Now showing items 41-60 of 8554

    • The Centrosomal Kinase Plk1 Localizes to the Transition Zone of Primary Cilia and Induces Phosphorylation of Nephrocystin-1

      Fox Chase Cancer Center (Temple University) (2012-06-11)
      Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.
    • Inhibiting Heat Shock Protein 90 (HSP90) Limits the Formation of Liver Cysts Induced by Conditional Deletion of Pkd1 in Mice

      Fox Chase Cancer Center (Temple University) (2014-12-04)
      Polycystic liver disease (PLD) occurs in 75–90% of patients affected by autosomal dominant polycystic kidney disease (ADPKD), which affects 1∶400–1,000 adults and arises from inherited mutations in the PKD1 or PKD2 genes. PLD can lead to bile duct obstructions, infected or bleeding cysts, and hepatomegaly, which can diminish quality of life. At present, no effective, approved therapy exists for ADPKD or PLD. We recently showed that inhibition of the molecular chaperone heat shock protein 90 (HSP90) with a small molecule inhibitor, STA-2842, induced the degradation of multiple HSP90-dependent client proteins that contribute to ADPKD pathogenesis and slowed the progression of renal cystogenesis in mice with conditional deletion of Pkd1. Here, we analyzed the effects of STA-2842 on liver size and cystic burden in Pkd-/- mice with established PLD. Using magnetic resonance imaging over time, we demonstrate that ten weeks of STA-2842 treatment significantly reduced both liver mass and cystic index suggesting selective elimination of cystic tissue. Pre-treatment cystic epithelia contain abundant HSP90; the degree of reduction in cysts was accompanied by inhibition of proliferation-associated signaling proteins EGFR and others, and induced cleavage of caspase 8 and PARP1, and correlated with degree of HSP90 inhibition and with inactivation of ERK1/2. Our results suggest that HSP90 inhibition is worth further evaluation as a therapeutic approach for patients with PLD.
    • Adaptors for disorders of the brain? The cancer signaling proteins NEDD9, CASS4, and PTK2B in Alzheimer’s disease

      Fox Chase Cancer Center (Temple University) (2014-07-23)
      No treatment strategies effectively limit the progression of Alzheimer’s disease (AD), a common and debilitating neurodegenerative disorder. The absence of viable treatment options reflects the fact that the pathophysiology and genotypic causes of the disease are not well understood. The advent of genome-wide association studies (GWAS) has made it possible to broadly investigate genotypic alterations driving phenotypic occurrences. Recent studies have associated single nucleotide polymorphisms (SNPs) in two paralogous scaffolding proteins, NEDD9 and CASS4, and the kinase PTK2B, with susceptibility to late-onset AD (LOAD). Intriguingly, NEDD9, CASS4, and PTK2B have been much studied as interacting partners regulating oncogenesis and metastasis, and all three are known to be active in the brain during development and in cancer. However, to date, the majority of studies of these proteins have emphasized their roles in the directly cancer relevant processes of migration and survival signaling. We here discuss evidence for roles of NEDD9, CASS4 and PTK2B in additional processes, including hypoxia, vascular changes, inflammation, microtubule stabilization and calcium signaling, as potentially relevant to the pathogenesis of LOAD. Reciprocally, these functions can better inform our understanding of the action of NEDD9, CASS4 and PTK2B in cancer.
    • A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer

      Fox Chase Cancer Center (Temple University) (2015-12-04)
      Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity.
    • Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization

      Fox Chase Cancer Center (Temple University) (2015-05-28)
      Background: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. Methods: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. Results: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. Conclusions: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.
    • Genomic insights into head and neck cancer

      Fox Chase Cancer Center (Temple University) (2016-06-03)
      Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is frequently impervious to curative treatment efforts. Similar to other cancers associated with prolonged exposure to carcinogens, HNSCCs often have a high burden of mutations, contributing to substantial inter- and intra-tumor heterogeneity. The heterogeneity of this malignancy is further increased by the rising rate of human papillomavirus (HPV)-associated (HPV+) HNSCC, which defines an etiological subtype significantly different from the more common tobacco and alcohol associated HPV-negative (HPV-) HNSCC. Since 2011, application of large scale genome sequencing projects by The Cancer Genome Atlas (TCGA) network and other groups have established extensive datasets to characterize HPV- and HPV+ HNSCC, providing a foundation for advanced molecular diagnoses, identification of potential biomarkers, and therapeutic insights. Some genomic lesions are now appreciated as widely dispersed. For example, HPV- HNSCC characteristically inactivates the cell cycle suppressors TP53 (p53) and CDKN2A (p16), and often amplifies CCND1 (cyclin D), which phosphorylates RB1 to promote cell cycle progression from G1 to S. By contrast, HPV+ HNSCC expresses viral oncogenes E6 and E7, which inhibit TP53 and RB1, and activates the cell cycle regulator E2F1. Frequent activating mutations in PIK3CA and inactivating mutations in NOTCH1 are seen in both subtypes of HNSCC, emphasizing the importance of these pathways. Studies of large patient cohorts have also begun to identify less common genetic alterations, predominantly found in HPV- tumors, which suggest new mechanisms relevant to disease pathogenesis. Targets of these alterations including AJUBA and FAT1, both involved in the regulation of NOTCH/CTNNB1 signaling. Genes involved in oxidative stress, particularly CUL3, KEAP1 and NFE2L2, strongly associated with smoking, have also been identified, and are less well understood mechanistically. Application of sophisticated data-mining approaches, integrating genomic information with profiles of tumor methylation and gene expression, have helped to further yield insights, and in some cases suggest additional approaches to stratify patients for clinical treatment. We here discuss some recent insights built on TCGA and other genomic foundations.
    • Identification of evolutionarily conserved DNA damage response genes that alter sensitivity to cisplatin

      Fox Chase Cancer Center (Temple University) (2016-11-15)
      Ovarian, head and neck, and other cancers are commonly treated with cisplatin and other DNA damaging cytotoxic agents. Altered DNA damage response (DDR) contributes to resistance of these tumors to chemotherapies, some targeted therapies, and radiation. DDR involves multiple protein complexes and signaling pathways, some of which are evolutionarily ancient and involve protein orthologs conserved from yeast to humans. To identify new regulators of cisplatin-resistance in human tumors, we integrated high throughput and curated datasets describing yeast genes that regulate sensitivity to cisplatin and/or ionizing radiation. Next, we clustered highly validated genes based on chemogenomic profiling, and then mapped orthologs of these genes in expanded genomic networks for multiple metazoans, including humans. This approach identified an enriched candidate set of genes involved in the regulation of resistance to radiation and/or cisplatin in humans. Direct functional assessment of selected candidate genes using RNA interference confirmed their activity in influencing cisplatin resistance, degree of γH2AX focus formation and ATR phosphorylation, in ovarian and head and neck cancer cell lines, suggesting impaired DDR signaling as the driving mechanism. This work enlarges the set of genes that may contribute to chemotherapy resistance and provides a new contextual resource for interpreting next generation sequencing (NGS) genomic profiling of tumors.
    • Phospho-T356RB1 predicts survival in HPV-negative squamous cell carcinoma of the head and neck

      Fox Chase Cancer Center (Temple University) (2015-05-29)
      Locally advanced squamous cell carcinoma of the head and neck (SCCHN) that is not associated with human papillomavirus (HPV) has a poor prognosis in contrast to HPV-positive disease. To better understand the importance of RB1 activity in HPV-negative SCCHN, we investigated the prognostic value of inhibitory CDK4/6 phosphorylation of RB1 on threonine 356 (T356) in archival HPV-negative tumor specimens from patients who underwent surgical resection and adjuvant radiation. We benchmarked pT356RB1 to total RB1, Ki67, pT202/Y204ERK1/2, and TP53, as quantified by automatic quantitative analysis (AQUA), and correlated protein expression with tumor stage and grade. High expression of pT356RB1 but not total RB1 predicted reduced overall survival (OS; P = 0.0295), indicating the potential relevance of post-translational phosphorylation. Paired analysis of The Cancer Genome Atlas (TCGA) data for regulators of this RB1 phosphorylation identified loss or truncating mutation of negative regulator CDKN2A (p16) and elevated expression of the CDK4/6 activator CCND1 (cyclin D) as also predicting poor survival. Given that CDK4/6 inhibitors have been most effective in the context of functional RB1 and low expression or deletion of p16 in other tumor types, these data suggest such agents may merit evaluation in HPV-negative SCCHN, specifically in cases associated with high pT356RB1
    • Systematic evaluation of underlying defects in DNA repair as an approach to case-only assessment of familial prostate cancer

      Fox Chase Cancer Center (Temple University) (2015-10-14)
      familial prostate cancer, whole exome sequencing, DNA damage response, genetic susceptibility to prostate cancer, case-only study
    • NSD1- and NSD2-damaging mutations define a subset of laryngeal tumors with favorable prognosis

      Fox Chase Cancer Center (Temple University) (2017-11-24)
      Squamous cell carcinomas of the head and neck (SCCHN) affect anatomical sites including the oral cavity, nasal cavity, pharynx, and larynx. Laryngeal cancers are characterized by high recurrence and poor overall survival, and currently lack robust molecular prognostic biomarkers for treatment stratification. Using an algorithm for integrative clustering that simultaneously assesses gene expression, somatic mutation, copy number variation, and methylation, we for the first time identify laryngeal cancer subtypes with distinct prognostic outcomes, and differing from the non-prognostic laryngeal subclasses reported by The Cancer Genome Atlas (TCGA). Although most common laryngeal gene mutations are found in both subclasses, better prognosis is strongly associated with damaging mutations of the methyltransferases NSD1 and NSD2, with findings confirmed in an independent validation cohort consisting of 63 laryngeal cancer patients. Intriguingly, NSD1/2 mutations are not prognostic for nonlaryngeal SCCHN. These results provide an immediately useful clinical metric for patient stratification and prognostication.
    • Combined inhibition of Aurora A and p21-activated kinase 1 as a new treatment strategy in breast cancer

      Fox Chase Cancer Center (Temple University) (2019-06-28)
      Purpose: The serine-threonine kinases Aurora A (AURKA) and p21-activated kinase 1 (PAK1) are frequently overexpressed in breast tumors, with overexpression promoting aggressive breast cancer phenotypes and poor clinical outcomes. Besides the well-defined roles of these proteins in control of cell division, proliferation, and invasion, both kinases support MAPK kinase pathway activation and can contribute to endocrine resistance by phosphorylating estrogen receptor alpha (ERα). PAK1 directly phosphorylates AURKA and its functional partners, suggesting potential value of inhibiting both kinases activity in tumors overexpressing PAK1 and/or AURKA. Here, for the first time, we evaluated the effect of combining the AURKA inhibitor alisertib and the PAK inhibitor FRAX1036 in preclinical models of breast cancer. Methods: Combination of alisertib and FRAX1036 was evaluated in a panel of 13 human breast tumor cell lines and BT474 xenograft model, with assessment of the cell cycle by FACS, and signaling changes by immunohistochemistry and Western blot. Additionally, we performed in silico analysis to identify markers of response to alisertib and FRAX1036. Results: Pharmacological inhibition of AURKA and PAK1 synergistically decreased survival of multiple tumor cell lines, showing particular effectiveness in luminal and HER2-enriched models, and inhibited growth and ERα-driven signaling in a BT474 xenograft model. In silico analysis suggested cell lines with dependence on AURKA are most likely to be sensitive to PAK1 inhibition. Conclusion: Dual targeting of AURKA and PAK1 may be a promising therapeutic strategy for treatment of breast cancer, with a particular effectiveness in luminal and HER2-enriched tumor subtypes.
    • An improved method of delivering a sclerosing agent for the treatment of malignant pleural effusion

      Fox Chase Cancer Center (Temple University) (2019-06-24)
      Background: Malignant pleural effusion (MPE) is a devastating sequela associated with cancer. Talc pleurodesis is a common treatment strategy for MPE but has been estimated to be unsuccessful in up to 20–50% of patients. Clinical failure of talc pleurodesis is thought to be due to poor dispersion. This monograph reports the development of a foam delivery system designed to more effectively coat the pleural cavity. Methods: C57BL/6 mice were injected with Lewis lung carcinoma (LL/2) cells intrapleurally to induce MPE. The mice then received either normal saline (NS) control, foam control (F), talc slurry (TS, 2 mg/g) or talc foam (TF, 2 mg/g). Airspace volume was evaluated by CT, lungs/pleura were collected, and percent fibrosis was determined. Results: The TF group had significantly better survival than the TS group (21 vs 13.5 days, p < 0.0001). The average effusion volume was less in the talc groups compared to the control group (140 vs 628 μL, p < 0.001). TF induced significant lung fibrosis (p < 0.01), similar to TS. On CT, TF significantly (p < 0.05) reduced loss of right lung volume (by 30–40%) compared to the control group. This was not seen with TS (p > 0.05). Conclusions: This report describes using a novel talc foam delivery system for the treatment of MPE. In the LL/2 model, mice treated with the TF had better survival outcomes and less reduction of lung volume than mice treated with the standard of care TS. These data provide support for translational efforts to move talc foam from animal models into clinical trials.
    • Tumor-targeted SN38 inhibits growth of early stage non-small cell lung cancer (NSCLC) in a KRas/p53 transgenic mouse model

      Fox Chase Cancer Center (Temple University) (2017-04-28)
      Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, with a 5-year survival of only ~16%. Potential strategies to address NSCLC mortality include improvements in early detection and prevention, and development of new therapies suitable for use in patients with early and late stage diagnoses. Controlling the growth of early stage tumors could yield significant clinical benefits for patients with comorbidities that make them poor candidates for surgery: however, many drugs that limit cancer growth are not useful in the setting of long-term use or in comorbid patients, because of associated toxicities. In this study, we explored the use of a recently described small molecule agent, STA-8666, as a potential agent for controlling early stage tumor growth. STA-8666 uses a cleavable linker to merge a tumor-targeting moiety that binds heat shock protein 90 (HSP90) with the cytotoxic chemical SN38, and has been shown to have high efficacy and low toxicity, associated with efficient tumor targeting, in preclinical studies using patient-derived and other xenograft models for pancreatic, bladder, and small cell lung cancer. Using a genetically engineered model of NSCLC arising from induced mutation of KRas and knockout of Trp53, we continuously dosed mice with STA-8666 from immediately after tumor induction for 15 weeks. STA-8666 significantly slowed the rate of tumor growth, and was well tolerated over this extended dosing period. STA-8666 induced DNA damage and apoptosis, and reduced proliferation and phosphorylation of the proliferation-associated protein ERK1/2, selectively in tumor tissue. In contrast, STA-8666 did not affect tumor features, such as degree of vimentin staining, associated with epithelial-mesenchymal transition (EMT), or downregulate tumor expression of HSP90. These data suggest STA-8666 and other similar targeted compounds may be useful additions to control the growth of early stage NSCLC in patient populations.
    • Microscopy-Based Automated Live Cell Screening for Small Molecules That Affect Ciliation

      Fox Chase Cancer Center (Temple University) (2019-02-12)
      The primary monocilium, or cilium, is a single antenna-like organelle that protrudes from the surface of most mammalian cell types, and serves as a signaling hub. Mutations of cilia-associated genes result in severe genetic disorders termed ciliopathies. Among these, the most common is autosomal dominant polycystic kidney disease (ADPKD); less common genetic diseases include Bardet–Biedl syndrome, Joubert syndrome, nephronophthisis, and others. Important signaling cascades with receptor systems localized exclusively or in part at cilia include Sonic Hedgehog (SHH), platelet derived growth factor alpha (PDGFRα), WNTs, polycystins, and others. Changes in ciliation during development or in pathological conditions such as cancer impacts signaling by these proteins. Notably, ciliation status of cells is coupled closely to the cell cycle, with cilia protruding in quiescent (G0) or early G1 cells, declining in S/G2, and absent in M phase, and has been proposed to contribute to cell cycle regulation. Because of this complex biology, the elaborate machinery regulating ciliary assembly and disassembly receives input from many cellular proteins relevant to cell cycle control, development, and oncogenic transformation, making study of genetic factors and drugs influencing ciliation of high interest. One of the most effective tools to investigate the dynamics of the cilia under different conditions is the imaging of live cells. However, developing assays to observe the primary cilium in real time can be challenging, and requires a consideration of multiple details related to the cilia biology. With the dual goals of identifying small molecules that may have beneficial activity through action on human diseases, and of identifying ciliary activities of existing agents that are in common use or development, we here describe creation and evaluation of three autofluorescent cell lines derived from the immortalized retinal pigmented epithelium parental cell line hTERT-RPE1. These cell lines stably express the ciliary-targeted fluorescent proteins L13-Arl13bGFP, pEGFP-mSmo, and tdTomato-MCHR1-N-10. We then describe methods for use of these cell lines in high throughput screening of libraries of small molecule compounds to identify positive and negative regulators of ciliary disassembly.
    • Prevalence of pathogenic variants in DNA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of early-onset renal cancer

      Fox Chase Cancer Center (Temple University) (2020-08-11)
      Pathogenic variants (PVs) in multiple genes are known to increase the risk of early-onset renal cancer (eoRC). However, many eoRC patients lack PVs in RC-specific genes; thus, their genetic risk remains undefined. Here, we determine if PVs in DNA damage response and repair (DDRR) genes are enriched in eoRC patients undergoing cancer risk assessment. Retrospective review of de-identified results from 844 eoRC patients, undergoing testing with a multi-gene panel, for a variety of indications, by Ambry Genetics. PVs in cancer-risk genes were identified in 12.8% of patients—with 3.7% in RC-specific, and 8.55% in DDRR genes. DDRR gene PVs were most commonly identified in CHEK2, BRCA1, BRCA2, and ATM. Among the 2.1% of patients with a BRCA1 or BRCA2 PV, < 50% reported a personal history of hereditary breast or ovarian-associated cancer. No association between age of RC diagnosis and prevalence of PVs in RC-specific or DDRR genes was observed. Additionally, 57.9% patients reported at least one additional cancer; breast cancer being the most common (40.1% of females, 2.5% of males). Multi-gene testing including DDRR genes may provide a more comprehensive risk assessment in eoRC patients. Further validation is needed to characterize the association with eoRC.
    • Tumor-Targeted Drug Conjugates as an Emerging Novel Therapeutic Approach in Small Cell Lung Cancer (SCLC)

      Fox Chase Cancer Center (Temple University) (2019-09-03)
      There are few effective therapies for small cell lung cancer (SCLC), a highly aggressive disease representing 15% of total lung cancers. With median survival <2 years, SCLC is one of the most lethal cancers. At present, chemotherapies and radiation therapy are commonly used for SCLC management. Few protein-targeted therapies have shown efficacy in improving overall survival; immune checkpoint inhibitors (ICIs) are promising agents, but many SCLC tumors do not express ICI targets such as PD-L1. This article presents an alternative approach to the treatment of SCLC: the use of drug conjugates, where a targeting moiety concentrates otherwise toxic agents in the vicinity of tumors, maximizing the differential between tumor killing and the cytotoxicity of normal tissues. Several tumor-targeted drug conjugate delivery systems exist and are currently being actively tested in the setting of SCLC. These include antibody-drug conjugates (ADCs), radioimmunoconjugates (RICs), small molecule-drug conjugates (SMDCs), and polymer-drug conjugates (PDCs). We summarize the basis of action for these targeting compounds, discussing principles of construction and providing examples of effective versus ineffective compounds, as established by preclinical and clinical testing. Such agents may offer new therapeutic options for the clinical management of this challenging disease in the future.
    • Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

      Fox Chase Cancer Center (Temple University) (2021-03-15)
      Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.
    • Synthetic Lethal Targeting of Mitotic Checkpoints in HPV-Negative Head and Neck Cancer

      Fox Chase Cancer Center (Temple University) (2020-01-28)
      Head and neck squamous cell carcinomas (HNSCC) affect more than 800,000 people annually worldwide, causing over 15,000 deaths in the US. Among HNSCC cancers, human papillomavirus (HPV)-negative HNSCC has the worst outcome, motivating efforts to improve therapy for this disease. The most common mutational events in HPV-negative HNSCC are inactivation of the tumor suppressors TP53 (>85%) and CDKN2A (>57%), which significantly impairs G1/S checkpoints, causing reliance on other cell cycle checkpoints to repair ongoing replication damage. We evaluated a panel of cell cycle-targeting clinical agents in a group of HNSCC cell lines to identify a subset of drugs with single-agent activity in reducing cell viability. Subsequent analyses demonstrated potent combination activity between the CHK1/2 inhibitor LY2606268 (prexasertib), which eliminates a G2 checkpoint, and the WEE1 inhibitor AZD1775 (adavosertib), which promotes M-phase entry, in induction of DNA damage, mitotic catastrophe, and apoptosis, and reduction of anchorage independent growth and clonogenic capacity. These phenotypes were accompanied by more significantly reduced activation of CHK1 and its paralog CHK2, and enhanced CDK1 activation, eliminating breaks on the mitotic entry of cells with DNA damage. These data suggest the potential value of dual inhibition of CHK1 and WEE1 in tumors with compromised G1/S checkpoints.
    • Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers

      Fox Chase Cancer Center (Temple University) (2022-03-25)
      Loss of expression or activity of the tumor suppressor PTEN acts similarly to an activating mutation in the oncogene PIK3CA in elevating intracellular levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), inducing signaling by AKT and other pro-tumorigenic signaling proteins. Here, we analyze sequence data for 34,129 colorectal cancer (CRC) patients, capturing 3,434 PTEN mutations. We identify specific patterns of PTEN mutation associated with microsatellite stability/instability (MSS/MSI), tumor mutational burden (TMB), patient age, and tumor location. Within groups separated by MSS/MSI status, this identifies distinct profiles of nucleotide hotspots, and suggests differing profiles of protein-damaging effects of mutations. Moreover, discrete categories of PTEN mutations display non-identical patterns of co-occurrence with mutations in other genes important in CRC pathogenesis, including KRAS, APC, TP53, and PIK3CA. These data provide context for clinical targeting of proteins upstream and downstream of PTEN in distinct CRC cohorts.
    • Targeting the Epidermal Growth Factor Receptor in EGFR-Mutated Lung Cancer: Current and Emerging Therapies

      Fox Chase Cancer Center (Temple University) (2021-06-24)
      Epidermal growth factor receptor-targeting tyrosine kinase inhibitors (EGFR TKIs) are the standard of care for patients with EGFR-mutated metastatic lung cancer. While EGFR TKIs have initially high response rates, inherent and acquired resistance constitute a major challenge to the longitudinal treatment. Ongoing work is aimed at understanding the molecular basis of these resistance mechanisms, with exciting new studies evaluating novel agents and combination therapies to improve control of tumors with all forms of EGFR mutation. In this review, we first provide a discussion of EGFR-mutated lung cancer and the efficacy of available EGFR TKIs in the clinical setting against both common and rare EGFR mutations. Second, we discuss common resistance mechanisms that lead to therapy failure during treatment with EGFR TKIs. Third, we review novel approaches aimed at improving outcomes and overcoming resistance to EGFR TKIs. Finally, we highlight recent breakthroughs in the use of EGFR TKIs in non-metastatic EGFR-mutated lung cancer.