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Summary of in vivo studies

Summary of RNA sequencing (RNA seq) data of the Met superfamily kinases in 4 breast PDX models.

A. MCF7 tumor growth rate in NOD/SCID mice implanted with beeswax E2 pellets, commercial 0.72 mg/60 days E2 pellets (Innovative Research of America) and control mice. Both, 0.72 mg/60 days E2 and beeswax E2 pellets equally supported estrogen dependent tumor growth. In contrast, control mice not supplemented with E2 pellets failed to grow tumors. B. Quantification of E2 plasma levels 10 days post implantation of E2 pellets in NOD/SCID mice. The beeswax pellets yielded mean E2 plasma levels of 84 pg/ml with very little variation, which are comparable plasma estrogen levels released from commercially available E2 pellets.

Purpose: Recent studies have demonstrated that short-form Ron (sfRon) kinase drives breast tumor progression and metastasis through robust activation of the PI3K pathway. We reasoned that upfront, concurrent inhibition of sfRon and PI3K might enhance the antitumor effects of Ron kinase inhibitor therapy while also preventing potential therapeutic resistance to tyrosine kinase inhibitors (TKI).Experimental Design: We used patient-derived breast tumor xenografts (PDX) as high-fidelity preclinical models to determine the efficacy of single-agent or dual Ron/PI3K inhibition. We tested the Ron kinase inhibitor ASLAN002 with and without coadministration of the PI3K inhibitor NVP-BKM120 in hormone receptor–positive [estrogen receptor (ER)+/progesterone receptor (PR)+] breast PDXs with and without PIK3CA gene mutation.Results: Breast PDX tumors harboring wild-type PIK3CA showed a robust response to ASLAN002 as a single agent. In contrast, PDX tumors harboring mutated PIK3CA demonstrated partial resistance to ASLAN002, which was overcome with addition of NVP-BKM120 to the treatment regimen. We further demonstrated that concurrent inhibition of sfRon and PI3K in breast PDX tumors with wild-type PIK3CA provided durable tumor stasis after therapy cessation, whereas discontinuation of either monotherapy facilitated tumor recurrence.Conclusions: Our work provides preclinical rationale for targeting sfRon in patients with breast cancer, with the important stipulation that tumors harboring PIK3CA mutations may be partially resistant to Ron inhibitor therapy. Our data also indicate that tumors with wild-type PIK3CA are most effectively treated with an upfront combination of Ron and PI3K inhibitors for the most durable response. Clin Cancer Res; 21(24); 5588–600. ©2015 AACR.

Representative co-IP Western blot showing interaction between sfRon and the p85a subunit of PI3K in MCF7-sfRon cells treated with DMSO or increasing amounts of ASALAN002, and in tumors harvested from NOD/SCID mice bearing HCI-011 (PIK3CAWT) PDX. Mice bearing PDXs received a single dose of ASLAN002 (50 mg/kg) or vehicle and tumors were harvested 3 h later. For co-immunprecipititation studies, whole-cell extracts were immunoprecipitated with anti-p85a antibody followed by Western blot with anti-Ron antibody. Results indicate that inhibition of sfRon in both MCF7 cells and PDX tumors leads to dissociation of p85a subunit of PI3K from sfRon. The mock IP represents the same conditions as the other IPs, but without inclusion of protein lysate.

Despite advances in understanding of ovarian cancer biology, the progress in translation of research findings into new therapies is still slow. It is associated in part with limitations of commonly used cancer models such as cell lines and genetically engineered mouse models that lack proper representation of diversity and complexity of actual human tumors. In addition, the development of

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here we explore a therapeutic strategy for PCa based on the ability of sh/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3’-UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell cycle genes. When tested in mice, the toxic shRNAs also inhibited castration resistant PCa and exhibited therapeutic efficacy in pre-established tumors. This multi-targeted RNAi approach may be a promising therapeutic strategy for PCa.

The sfRon kinase is an important therapeutic target in ovarian cancer that contributes to prominent tumor growth and disease progression. We reasoned that a multi-kinase inhibition of sfRon pathway might be an effective strategy to achieve a sustained anti-tumor response, while simultaneously preventing treatment resistance. We performed a detailed dissection of sfRon signaling in vitro and demonstrated that S6K1 is a key component of a multi-kinase targeting strategy in sfRon expressing ovarian tumors. We selected AD80 compound that targets several kinases within sfRon pathway including AKT and S6K1, and compared its efficacy with inhibitors that selectively target either sfRon or PI3 kinase. Using human ovarian xenografts and clinically relevant patient-derived xenografts (PDXs), we demonstrated that in vivo treatment with single agent AD80 shows superior efficacy to a standard-care chemotherapy (cisplatin/paclitaxel), or to the direct inhibition of sfRon kinase by BMS777607. Our findings indicate that ovarian tumors expressing sfRon are most effectively treated with multi-kinase inhibitors simultaneously targeting AKT and S6K1, such as AD80, which results in long-term anti-tumor response and prevents metastasis development.

We discovered that the overexpression of mitochondrial enzyme succinate dehydrogenase (SDHA) is particularly prevalent in ovarian carcinoma and promotes highly metabolically active phenotype. Succinate dehydrogenase deficiency has been previously studied in some rare disorders. However, the role of SDHA upregulation and its impact on ovarian cancer metabolism has never been investigated, emphasizing the need for further research. We investigated the functional consequences of SDHA overexpression in ovarian cancer. Using proteomics approaches and biological assays, we interrogated protein content of metabolic pathways, cell proliferation, anchorage-independent growth, mitochondrial respiration, glycolytic function, and ATP production rates in those cells. Lastly, we performed a drug screening to identify agents specifically targeting the SDHA overexpressing tumor cells. We showed that SDHA overexpressing cells are characterized by enhanced energy metabolism, relying on both glycolysis and oxidative phosphorylation to meet their energy needs. In addition, SDHA-high phenotype was associated with cell vulnerability to glucose and glutamine deprivation, which led to a substantial reduction of ATP yield. We also identified an anti-metabolic compound shikonin with a potent efficacy against SDHA overexpressing ovarian cancer cells. Our data underline the unappreciated role of SDHA in reprogramming of ovarian cancer metabolism, which represents a new opportunity for therapeutic intervention.

High-grade serous ovarian carcinoma (HGSOC) is one of the deadliest types of cancer in the US. Occurrence of sporadic HGSOC is associated with high genomic instability, which causes amplification and deletion of several different genes. BRD4 is one of the most frequently amplified genes in ovarian carcinoma patients. BRD4 amplification leads to overexpression of BRD4 proteins including long and short BRD4 isoforms (BRD4-L and BRD4-S, respectively), and is associated with poor patient prognosis. Recently, it has been shown that the short isoform BRD4-S contributes to oncogenic properties of several tumor types; however, little is known about its role in ovarian cancer. Therefore, in order to interrogate the function of different BRD4 isoforms in HGSOC, we generated ovarian cancer cell lines stably overexpressing BRD4 isoforms (BRD4-L and BRD-S). Our data revealed that overexpression of BRD4-S in ovarian carcinoma cell lines induces a highly aggressive tumor phenotype in vitro and in vivo. Ovcar4-BRD4-S cells showed significantly higher cell proliferation rate and increased anchorage-independent growth when compared with parental cell lines. In addition, Ovcar4-BRD4-S presents a high level of DNA damage reflected by increased pH2AX foci. We also observed a disruption of cell cycle in Ovcar4-BRD4-S cells associated with a higher number of cells arrested in G2/M phase and increased population of polyploid cells. In vivo experiments demonstrated that BRD4-S expressing tumors grow significantly faster than parental tumors. BRD4-S overexpressing tumors are also very resistant to DNA-damage agents including standard of care platinum drugs. Finally, our preliminary drug screening demonstrated that Ovcar4-BRD4-S cells are highly sensitive to Wee1 inhibitor MK-1775. Furthermore, the combination of MK-1775 with paclitaxel synergistically increased the antitumor effect. In summary, our studies indicate that amplification of the BRD4 gene in HGSOC patients might lead to an increase in splicing of BRD4-S transcript, which in consequence promotes highly aggressive and platinum-resistant tumors. Precision therapies targeting BRD4 amplified tumors, such as Wee1 inhibitors in combination with paclitaxel hold great promise to improve ovarian cancer treatment and reduce chemotherapy resistance, which is a critical problem for eradication of this aggressive tumor. Citation Format: Ana Luiza Drumond-Bock, Luyao Wang, Lin Wang, Magdalena Cybula, Maria Rostworowska, Magdalena Bieniasz. Overexpression of the short isoform of Brd4 in ovarian carcinoma leads to highly aggressive tumor phenotype [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 LB542.

The reprogramming of cellular metabolism is an important hallmark of cancer allowing tumor cells to survive and proliferate. Recent data revealed that subsets of ovarian cancer with invasive and metastatic phenotype are highly metabolically active with increased mitochondrial respiration. To better understand the metabolic profiles of ovarian cancer, we utilized patient-derived xenografts (PDXs) derived from high-grade serous ovarian carcinoma (HGSOC), and performed comprehensive analysis of metabolic pathways by Reverse Phase Protein Array (RPPA). The RPPA analysis revealed PDX subsets with high or low mitochondrial activity and metabolism, and we identified succinate dehydrogenase (SDHA) to be a top differentially expressed metabolic protein between those PDX subsets. SDHA is a mitochondrial enzyme that participates in the TCA cycle and the electron transport chain, which are both essential for cellular metabolism. Analysis of the TCGA data demonstrated that SDHA gene alterations are highly prevalent in HGSOC patients (19% cases). Despite these findings, the impact of SDHA amplification on cancer biology is poorly understood. To assess the effect of SDHA overexpression on energy metabolism, we used SDHA-overexpressing ovarian cancer cell lines and measured cellular respiration by Seahorse. The results revealed that the overexpression of SDHA leads to a significant increase in basal and maximal respiration shifting cellular metabolism towards increased oxidative phosphorylation in mitochondria. Further, we determined if SDHA overexpression redirects cellular metabolism in response to deprivation of selected nutrients. In the conditions with low glucose or glutamine, SDHA overexpressing cells significantly increased basal respiration when compared with controls. In galactose medium that disrupts glycolysis, the SDHA lost its ability to induce cellular respiration, which indicates that SDHA relays on glycolysis to increase mitochondrial respiration. Together, our data indicate that SDHA requires glycolysis to fuel TCA cycle and and support cellular respiration. Next, we performed in vitro drug screening and found that SDHA overexpressing cells are highly sensitive to glycolysis inhibitor Shikonin. Our pilot in vivo data showed that Shikonin significantly reduces growth of SDHA-amplified tumors. In summary, our studies indicate that SDHA upregulation plays a role in reprogramming of cellular metabolism and pathogenesis of ovarian cancer. Moreover, the unique metabolic state of ovarian cancer associated with SDHA amplification could be successfully targetable offering a potential new treatment strategy for ovarian cancer patients. Citation Format: Magdalena Cybula, Maria Rostworowska, Lin Wang, Summer Wang, Ana Luiza Drumond-Bock, Magdalena Bieniasz. The role of succinate dehydrogenase in ovarian cancer metabolism [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 LB125.

Objective Mebendazole and other anti-parasitic drugs are being used off-prescription based on social media and unofficial accounts of their anti-cancer activity. The purpose of this study was to conduct a controlled evaluation of mebendazole's therapeutic efficacy in cell culture and in vivo models of ovarian cancer. The majority of ovarian cancers harbor p53 null or missense mutations, therefore the effects of p53 mutations and a mutant p53 reactivator, PRIMA-1MET (APR246) on mebendazole activity were evaluated. Methods Mebendazole was evaluated in cisplatin-resistant high grade serous stage 3C ovarian cancer patient derived xenograft (PDX) models: PDX-0003 (p53 null) and PDX-0030 (p53 positive), and on ovarian cancer cell lines: MES-OV (p53 R282W), ES2 (p53 S241F), A2780 (p53 wild type), SKOV3 parental (p53 null) and isogenic sublines, SKOV3 R273H p53 and SKOV3 R248W p53. Drug synergy and mechanisms were evaluated in cell cultures using isobolograms, clonogenic assays and western blots. Prevention of tumor establishment was studied in a MES-OV orthotopic model. Results Mebendazole inhibited growth of ovarian cancer cell cultures at nanomolar concentrations and PDXs at doses up to 50 mg/kg, and reduced orthotopic tumor establishment at 50 mg/kg. The mechanism of mebendazole was associated with p53-independent induction of p21 and tubule depolymerization. PRIMA-1MET also inhibited tumor establishment and worked synergistically with mebendazole in cell culture to inhibit growth and induce intrinsic apoptosis through a p53- and tubule destabilization-independent mechanism. Conclusion This work demonstrates the therapeutic potential of repurposing mebendazole and supports clinical development of mebendazole for ovarian cancer therapy and maintenance.

Auroliposome represents a highly effective siRNA delivery platform for in vitro and in vivo application., Gene silencing using small-interfering RNA (siRNA) is a viable therapeutic approach; however, the lack of effective delivery systems limits its clinical translation. Herein, we doped conventional siRNA-liposomal formulations with gold nanoparticles to create “auroliposomes,” which significantly enhanced gene silencing. We targeted MICU1, a novel glycolytic switch in ovarian cancer, and delivered MICU1-siRNA using three delivery systems—commercial transfection agents, conventional liposomes, and auroliposomes. Low-dose siRNA via transfection or conventional liposomes was ineffective for MICU1 silencing; however, in auroliposomes, the same dose gave >85% gene silencing. Efficacy was evident from both in vitro growth assays of ovarian cancer cells and in vivo tumor growth in human ovarian cell line—and patient-derived xenograft models. Incorporation of gold nanoparticles shifted intracellular uptake pathways such that liposomes avoided degradation within lysosomes. Auroliposomes were nontoxic to vital organs. Therefore, auroliposomes represent a novel siRNA delivery system with superior efficacy for multiple therapeutic applications.

Short-form Ron (sfRon) is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase. In contrast to Ron, which has been shown to be an important player in many cancers, little is known about the role of sfRon in cancer pathogenesis. Here we report the striking discovery that sfRon expression is required for development of carcinogen-induced malignant ovarian tumors in mice. We also show that sfRon is expressed in several subtypes of human ovarian cancer including high-grade serous carcinomas, which is in contrast to no detectable expression in healthy ovaries. In addition, we report that introduction of sfRon into OVCAR3 cells resulted in epithelial-to-mesenchymal transition, activation of the PI3K and PDK1 pathway, and inhibition of the MAPK pathway. We demonstrated that sfRon confers an aggressive cancer phenotype in vitro characterized by increased proliferation and migration, and decreased adhesion of ovarian cancer cells. Moreover, the in vivo studies show that OVCAR3 tumors expressing sfRon exhibit significantly more robust growth and spreading to the abdominal cavity when compared with the parental sfRon negative OVCAR3 cells. These data suggest that sfRon plays a significant role in ovarian cancer initiation and progression, and may represent a promising therapeutic target for ovarian cancer treatment.

Purpose: Recent studies have demonstrated that short-form Ron (sfRon) kinase drives breast tumor progression and metastasis through robust activation of the PI3K pathway. We reasoned that upfront, concurrent inhibition of sfRon and PI3K might enhance the antitumor effects of Ron kinase inhibitor therapy while also preventing potential therapeutic resistance to tyrosine kinase inhibitors (TKI). Experimental Design: We used patient-derived breast tumor xenografts (PDX) as high-fidelity preclinical models to determine the efficacy of single-agent or dual Ron/PI3K inhibition. We tested the Ron kinase inhibitor ASLAN002 with and without coadministration of the PI3K inhibitor NVP-BKM120 in hormone receptor–positive [estrogen receptor (ER)+/progesterone receptor (PR)+] breast PDXs with and without PIK3CA gene mutation. Results: Breast PDX tumors harboring wild-type PIK3CA showed a robust response to ASLAN002 as a single agent. In contrast, PDX tumors harboring mutated PIK3CA demonstrated partial resistance to ASLAN002, which was overcome with addition of NVP-BKM120 to the treatment regimen. We further demonstrated that concurrent inhibition of sfRon and PI3K in breast PDX tumors with wild-type PIK3CA provided durable tumor stasis after therapy cessation, whereas discontinuation of either monotherapy facilitated tumor recurrence. Conclusions: Our work provides preclinical rationale for targeting sfRon in patients with breast cancer, with the important stipulation that tumors harboring PIK3CA mutations may be partially resistant to Ron inhibitor therapy. Our data also indicate that tumors with wild-type PIK3CA are most effectively treated with an upfront combination of Ron and PI3K inhibitors for the most durable response. Clin Cancer Res; 21(24); 5588–600. ©2015 AACR.

BACKGROUND: The 5-year survival rate of patients with recurrent epithelial ovarian cancer is only 30%, in part due to relapse and resistance to first-line platinum-based chemotherapy. Even with marked progress toward understanding ovarian cancer biology, the translation of research findings into new therapies is still an enormous barrier to progress. Recent data suggests a 90% failure rate for new oncology drugs in the clinic. Development and preclinical testing of new cancer therapies has been limited by the availability of clinically relevant models that recapitulate the molecular and phenotypic characteristics of primary ovarian cancers. To overcome this barrier, we have initiated collaborative project between Stephenson Cancer Center (SCC) and Oklahoma Medical Research Foundation (OMRF) with the goal to generate a biobank of patient-derived xenograft (PDX) models derived directly from ovarian cancer patients undergoing therapy. RESULTS: PDX tumor models are generated in the Patient-Derived Xenograft and Preclinical Therapeutics (PDX-PCT) Core at OMRF and used to improve preclinical evaluation of new drugs towards more personalized medicine. We have collected blood samples and tumor tissues from consenting patients having primary debulking surgery at Stephenson Cancer Center since 2015 (>140 unique patients). Fresh tumor tissue is minced into small tumor chunks (~2 mm) and implanted into immunocompromised mice. PDXs are expanded for 3-4 passages and characterized based on tumor type, histology and molecular characteristics. Immunohistochemical and molecular analysis showed that histology of the original patient tumors as well as the expression of commonly used markers for high-grade serous ovarian cancer such as cytokeratin, PAX8 and WT1 are perfectly maintained in the derivative PDX lines. Genotyping of patients' tumors and their corresponding PDXs using Illumina's Exome Array revealed that single nucleotide polymorphism (SNP) profile found in the original tumors is perfectly maintained in PDX lines. One of the PDX limitations is that human tumor stromal and immune cells are being replaced with mouse equivalents upon in vivo propagation of PDXs, which negatively affects quality of genomic or proteomic analyses. To overcome this challenge, we optimized a method to deplete mouse cells from PDX tumors by magnetic cells separation. Our data showed that ovarian cancer PDXs contain around 28-54% of human tumor cells, where the rest are mouse cells. We also optimized protocols to utilize pure population of human tumor cells isolated from PDXs to transduce these with lentiviral vectors expressing GFP and/or luciferase to generate luciferized PDXs. Luciferized ovarian PDXs are excellent models used for non-invasive orthotopic tumor growth and drug response monitoring. CONCLUSIONS: Collection of our PDXs illustrates heterogeneity and diversity of human ovarian tumors. However, each PDX maintains essential molecular features of the original patients' tumor. Our goal is to generate clinically faithful ovarian tumor models that will provide a platform to learn more about cancer biology and to screen these for new better therapies. Citation Format: Magdalena Cybula, Katherine Moxley, Lin Wang, Luyao Wang, Magdalena Bieniasz. UTILIZATION OF PATIENT-DERIVED TUMOR XENOGRAFT (PDX) MODELS IN ONCOLOGY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-021.

Lung cancer is a particular challenge in oncology. More than 1 million new cases occur worldwide every year and despite many clinical trials and modern diagnostic techniques, long-term survival rate has only marginally improved. The aim of the current research is to explore new molecular prognostic factors and identify new targets for anticancer therapy. Current evidence shows that angiogenesis is controlled by several angiogenic factors including VEGF and BMP-2. It has been also demonstrated that VEGF plays a key role in this process that is essential in carcinogenesis. Our study has shown that the expressions of the VEGF, BMP-2 and BMP-4 mRNAs were significantly higher (7.1-fold, 25.6-fold and 2.3-fold, respectively) in lung cancer samples than in adjacent normal lung tissues (real-time RT-PCR). Analysis based on the Pearson's correlation coefficient indicated the positive correlation between VEGF and BMP-2 gene expression, whereas no significant correlation between VEGF and BMP-4 gene expression was found. The mean+/-standard deviation serum level of VEGF was 423+/-136 pg/ml. Significant differences in the serum levels of VEGF between patients with T1 tumors and patients with T2, T3 or T4 tumors were observed. Patients with T2, T3 and T4 tumors, respectively, had 1.6-fold, 1.8-fold and 2.3-fold greater serum levels of VEGF than their peers with T1 tumors. In current study patients homozygous for the 936T allele of the +936C/T VEGF gene polymorphism had 12-fold lower VEGF gene expression and 1.3-fold lower VEGF serum level than patients homozygous for the 936C allele. In conclusion, our findings underline the importance of the two angiogenic factors namely VEGF and BMP-2 as well as +936C/T VEGF gene polymorphism in the evaluation of lung cancer patients.

BACKGROUND: Although 70–80% of women respond to standard platinum-based chemotherapy, a majority of patients will develop recurrent platinum-resistant disease. Novel treatment approaches directed against key oncogenic drivers and/or the use of novel drug combinations to prevent/overcome platinum resistance are promising opportunities to combat this disease. sfRon is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase, the latter of which has been investigated in cancer for several years. Unlike full-length Ron, the sfRon protein lacks the extracellular ligand-binding domain, but organizes into a constitutively active transmembrane protein. Our in vitro and in vivo studies indicate that sfRon expression is associated with the acquisition of aggressive and invasive ovarian tumor phenotype. Such data carries additional importance because sfRon does not require ligand binding for activity, it may not be targetable using standard approaches such as antibody inhibitors. Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, might have great therapeutic benefit in ovarian tumors that express sfRon. RESULTS: Our lab has recently made the striking and unexpected discovery that sfRon expression is necessary for development of carcinogen-driven ovarian tumors in mice. Our study involved treating wild type mice vs. mice with a specific genetic deletion of sfRon (ΔsfRon) with DMBA, a known carcinogen. The study revealed that lack of sfRon completely protected the mice from ovarian cancer. The frequency of ovarian tumors was 25% in wild-type mice vs. 0% in ΔsfRon mice (p CONCLUSION: Our data implicate sfRon in ovarian cancer pathogenesis, and indicate that Ron kinase inhibitors targeting both Ron and sfRon may not only suppress ovarian tumor progression, but also have potential to overcome resistance to chemotherapy. These data could have significant impact on future clinical trials for both Ron kinase inhibitors and platinum compounds, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Katherine Moxley, Luyao Wang, Alana L. Welm. SHORT-FORM RON KINASE AS A NOVEL THERAPEUTIC TARGET IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-048.

Background: Although 70–80% of women respond to standard platinum-based chemotherapy, a majority of patients will develop recurrent platinum-resistant disease. Novel treatment approaches directed against key oncogenic drivers and/or the use of novel drug combinations to prevent/overcome platinum resistance are promising opportunities to combat this disease. Our lab has developed a strong interest in short-form Ron (sfRon) in the context of ovarian cancer pathogenesis. sfRon is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase, the latter of which has been investigated in cancer for several years. Unlike full-length Ron, the sfRon protein lacks the extracellular ligand-binding domain, but organizes into a constitutively-active transmembrane protein. Our in vitro and in vivo studies indicate that sfRon expression has more potent oncogenic activity than full-length Ron, presumably because of the constitutive kinase activity of the sfRon protein. Such data carries additional importance because sfRon does not require ligand binding for activity, appears to function in a manner distinct from full length Ron, and may not be targetable using standard approaches such as antibody inhibitors (e.g. RON8, which is now being tested in clinical trials). Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, might have great therapeutic benefit in ovarian tumors that express sfRon. Results: Our lab has recently made the striking and unexpected discovery that sfRon expression is necessary for development of carcinogen-driven ovarian tumors in mice. Our study involved treating wild type mice vs. mice with a specific genetic deletion of sfRon (ΔsfRon) with DMBA, a known carcinogen. The study revealed that lack of sfRon completely protected the mice from ovarian cancer. The frequency of ovarian tumors was 25% in wild-type mice vs. 0% in ΔsfRon mice (p Based on these findings supporting the role of sfRon in ovarian cancer, we aimed to investigate the expression of sfRon in primary ovarian samples. Our studies using ovarian tumors from patients revealed that sfRon becomes overexpressed in several different subtypes of ovarian cancer, with no expression in healthy ovarian tissue. Our studies revealed that introduction of sfRon into OVCAR-3 cells resulted in increased proliferation of tumor cells in vitro, and accelerated tumor growth in vivo as compared with parental OVCAR-3 cells. Our further studies aimed to investigate the potential clinical relevance of total Ron expression (Ron and sfRon) in a large cohort of high-grade serous ovarian cancer (HG-SOC) patients, with a particular focus on response to platinum drugs. Our data demonstrated that in the population of HG-SOC patients, Ron and/or sfRon are expressed in 86/126 (68%) of all cases, and high expression of Ron and/or sfRon was detected in 27/126 (21%) of all cases. Importantly, we also observed that significantly more patients in the cisplatin refractory/resistant group express high levels of Ron receptors (Ron and sfRon), compared to patients in the cisplatin sensitive group. Conclusion: Our data implicate sfRon in ovarian cancer pathogenesis, and indicate that Ron kinase inhibitors targeting both Ron and sfRon may not only suppress ovarian tumor progression, but also have potential to overcome resistance to chemotherapy. These data could have significant impact on future clinical trials for both Ron kinase inhibitors and platinum compounds, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Alana L. Welm. Short-form Ron kinase as a novel therapeutic target in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A20.

Receptor tyrosine kinases (RTKs) have been the subject of intense investigation due to their widespread deregulation in cancer and the prospect of developing targeted therapeutics against these proteins. The Ron RTK has been implicated in tumor aggressiveness and is a developing target for therapy, but its function in tumor progression and metastasis is not fully understood. We examined Ron activity in human breast cancers and found striking predominance of an activated Ron isoform known as short-form Ron (sfRon), whose function in breast tumors has not been explored. We found that sfRon plays a significant role in aggressiveness of breast cancer in vitro and in vivo. sfRon expression was sufficient to convert slow-growing, nonmetastatic tumors into rapidly growing tumors that spontaneously metastasized to liver and bones. Mechanistic studies revealed that sfRon promotes epithelial-mesenchymal transition, invasion, tumor growth, and metastasis through interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K activity, or introduction of a single mutation in the p85 docking site on sfRon, completely eliminated the ability of sfRon to promote tumor growth, invasion, and metastasis. These findings reveal sfRon as an important new player in breast cancer and validate Ron and PI3K as therapeutic targets in this disease.

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fcγ2a) to deliver the antigenic cassette to the activating Fcγ receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fcγ2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fcγ2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

Given the recent findings on the importance of CD38 signaling in the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), we hypothesized that single nucleotide polymorphisms (SNP) in the CD38 gene may be related to B-CLL risk. We evaluated two potentially functional CD38 SNPs, intronic rs6449182 (184C>G) and missense rs1800561 (418C>T, Arg140Trp) in two hospital-based case-control studies (study A and validation study B). Genotyping was done using PCR-based assays in a total of 460 Polish Caucasian patients with B-CLL and 503 age-matched and gender-matched controls. We found that frequencies of both variant alleles (rs6449182 G and rs1800561 T) were significantly higher in B-CLL. In study A, logistic regression analysis revealed an association between B-CLL and genotypes: rs6449182 CG [odds ratio (OR), 3.57; 95% confidence interval (95% CI), 2.4-5.3], rs6449182 GG (OR, 5.2; 95% CI, 2.36-11.5), and rs1800561 CT (OR, 6.72; 95% CI, 1.5-30.1), although no homozygous rs1800561 TT genotype was detected in either study. These results were confirmed in study B, which showed an association between B-CLL and genotypes rs6449182 CG (OR, 4.00; 95% CI, 2.7-6.0), rs6449182 GG (OR, 12.84; 95% CI, 4.3-38.7), and rs1800561 CT (OR, 10.12; 95% CI, 1.3-81.6), and in the combined analysis of both studies. We also observed that rs6449182 G carriers had more advanced clinical stage (P = 0.002) and tended to be younger at diagnosis (P = 0.056). Furthermore, we found higher CD38 transcript levels and higher proportions of CD38-positive cells in carriers of rs6449182 G and rs1800561 T alleles (P < 0.05 for all comparisons). In conclusion, our data show that CD38 SNPs may affect CD38 expression and contribute to the increased risk of B-CLL carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2009;18(3):945–53)

In this study we examined the effects of exogenous nitric oxide (sodium nitroprusside, SNP) and hydrogen peroxide (H2O2) on the expression level of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVEC). The expression of selected genes involved in fibrynolysis under the influence of oxidative stress was analyzed at the levels of mRNA, protein, and promoter activity. The results of the conducted studies revealed that oxidative stress in endothelial cells causes a significant increase in PAI-1 and u-PAR expression and a moderate increase in t-PA and u-PA expression at all of the investigated levels. We attempted to elucidate the molecular signaling mechanisms by which SNP and H2O2 regulate expression of the respective fibrinolytic factors. Therefore, we tested the protein levels of AP-1, NF-κB, and HIF-1 and their DNA-binding activity in endothelial cells subjected to oxidative stress. We found strong correlation between AP-1, NF-κB, and HIF-1 in the contribution of regulation of selected genes. In addition, we also found that the inhibition of PAI-1 synthesis by antisense oligonucleotide to PAI-1 mRNA results in markedly increased u-PAR expression and that NF-κB and AP-1 are involved in this regulation.

Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are one of the molecules that have become a topic of great interest among scientists studying lung cancers. There is a distinct tendency toward higher expression of selected MMP and TIMP in tumor lung tissue. Furthermore, there is a significant correlation between high expression of TIMP-1 or MMP-2 in lung cancer and shortened survival and between high expression of TIMP-1 or MMP-7 in lung cancer and higher stage of disease. There have been only a few articles about the role of bone morphogenetic proteins (BMP) in lung cancer pathogenesis published so far in which BMP-2 or BMP-4 were overexpressed. It was also shown that BMP-2 stimulates tumor growth while BMP-4 inhibits it. This article is mainly concentrated on the expression of MMP, TIMP and BMP in lung cancers, but also it shows the significance of these proteins.

Introduction: Receptor tyrosine kinases (RTKs) and their pleiotropic effectors are key factors in metastasis. Met and Ron are RTKs that have been shown to contribute to tumor growth and metastasis in both animal models and in patients. Ron is over-expressed in many epithelial cancers, but not usually amplified or mutated. Recently, an alternative Ron isoform known as “short-form” Ron (sfRon) was associated with aggressive phonotype of breast cancer. sfRon is a 55-kD N-terminally truncated form of Ron. It lacks most of the extracellular domain of Ron, but contains intact transmembrane and cytoplasmic domains. The expression of this alternative Ron isoform yields constitutively active protein. sfRon plays a crucial role in the aggressiveness of breast cancer; its expression significantly promotes tumor growth and spontaneous metastasis to distant organs. We observed that sfRon strongly signals through the PI3K pathway and that interaction between sfRon and PI3K is required for sfRon function. Blocking the interaction between sfRon and PI3K, or directly blocking PI3K activity, thoroughly abrogates the ability of sfRon to confer aggressive tumor behavior in vitro. The PI3K signaling cascade regulates many cellular functions that are mainly associated with cell proliferation, survival, and migration. PI3K is activated in a wide variety of human malignancies including breast cancer, resulting in tumor development and progression. The PI3K pathway has been identified as an important target in breast cancer research for a number of years. In our study we utilized NVP-BEZ235 and NVP-BKM120, compounds provided by Novartis. NVP-BEZ235 is a dual PI3K/mTOR inhibitor and NVP-BKM120 is a pan-PI3K inhibitor. Both compounds exhibit potent antiproliferative and proapoptotic activities in a variety of tumor cell lines as well as in animal models. Currently, NVP-BKM120 and NVP-BEZ235 compounds are undergoing phase I/II clinical trials in patients with cancer, including breast malignancy. For in vivo studies, we used highly tumorigenic and invasive MCF7 breast cancer cells. Our cell line overexpresses sfRon and is characterized by high levels of active PI3K. We have shown before that sfRon overexpression triggers activation of PI3K signaling, which is necessary for sfRon aggressive behavior. Our goal is to test whether inhibition of PI3K pathway, which signals downstream from sfRon, will have any effect on this aggressive tumor growth in vivo. Methods: Three groups of NOD/SCID female mice were injected orthotopically with 1x106 sfRon-overexpressing MCF7 cells suspended in Matrigel. Treatment was initiated when tumors reached 500 mm3 in size. Mice were treated for 3 weeks daily by oral gavage with 45 mg/kg of NVP-BEZ235, 60 mg/kg of NVP-BKM120, or vehicle. Tumor size was monitored every three days with caliper measurements. Treatment continued until mice had grown tumors up to the size limit. Results: The data showed that both drugs significantly inhibited mammary tumor growth in comparison to mice treated with vehicle. We observed the most dramatic inhibitory effect on tumor growth in the group of mice treated with NVP-BKM120. Three weeks after treatment initiation, NVP-BKM120-treated mice had an average tumor size of 405 mm3. Meanwhile, mice treated with NVP-BEZ235 and vehicle had tumors that averaged 1182 mm3 and 3007 mm3, respectively. Conclusion: Our data shows that PI3K pathway inhibitors are very well tolerated by mice and are excellent in the inhibition of tumor growth driven by sfRon, especially NVP-BKM120. NVP-BEZ235 and NVP-BKM120 compounds hold great promise in the treatment of breast cancer overexpressing sfRon with strongly activated PI3K. Citation Format: Parvathi Radhakrishnan, Magdalena Bieniasz, Alana Welm. Phosphatidylinositol 3 kinase (PI3K) inhibitors as therapeutic agents for breast tumors overexpressing sfRon. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B053.

Since its discovery in 1993, various investigations have explored different roles of RON receptor tyrosine kinase in cancer pathogenesis. RON (also known as MST1R), a receptor tyrosine kinase with significant homology to c-Met, exhibits increased expression in at least 47% of human breast cancers, and deregulated expression of the RON signaling axis has been correlated with reduced metastasis-free survival. RON is known to activate multiple signaling pathways in vitro, including MAPK, phosphatidylinositol-3 kinase (PI3K), Src tyrosine kinase, phospholipase C-γ (PLC-γ) and β-catenin, largely through direct binding of SH2-domain containing adaptor proteins to the C-terminal docking site. However, the relationships of each of these signal transduction pathways to specific biologic responses relevant to cancer progression and metastasis are still poorly defined. To this end, we have taken advantage of a strategy used for c-Met in order to increase the binding affinity of individual adaptors to the docking site, which promotes signaling through one pathway at the expense of the others. The C-terminal part of RON contains two tandem tyrosines in a degenerate motif that when phosphorylated bind different SH2-containing molecules in a competitive manner. To amplify the signaling through particular pathways, we mutated the multifunctional docking site of RON to optimal binding motifs for PI3K, Src, Grb2 or PLC-γ. Mutations were introduced to the docking site using pMSCV retroviruses expressing human wild type Ron cDNA. As a negative control, two essential tyrosines in the docking site were replaced by phenylalanine to eliminate the transforming capability of RON. Viral transduction of the generated mutants or wild type RON was performed in MCF-7 and T47D breast cancer cells and LoVo colon cancer cells. Analysis of mRNA and protein showed the same expression levels among the mutants and wild type infected cells, indicating similar stability of the mutants compared with WT-RON. We report an analysis of RON signaling through the aforementioned pathways in the presence and absence of its ligand, macrophage stimulating protein (MSP). Our data indicate that the MAPK, PI3K, PLC-γ and Src pathways are differentially activated in the various cellular contexts we tested. Moreover, the activation of each signaling pathway occurs with different kinetics and with differential responses to MSP concentration. Citation Format: Najme Faham, Magdalena Bieniasz, Alana Welm. Contributions of RON downstream signaling to breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A129.

Background: Our recent data revealed striking predominance of an alternatively transcribed form of the Ron receptor tyrosine kinase, known as short-form Ron (sfRon), in breast cancer. sfRon is the major active Ron isoform in breast tumors from patients, and has remarkable effects on metastasis. sfRon lacks the extracellular ligand-binding domain, but organizes into a constitutively-active receptor. Our in vitro studies revealed that overexpression of sfRon in breast cancer cell lines promotes aggressive phenotypes including an epithelial-mesenchymal transition and invasion through basement membranes. We observed that sfRon strongly signals through the PI3K pathway and that interaction between sfRon and PI3K was required for sfRon function. In vivo studies revealed striking results that expression of sfRon was sufficient to convert slow growing non-metastatic tumors into fast growing tumors that spontaneously metastasized from the mammary gland to liver, spleen and bones. An increased understanding of how sfRon contributes to tumor progression and metastasis is critical. Such data carries additional importance because sfRon does not require ligand binding for activity, appears to function in a manner distinct from full length Ron, and may not be targetable using standard approaches such as antibody inhibitors (e.g. RON8, which is now being tested in clinical trials). Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, would be preferable and that inhibition of downstream PI3K might also be effective in breast tumors expressing sfRon. Results: Through collaborations with various pharmaceutical companies we have obtained several compounds that specifically target Ron/sfRon kinase (e.g. ASLAN002) or PI3 kinase (e.g. BKM120). Our in vitro data demonstrate that ASLAN002 inhibits the activity of sfRon kinase in MCF7 breast cancer cells. The drug also completely blocked PI3K signaling downstream of sfRon. Our studies also showed BKM120 to be a potent inhibitor of PI3K pathway in sfRon-expressing breast cancer cells, effective in doses as low as 250 nM. We have also shown that, while the RON8 antibody inhibits activity of full length Ron kinase, it is not effective against sfRon activity. Based on these promising in vitro results, we extended our work to in vivo assays. We used NOD/SCID mice to orthotopically transplant sfRon-expressing MCF7 cells. After tumors reached 200mm3 we initiated treatment every other day with vehicle control, RON8 antibody, ASLAN002, BKM120, or ASLAN002+BKM120 combination therapy. Our data showed that although the RON8 antibody designed to target Ron is not effective against tumors expressing sfRon, monotherapy with ASLAN002 or BKM120 significantly inhibited tumor growth. However, the most effective treatment regimen was ASLAN002+BKM120 combination therapy. Most importantly, we also observed reduced formation of metastasis in mice treated with ASLAN002, BKM120, or the combination therapy compared to vehicle controls (% of spontaneous metastasis: 44%, 44%, and 33% vs 75%, respectively). Conclusion: Our experimental approach supports the novel concept to use Ron and PI3K pathway inhibitors as potential new therapeutic strategies in breast cancer. These data could have significant impact on future clinical trials for both Ron and PI3K inhibitors, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Najme Faham, Alana L. Welm. The sfRon kinase as a potential novel target for breast cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A049.

To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of (125)I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.