mutations were compared between both histologic subtypes of the same tumor sample to assess for clonality. Results: 10 patients were identified with mixed histologies and subjected to LCM. Extracted DNA was of sufficient quality to sequence both histologies in 9 patients. Thus far, 4 patients samples have been sequenced and analyzed. In one patient no TP53 mutation was identified. In the remaining 3, a single mutation was found, and was identical in both histologic sections. All mutations were missense. The TP53 mutations identified in these samples had all previously been described, supporting the validity of identified mutations. These findings support additional sequencing of the remaining 5 patients. Conclusions: These analyses suggest that the separate components of mixed histology epithelial ovarian cancer harbor the same early TP53 mutations - supporting their clonality. This supports the hypothesis that that mixed histology EOC tumors originate from the same tumor initiating cell, and that not all histologic subtypes of non-mixed ovarian cancers may have separate origins. Citation Format: Britt K. Erickson, Zachary C. Dobbin, Eun-Hee Shim, Ronald D. Alvarez, Michael G. Conner, Charles N. Landen. Identical TP53 mutations support a common origin for mixed histology epithelial ovarian 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 1193. doi:10.1158/1538-7445.AM2013-1193

Background: Cancer stem cells (CSCs) have been implicated in the development and recurrence of chemoresistant tumors in multiple malignancies. Previous research has suggested that targeting stem cell pathways, such as Notch and Hedgehog, may aid in overcoming the chemoresistant properties of these cells. However, attempts to target the Notch and Hedgehog pathways synergistically have had mixed results using different inhibitors, and the mechanism through which these inhibitors act is not fully known. Objective: To explore the potential synergy of concurrent targeting of the Notch and Hedgehog pathways and the mechanism through which this may occur. Methods: The Notch pathway was targeted with GSI-1 and GSI-XXI, and the Hedgehog pathway was targeted with the Smoothened small molecule inhibitor LDE225. The proteasome inhibitor Bortezomib was used alone and in combination with LDE225. Effects of these drugs on three pairs of chemosensitive and chemoresistant ovarian cancer cell lines were examined: SKOV3ip1 and SKOV3TRip2 (taxane-resistant), HeyA8 and HeyA8MDR (multi-drug resistant), and A2780ip2/A2780cp20 (platinum resistant). Cell viability was determined by the MTT assay. PI staining and PARP cleavage were used to assess cell cycle arrest and apoptosis. Proteasome activity was determined by the Proteasome-glo chymotrypsin-like cell-based assay. Results: GSI-1 significantly decreased cell viability alone and synergized with LDE225 in SKOV3TRip2 cells. However, GSI-XXI did not reduce cell viability or exhibit synergy with LDE225. In addition, specific inhibition of Notch via siRNA-mediated downregulation did not result in synergy with LDE225, suggesting a Notch-independent mechanism of synergy with GSI-1. To elucidate differences between GSI-1 and GSI-XXI, we examined effects of these drugs on the proteasome. GSI-1 inhibited the proteasome to the same extent as the conventional proteasome inhibitor bortezomib, while GSI-XXI did not affect proteasome activity. Combination treatment with bortezomib and LDE225 also resulted in synergy in SKOV3TRip2 cells. Monotherapy with both GSI-1 and bortezomib decreased transcription of the hedgehog target genes Patched, Gli1, and Gli2. This effect was enhanced when these drugs were combined with LDE225. Combination therapy also resulted in arrest of cells in the G2/M and S phases (60% of cells in S/G2/M with combination therapy vs. 38% of control, p Conclusion: The Notch inhibitor GSI-1 acts synergistically with Hedgehog inhibition through a Notch-independent decrease in proteasome activity. Similar synergy was noted with the proteasome inhibitor bortezomib. This synergistic combination appears to be working through S/G2/M phase arrest. Combined Hedgehog and proteasome inhibition offers a promising approach to targeting chemoresistant cells in ovarian malignancies. Citation Format: Mata R. Burke, Adam Steg, Dae Hoon Jeong, Zachary C. Dobbin, Charles N. Landen. GSI-1 synergizes with LDE225 in ovarian cancer cells by inhibiting the proteasome. [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 3423. doi:10.1158/1538-7445.AM2013-3423

5036 Background: Current xenograft and transgenic models of ovarian cancer are mainly homogeneous and poorly predict response to therapy. Use of patient tumors may represent a better model for tumor biology and offer potential to test personalized medicine approaches, but poor take rates and questions of recapitulation of patient tumors have limited this approach. We have developed a protocol for improved feasibility of such a model and examined its similarity to the patient tumor. Methods: Under IRB and IACUC approval, 23 metastatic ovarian cancer samples were collected at the time of tumor reductive surgery. Samples were implanted either subcutaneously (SQ), intraperitoneally (IP), in the mammary fat pad (MFP), or in the subrenal capsule (SRC) and monitored for tumor growth. Cohorts from 8 xenolines were treated with combined carboplatin and paclitaxel or vehicle, and response to therapy compared between xenografts and patients. Expression of tumor-initiating cell (TIC) markers ALDH1, CD133, and CD44 was assessed by immunohistochemistry in tumors from patients and treated and untreated xenografts. Results: At least one SQ implanted tumor developed in 91.3% of xenografts, significantly higher than in the MFP (63.6%), IP (23.5%), or SRC (8%). Xenografts were similar in expression of putative TIC’s compared to patient tumors. The patients and the xenografts also have similar responses to chemotherapy in that xenografts from patients with a partial response responded more slowly than those from patients achieving a complete response (45 vs 21 days, p=.004). Treated xenografts were more densely composed of TICs. ALDH1 increased to 36.1% from 16.2% (p=0.002) and CD133 increased to 33.8% from 16.2% (p=0.026). Conclusions: Xenoline development can be achieved at a high rate when tumors collected from metastatic sites are implanted SQ. These xenografts are similar to patient tumors with regard to chemotherapy response and TIC expression.. This model may be a more accurate model for in vivo pre-clinical studies as compared to current models. Also, as treated xenografts become chemoresistant, this model is well positioned to evaluate targeted therapies aimed at the most aggressive populations in a heterogeneous tumor.