Erica A. Golemis, PhD
Office Phone: 215-728-2860
1. Evaluation of HSP90-inhibitor drug conjugates (HDCs) to treat small cell lung cancerAnna Gaponova, Anna Nikovona & Alexander Deneka, in collaboration with Hossein Borghaei & James Duncan
Small cell lung cancer (SCLC) has a 5 year survival rate of only 6%, and 30,000 new cases are diagnosed in the United States annually. No protein-targeted therapies have proven effective; rather, carboplatin and etoposide for initial treatment are followed by topotecan or irinotecan (topoisomerase I inhibitors) after relapse. To improve the therapeutic efficacy of cytotoxic agents while eliminating dose-limiting toxicity, the novel compound STA-12-8666 combines a targeting moiety that binds active heat shock protein 90 (HSP90) with a cleavable linker attached to SN-38, the active metabolite of irinotecan. As HSP90 is strongly induced and activated in tumors agents binding HSP90 are concentrated in tumors at levels >30-fold than in normal tissue. Cleavage of the linker within the tumor provides time-release of SN-38 at high local concentration, while significantly limiting drug exposure and toxicity in non-transformed issue. The goal for this work was to evaluate STA-12-8666 for potential use as a new second line monotherapy, or as adjuvant in the frontline setting. Data in sum suggest exceptional potency of STA-12-8666 as a monoagent in multiple SCLC models, with three weekly doses resulting in clearance of palpable tumors, with durable effect (>2 months). Low dose STA-12-8666 substantially augmented the activity of other chemotherapy agents, such as carboplatin. Higher dose STA-12-8666 was effective at controlling or eliminating tumors pre-treated with and resistant to irinotecan or platinum compounds. No weight loss or toxicities were observed. Profiling of tumors treated for short periods with STA-12-8666 showed robust induction of intratumoral toxicity. Candidate response biomarkers are under investigation.These results indicate that STA-12-8666 is a promising therapy in both frontline and salvage settings for treating patients with this disease. These data strongly support the evaluation of this compound in Phase I/II clinical trials.
2. Analysis of pro-survival signaling networks and epithelial-to-mesenchymal transition, to predict effective therapeutic strategiesTim Beck & Ilya Serebriiskii, in collaboration with David Proia
Epithelial-to-mesenchymal transition (EMT) is a critical process during development and wound healing, and is commonly re-activated in primary tumors, promoting tissue invasion and therapy resistance. HSP90 is a molecular chaperone with a plethora of ‘clients’, many of which are also involved in cell proliferation and EMT and function within the EGFR and TGF-ß signaling cascades; many cancer cells upregulate HSP90 and are dependent on its action. HSP90 inhibitors such as ganetespib are showing considerable promise in the clinic. In this project, we have used RNAi screening to identify genes influencing lung cancer cell resistance to HSP90 inhibition. Interestingly, depletion of specific members of the TGF- superfamily previously not thought to be expressed or active in lung cancer cells strongly sensitized cells to HSP90 inhibition in RAS-mutated and EML4-ALK expressing cell lines. Mechanistically, depressed activity of NF-κB and AKT, both critical EMT and survival regulators, was observed. Further detailed investigation of the mechanism of sensitization is in progress to establish how this observation can be exploited therapeutically.
3. New regulators of cisplatin resistance in head and neck cancersIlya Serebriiskii & Anna Gaponova, in collaboration with Ranee Mehra
Squamous cell carcinomas of the head and neck (SCCHN) are a heterogeneous group of cancers with global incidence 550 000 worldwide per year and an annual mortality rate of 271,000. Use of tobacco and alcohol, UV light, particular chemicals used in certain workplaces have been implicated as causative risk factors in the pathogenesis of SCCHN. A major problem in treatment of SCCHN is the emergence of resistance to radio- and chemotherapy. Cisplatin and ionizing radiation are commonly used for treatment of SCCHN. Both have similar effects on DNA, causing formation of double-strand breaks (DSBs), which in turn activates DNA Damage Response (DDR) signaling pathways. A better understanding of the cellular response to DNA damage would be helpful in improving disease management.
To gain insight into human DDR, we took advantage of the fact that some of the signaling pathways involved in this process are evolutionarily ancient, and similar in yeast and human cells. We performed bioinformatic analysis which identified 122 human genes with orthologs that regulate resistance to cisplatin or related DNA-damaging agents in Saccharomyces cerevisiae. To evaluate candidates for human regulators of DDR identified through this process, we then used siRNA knockdown in a panel of SCCHN cell lines, to determine if they regulated cell sensitivity to cisplatin. Among positive, validated new DDR regulators, we have subsequently measured effect of gene knockdown on formation of phosphorylated γH2AX histone foci formation (a sensitive marker for DSBs), for expression and activation of previously known DDR-associated proteins, and for effects on cell cycle progression. Analysis is in progress.
4. Constitutional defects in suppression of DNA double strand breaks (DSB) in familial colon cancerSanjeevani Arora & Ilya Serebriiskii, in collaboration with Tim Yen, Michael Hall & Roland Dunbrack, Jr
Despite progress in screening and treatment, colorectal cancer (CRC) remains the second most common fatal cancer in the United States. Some 20-30% of such cases are familial (FCRC). Genetic predispositions have been defined for some 25% of FCRC (including Lynch syndrome, Familial Adenomatous Polyposis (FAP), and MutYH polyposis). However, most FCRC remains formally undiagnosed, reflecting serious gaps in our understanding of disease pathogenesis.
We hypothesized that a substantial subset of uFCRC would resemble other subsets of FCRC in being caused by genetic instability but comprise a specific new class: constitutional genomic instability (CGI), due to an excess of double-strand DNA breaks (DSBs) and chromosomal defects. We further hypothesized that the responsible lesions would be heterozygous, resulting in haploinsufficiency, dominant negative effects, and/or important genetic interactions.
The aim of this project is to genetically define cases of undefined familial colorectal cancer (uFCRC). As a resource to test these ideas, we assessed individuals in the Fox Chase Cancer Center Risk Assessment Program (RAP). Exome-sequencing analysis was combined with experimental assays using patient primary lymphocytes to provide evidence for the existence of a broad constitutional genomic instability (CGI) in uFCRC due to a defect in suppression of DNA double-strand breaks (DSBs). Alterations in candidate genes are interrogated for impact on genome, protein, and cell function using bioinformatics, specialized molecular modeling, and molecular biology assays. Based on our experience on data mining for model organisms, we also identified candidate disruptive mutations in genes lacking extensive prior functional characterization in humans. Finally, we analyzed the expression of these genes in the context of larger databases for sporadic CRC in the TCGA/cBioPortal, and the prevalence of these alterations in general population using 1000 genomes database. The results from these studies would help identify novel genetic subsets of FCRC that could potentially help predict risk within families and improve screening and diagnosis, and substantially enlarge consideration of possible risk factors for FCRC.
5. Novel regulatory mechanism in Wee1 modulating DNA damage repair responsesSanjeevani Arora
Wee1 is a central inhibitor of mitotic entry, and functions by inactivating Cyclin dependent kinase 1 (Cdk1). Wee1 inhibitors are undergoing clinical development, to augment the efficacy of cytotoxic chemotherapeutic agents by driving tumor cells into lethal mitoses. Recent studies suggest that Wee1 also has a role in S phase progression, but the mechanism and importance of this activity are poorly understood. In previous work, we identified a conserved regulatory region of Wee1 that presents overlapping binding sites for cyclin A/Cdk complexes (RxL, residues 182-184) and the nuclear exporter Crm1 (nuclear export sequence (NES), residues 175-184). Using knockin reagents that specifically disrupt this motif, we are studying the regulation of Wee1 export through phosphorylation near the NES, and how it differentially impacts response to different DNA damaging agents.
6. Contrasting signaling in cancer and polycystic kidney disease: insights to improve therapyAnna Nikovona, Alexander Deneka & Anna Gaponova, in collaboration with Stefan Somlo
Autosomal dominant polycystic kidney disease (ADPKD) affects about 1:500 individuals and typically manifests in middle age. 50% of affected individuals develop progressive cyst formation and ultimate loss of renal function, culminating in end stage renal disease that requires dialysis or kidney transplant as treatment. Genetically, ADPKD is caused by mutational inactivation of polycystins 1 and 2 (PC1 and PC2, encoded by PKD1 and PKD2). Importantly, on a molecular level, ADPKD has many features in common with cancer, with elevated activity of proteins such as HER2, SRC, ERK, and mTOR important in disease pathogenesis. However, in spite of many similarities, ADPKD cysts do not metastasize. We view comparative study of ADPKD and cancer as having two potential benefits: better understanding of the basis of metastasis, and better ability to apply targeted therapies.
The proteins PC1 and PC2 function at the cilium, a small antenna-like structure protruding from the cell surface that receives and interprets mechanical and soluble signals that influence cell growth. An increasing number of mutations in genes regulating ciliary function have been found to impact cystogenesis. We have recently shown that the oncogenes NEDD9/HEF1 and its partner Aurora-A (AURKA) have multiple properties relevant to PKD. NEDD9 activation of AURKA at the ciliary basal body controls ciliary disassembly. NEDD9 and AURKA also bind and regulate the activity of the PC2 calcium channel (influencing cytoplasmic Ca2+), and are abundant and periodically active in normal kidneys, with AURKA expression and activity elevated in early PKD-associated renal cysts.
We have used a conditional PKD1-floxed mouse model crossed to a NEDD9 knockout mouse model to investigate the effect of eliminating NEDD9 expression on cyst formation in vivo. We used a number of different approaches to study in vivo cystogenesis, including magnetic resonance imaging (MRI), scanning electron microscopy (SEM), and immunohistochemical analysis. To more exactly analyze relevant signaling changes, we are further studying the role of NEDD9 in cystogenesis using primary kidney cells isolated from Pkd1-/- and Pkd1-/-Nedd9-/- mice.
This cystogenic effect is associated with striking morphological defects in the cilia of Pkd1-/-;Nedd9-/- mice, associated with specific loss of ciliary localization of adenylase cyclase III in the doubly mutant genotype. Ciliary phenotypes imply a failure of Aurora-A activation: compatible with this idea, Pkd1-/-;Nedd9-/- mice had ciliary resorption defects, and treatment of Pkd1-/- mice with a clinical Aurora-A kinase inhibitor exacerbated cystogenesis. In addition, activation of the ADPKD-associated signaling effectors Src, Erk, and the mTOR effector S6 was enhanced, and Ca2+ response to external stimuli reduced, in Pkd1-/-;Nedd9-/- versus Pkd1-/- mice.
Together, these results indicated an important modifier action of Nedd9 on ADPKD pathogenesis involving failure to activate Aurora-A. As important implications, clinical inhibitors of Aurora-A also intensified ADPKD induced by mutation of PKD1, suggesting caution in use of these agents.Combination therapy of alisertib and erlotinib, an inhibitor of EGFR-dependent proliferative signaling, is currently showing promise in clinical trials. To better understand the action of Aurora-A and its inhibitors, we have explored the interaction of alisertib and erlotinib in an ADPKD model. Results suggest that erlotinib restrained cystogenesis, and limited the negative effects associated with alisertib used as a single agent in an ADPKD setting.We are working to define the mechanism of this interaction, assessing inhibition of direct drug targets and Aurora-A and EGFR-dependent signaling pathways. We are also exploring the role of this combination in regulation of ciliary morphology, Ca2+, and canonical Aurora-A functions in mitosis.
Finally, many of the proteins that are abnormally active in ADPKD are clients of HSP90, similar to the situation in cancer. Excitingly, we have recently shown that inhibition of the chaperone HSP90 effectively slows cystogenesis in Pkd1-/- and Pkd2-/- mice. We are currently investigating the role of HSP90 inhibition in regulating ciliary dynamics, and evaluating if combining HSP90 inhibitor with a glycolytic inhibitor, 2-deoxyglucose (2DG) that has also been proposed as a therapeutic agent for ADPKD, would have a synergistic action in controlling this disease.