Extraordinary advancements in sequencing technology have made what was once a decade-long multi-i... more Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting.We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized Onco-Genomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/ normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequenc-ing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homo-zygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscapeof an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level. The Oncologist 2014;19:623–630 Implications for Practice: We show that use of next-generation sequencing in clinical settings is practical and can benefit patients because of the ability to define tumors genetically.
The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activ... more The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCa and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1 S102 phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1 S102 and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565) and prostate (PC3, LNCap) cancer cells was inhibited by ,90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics.
Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery.... more Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery. We have developed a computational drug repositioning pipeline to perform large-scale molecular docking of small molecule drugs against protein drug targets, in order to map the drug-target interaction space and find novel interactions. Our method emphasizes removing false positive interaction predictions using criteria from known interaction docking, consensus scoring, and specificity. In all, our database contains 252 human protein drug targets that we classify as reliable-for-docking as well as 4621 approved and experimental small molecule drugs from DrugBank. These were cross-docked, then filtered through stringent scoring criteria to select top drug-target interactions. In particular, we used MAPK14 and the kinase inhibitor BIM-8 as examples where our stringent thresholds enriched the predicted drug-target interactions with known interactions up to 20 times compared to standard score thresholds. We validated nilotinib as a potent MAPK14 inhibitor in vitro (IC50 40 nM), suggesting a potential use for this drug in treating inflammatory diseases. The published literature indicated experimental evidence for 31 of the top predicted interactions, highlighting the promising nature of our approach. Novel interactions discovered may lead to the drug being repositioned as a therapeutic treatment for its off-target's associated disease, added insight into the drug's mechanism of action, and added insight into the drug's side effects.
In an attempt to assess potential treatment options, whole-genome and transcriptome sequencing we... more In an attempt to assess potential treatment options, whole-genome and transcriptome sequencing were performed on a patient with an unclassifiable small lymphoproliferative disorder. Variants from genome sequencing were prioritized using a combination of comparative variant distributions in a spectrum of lymphomas, and meta-analyses of gene expression profiling. In this patient, the molecular variants that we believe to be most relevant to the disease presentation most strongly resemble a diffuse large B-cell lymphoma (DLBCL), whereas the gene expression data are most consistent with a low-grade chronic lymphocytic leukemia (CLL). The variant of greatest interest was a predicted NOTCH2-truncating mutation, which has been recently reported in various lymphomas. [Supplemental material is available for this article.]
Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormon... more Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44 + cells. Luteolin's efficacy against drugresistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched
designed this study. H.Z. designed and performed biology experiments. J.Q. and J.E.B designed and... more designed this study. H.Z. designed and performed biology experiments. J.Q. and J.E.B designed and synthesized JQEZ5, JQEZ6 and JQEZ23.. performed all sequencing and sequencing data analysis. J.P. and J.Q. performed biochemical assays. A.J.F. and J.Q. performed computational modeling.
STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer... more STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether inactivation of tumor suppressor genes such as STK11/LKB1 exert similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T cell suppressive effects, along with a corresponding increase in the expression of T cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/ LKB1 inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1 targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL-6 neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1 mutated tumors with PD-1 targeting antibody therapies.
Recent next-generation sequencing studies have generated a comprehensive overview of the genomic ... more Recent next-generation sequencing studies have generated a comprehensive overview of the genomic landscape of Human Papillomavirus (HPV)-associated cancers. This review summarizes these findings to provide insight into the tumor biology of these cancers and potential therapeutic opportunities for HPV-driven malignancies. In addition to the tumorigenic properties of the HPV oncoproteins, integration of HPV DNA into the host genome is suggested to be a driver of the neoplastic process. Integration may confer a growth and survival advantage via enhanced expression of viral oncoproteins, alteration of critical cellular genes, and changes in global promoter methylation and transcription. Alteration of cellular genes may lead to loss of function of tumor suppressor genes, enhanced oncogene expression, loss of function of DNA repair genes, or other vital cellular functions. Recurrent integrations in RAD51B, NR4A2, and TP63, leading to aberrant forms of these proteins, are observed in both HPV-positive head and neck squamous cell carcinoma (HNSCC) and cervical carcinoma. Additional genomic alterations, independent of integration events, include recurrent PIK3CA mutations (and aberrations in other members of the PI3K pathway), alterations in receptor tyrosine kinases (primarily FGFR2 and FGFR3 in HPVpositive HNSCC, and ERBB2 in cervical squamous cell carcinoma), and genes in pathways related to squamous cell differentiation and immune responses. A number of the alterations identified are potentially targetable, which may lead to advances in the treatment of HPV-associated cancers.
Purpose-A rare 5% of cutaneous squamous cell carcinomas metastasize, lack FDA-approved therapies,... more Purpose-A rare 5% of cutaneous squamous cell carcinomas metastasize, lack FDA-approved therapies, and carry a poor prognosis. Our aim was to identify recurrent genomic alterations in this little-studied population of metastatic cSCCs.
Genetically engineered mouse models of lung cancer have demonstrated an important role in underst... more Genetically engineered mouse models of lung cancer have demonstrated an important role in understanding the function of novel lung cancer oncogenes and tumor suppressor genes identified in genomic studies of human lung cancer. Further, these models are important platforms for preclinical therapeutic studies. Here, we generated a mouse model of lung adenocarcinoma driven by mutation of the Discoidin Domain Receptor 2 (DDR2) gene combined with loss of TP53. DDR2 L63V ;TP53 L/L mice developed poorly differentiated lung adenocarcinomas in all transgenic animals analyzed with a latency of 40-50 weeks and a median survival of 67.5 weeks. Mice expressing wild-type DDR2 with combined TP53 loss did not form lung cancers. DDR2 L63V ; TP53 L/L tumors displayed robust expression of DDR2 and immunohistochemical markers of lung adenocarcinoma comparable to previously generated models though also displayed concomitant expression of the squamous cell markers p63 and SOX2. Tumor-derived cell lines were not solely DDR2 dependent and displayed up-regulation of and partial dependence on MYCN. Combined treatment with the multitargeted DDR2 inhibitor dasatinib and BET inhibitor JQ1 inhibited tumor growth in vitro and in vivo. Together, these results suggest that DDR2 mutation can drive lung cancer initiation in vivo and provide a novel mouse model for lung cancer therapeutics studies.
Somatic alterations of Fibroblast Growth Factor Receptors (FGFRs) have been described in a wide r... more Somatic alterations of Fibroblast Growth Factor Receptors (FGFRs) have been described in a wide range of malignancies. A number of anti-FGFR therapies are currently under investigation in clinical trials for subjects with FGFR gene amplifications, mutations and translocations. Here, we develop cell line models of acquired resistance to FGFR inhibition by exposure of cell lines harboring FGFR3 gene amplification and translocation to the selective FGFR inhibitor BGJ398 and multi-targeted FGFR inhibitor ponatinib. We show that the acquisition of resistance is rapid, reversible and characterized by an epithelial to mesenchymal transition (EMT) and a switch from dependency on FGFR3 to ERBB family members. Acquired resistance was associated with demonstrable changes in gene expression including increased production of ERBB2/3 ligands which were sufficient to drive resistance in the setting of FGFR3 dependency but not dependency on other FGFR family members. These data support the concept that activation of ERBB family members is sufficient to bypass dependency on FGFR3 and suggest that concurrent inhibition of these two pathways may be desirable when targeting FGFR3 dependent cancers.
Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Bar... more Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-kB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to con-stitutively activate NF-kB signalling, and we observed mutual exclusivity among tumours with somatic NF-kB pathway aberrations and LMP1-overexpression, suggesting that NF-kB activation is selected for by both somatic and viral events during NPC pathogenesis.
Substrates and inhibitors of chromatin-modifying enzymes are generated in intermediary metabolism... more Substrates and inhibitors of chromatin-modifying enzymes are generated in intermediary metabolism, so changes in nutrient availability and utilization can influence epigenetic regulation 1,2. Importantly, recent studies have indicated that the interplay between metabolism and epigenetics can serve as a programmed switch in cell states. For example, mouse embryonic stem cell differentiation is promoted by succinate-mediated inhibition of histone demethylases (HDMs) and TET DNA demethylases 3 , or by decreased S-adenosyl-methionine (SAM) levels leading to loss of histone H3K4 methylation 4. Moreover, aberrant metabolic activity can produce pathological effects by altering chromatin regulation. Most notably, mutations in the genes encoding the isocitrate dehydrogenase (IDH)1 and IDH2 enzymes lead to the generation of 2-hydroxyglutarate, which inhibits HDMs and TETs and thereby alters DNA and histone methylation—changes that have been implicated in overriding cell differentiation and promoting tumorigenesis 5. Whether this paradigm extends more generally to other oncogenic mutations remains unclear, and this question has implications for understanding cancer pathogenesis and developing improved treatments. Here, we demonstrate that dynamic exchange between metabolism and chromatin regulation contributes to pancreatic tumorigenesis driven by mutation of the LKB1 serine–threonine kinase. LKB1 is mutationally inactivated in a range of sporadic cancers, including pancreatic carcinomas 6–8. Additionally, germline mutations in LKB1 cause Peutz-Jeghers syndrome, which comprises gastrointesti-nal polyps and a high incidence of gastrointestinal tract carcinomas (for example, an approximately 100-fold increase in pancreatic cancer) 9,10. Cancers with LKB1 mutations tend to exhibit aggressive clinical features and different therapeutic sensitivity from cancers without these mutations 11–14. LKB1 directly activates a family of 14 kinases related to AMP-activated protein kinase (AMPK), many of which are coupled to nutrient sensing and broadly reprogram cell metabolism 15. Thus, metabolic rewiring is thought to be a driver of tumorigenesis after LKB1 loss. We now identify an LKB1-regulated program that links metabolic alterations to control of the epigenome and is involved in malignant growth. Our results provide evidence that coupled metabolic and epige-netic states have a more general role in cancer pathogenesis and suggest therapeutic strategies that could target these intersecting processes. Synergy between LKB1 and KRAS mutations LKB1 inactivation frequently coincides with mutations in the RAS–RAF pathway in human cancers and these genetic alterations cooperate to drive tumorigenesis in genetically engineered mouse models (GEMMs) 6,11,14,16. We examined the interactions between oncogenic KRAS G12D and deletion of LKB1 in adult pancreatic ducts using a tamoxifen-inducible GEMM (Extended Data Fig. 1a). The combined alterations resulted in pancreatic cancers by 20–25 weeks, whereas the individual mutations had no pathological effects at this age (Fig. 1a and Extended Data Fig. 1b). To investigate the mechanisms of tumorigenesis, we isolated primary pancreatic ductal epithelial cells from mice with conditional KRAS G12D and LKB1 alleles (n = 2 lines per genotype) and transduced them with adenoviruses expressing Cre and/or Flp recom-binase to generate KRAS G12D/+ , LKB1 −/− and KRAS G12D/+ ;LKB1 −/− cells (K, L and KL cells, respectively) as well as wild-type parental lines (Extended Data Fig. 1c). Only KL cells were tumorigenic following injection into severe combined immunodeficient (SCID) mice or growth in soft agar, and tumorigenicity was blocked by restoration of Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine–glycine– one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.
Given the success of targeted agents in specific populations it is expected that some degree of m... more Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted "panel" sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from wholegenome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate "drivers." Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.
The perceived inefficiency of pharmaceutical drug development has been widely discussed . Only 20... more The perceived inefficiency of pharmaceutical drug development has been widely discussed . Only 20 to 30 new chemical entities (NCEs: drugs not containing a previously approved active ingredient) are approved per year in the US , and each successful NCE requires an average of US$1.78 billion and 13.5 years from discovery to market . Although estimates of drug discovery costs vary (a recent study suggested that the minimum cost of developing an NCE is US$204 million [6]), it is important to note that these estimates do not yet account for drug failures. Given that only 11% of drugs investigated in clinical trials are eventually approved [3], the actual cost of drug development is much higher than the published estimates.
We describe cisRED, a database for conserved regulatory elements that are identified and ranked b... more We describe cisRED, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (www.cisred. org). The database and high-throughput predictive
We have developed a computational pipeline for the prediction of protein-small molecule interacti... more We have developed a computational pipeline for the prediction of protein-small molecule interactions and have applied it to the drug repositioning problem through a large-scale analysis of known drug targets and small molecule drugs. Our pipeline combines forward and inverse docking, the latter of which is a twist on the conventional docking procedure used in drug discovery: instead of docking many compounds against a specic target to look for potential inhibitors, one compound is docked against many proteins to search for potential targets. We collected an extensive set of 1,055 approved small molecule drugs and 1,548 drug target binding pockets (representing 78 unique human protein therapeutic targets) and performed a large-scale docking using ICM software to both validate our method and predict novel protein-drug interactions. For the 37 known protein-drug interactions in our data set that have a known structure complex, all docked conformations were within 2.0 A of the solved conformation, and 30 of these had a docking score passing the typical ICM score threshold. Out of the 237 known protein-drug interactions annotated by DrugBank, 74 passed the score threshold, and 52 showed the drug docking to another protein with a better docking score than to its known target. These protein targets are implicated in human diseases, so novel protein-drug interactions discovered represent potential novel indications for the drugs. Our results highlight the promising nature of the inverse docking method for identifying potential novel therapeutic uses for existing drugs.
Background Gastrointestinal carcinomas are genomically complex cancers that are lethal in the met... more Background Gastrointestinal carcinomas are genomically complex cancers that are lethal in the metastatic setting. Whole-genome and transcriptome sequencing allow for the simultaneous characterization of multiple oncogenic pathways.
Extraordinary advancements in sequencing technology have made what was once a decade-long multi-i... more Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting.We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized Onco-Genomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/ normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequenc-ing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homo-zygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscapeof an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level. The Oncologist 2014;19:623–630 Implications for Practice: We show that use of next-generation sequencing in clinical settings is practical and can benefit patients because of the ability to define tumors genetically.
The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activ... more The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCa and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1 S102 phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1 S102 and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565) and prostate (PC3, LNCap) cancer cells was inhibited by ,90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics.
Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery.... more Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery. We have developed a computational drug repositioning pipeline to perform large-scale molecular docking of small molecule drugs against protein drug targets, in order to map the drug-target interaction space and find novel interactions. Our method emphasizes removing false positive interaction predictions using criteria from known interaction docking, consensus scoring, and specificity. In all, our database contains 252 human protein drug targets that we classify as reliable-for-docking as well as 4621 approved and experimental small molecule drugs from DrugBank. These were cross-docked, then filtered through stringent scoring criteria to select top drug-target interactions. In particular, we used MAPK14 and the kinase inhibitor BIM-8 as examples where our stringent thresholds enriched the predicted drug-target interactions with known interactions up to 20 times compared to standard score thresholds. We validated nilotinib as a potent MAPK14 inhibitor in vitro (IC50 40 nM), suggesting a potential use for this drug in treating inflammatory diseases. The published literature indicated experimental evidence for 31 of the top predicted interactions, highlighting the promising nature of our approach. Novel interactions discovered may lead to the drug being repositioned as a therapeutic treatment for its off-target's associated disease, added insight into the drug's mechanism of action, and added insight into the drug's side effects.
In an attempt to assess potential treatment options, whole-genome and transcriptome sequencing we... more In an attempt to assess potential treatment options, whole-genome and transcriptome sequencing were performed on a patient with an unclassifiable small lymphoproliferative disorder. Variants from genome sequencing were prioritized using a combination of comparative variant distributions in a spectrum of lymphomas, and meta-analyses of gene expression profiling. In this patient, the molecular variants that we believe to be most relevant to the disease presentation most strongly resemble a diffuse large B-cell lymphoma (DLBCL), whereas the gene expression data are most consistent with a low-grade chronic lymphocytic leukemia (CLL). The variant of greatest interest was a predicted NOTCH2-truncating mutation, which has been recently reported in various lymphomas. [Supplemental material is available for this article.]
Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormon... more Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44 + cells. Luteolin's efficacy against drugresistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched
designed this study. H.Z. designed and performed biology experiments. J.Q. and J.E.B designed and... more designed this study. H.Z. designed and performed biology experiments. J.Q. and J.E.B designed and synthesized JQEZ5, JQEZ6 and JQEZ23.. performed all sequencing and sequencing data analysis. J.P. and J.Q. performed biochemical assays. A.J.F. and J.Q. performed computational modeling.
STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer... more STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether inactivation of tumor suppressor genes such as STK11/LKB1 exert similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T cell suppressive effects, along with a corresponding increase in the expression of T cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/ LKB1 inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1 targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL-6 neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1 mutated tumors with PD-1 targeting antibody therapies.
Recent next-generation sequencing studies have generated a comprehensive overview of the genomic ... more Recent next-generation sequencing studies have generated a comprehensive overview of the genomic landscape of Human Papillomavirus (HPV)-associated cancers. This review summarizes these findings to provide insight into the tumor biology of these cancers and potential therapeutic opportunities for HPV-driven malignancies. In addition to the tumorigenic properties of the HPV oncoproteins, integration of HPV DNA into the host genome is suggested to be a driver of the neoplastic process. Integration may confer a growth and survival advantage via enhanced expression of viral oncoproteins, alteration of critical cellular genes, and changes in global promoter methylation and transcription. Alteration of cellular genes may lead to loss of function of tumor suppressor genes, enhanced oncogene expression, loss of function of DNA repair genes, or other vital cellular functions. Recurrent integrations in RAD51B, NR4A2, and TP63, leading to aberrant forms of these proteins, are observed in both HPV-positive head and neck squamous cell carcinoma (HNSCC) and cervical carcinoma. Additional genomic alterations, independent of integration events, include recurrent PIK3CA mutations (and aberrations in other members of the PI3K pathway), alterations in receptor tyrosine kinases (primarily FGFR2 and FGFR3 in HPVpositive HNSCC, and ERBB2 in cervical squamous cell carcinoma), and genes in pathways related to squamous cell differentiation and immune responses. A number of the alterations identified are potentially targetable, which may lead to advances in the treatment of HPV-associated cancers.
Purpose-A rare 5% of cutaneous squamous cell carcinomas metastasize, lack FDA-approved therapies,... more Purpose-A rare 5% of cutaneous squamous cell carcinomas metastasize, lack FDA-approved therapies, and carry a poor prognosis. Our aim was to identify recurrent genomic alterations in this little-studied population of metastatic cSCCs.
Genetically engineered mouse models of lung cancer have demonstrated an important role in underst... more Genetically engineered mouse models of lung cancer have demonstrated an important role in understanding the function of novel lung cancer oncogenes and tumor suppressor genes identified in genomic studies of human lung cancer. Further, these models are important platforms for preclinical therapeutic studies. Here, we generated a mouse model of lung adenocarcinoma driven by mutation of the Discoidin Domain Receptor 2 (DDR2) gene combined with loss of TP53. DDR2 L63V ;TP53 L/L mice developed poorly differentiated lung adenocarcinomas in all transgenic animals analyzed with a latency of 40-50 weeks and a median survival of 67.5 weeks. Mice expressing wild-type DDR2 with combined TP53 loss did not form lung cancers. DDR2 L63V ; TP53 L/L tumors displayed robust expression of DDR2 and immunohistochemical markers of lung adenocarcinoma comparable to previously generated models though also displayed concomitant expression of the squamous cell markers p63 and SOX2. Tumor-derived cell lines were not solely DDR2 dependent and displayed up-regulation of and partial dependence on MYCN. Combined treatment with the multitargeted DDR2 inhibitor dasatinib and BET inhibitor JQ1 inhibited tumor growth in vitro and in vivo. Together, these results suggest that DDR2 mutation can drive lung cancer initiation in vivo and provide a novel mouse model for lung cancer therapeutics studies.
Somatic alterations of Fibroblast Growth Factor Receptors (FGFRs) have been described in a wide r... more Somatic alterations of Fibroblast Growth Factor Receptors (FGFRs) have been described in a wide range of malignancies. A number of anti-FGFR therapies are currently under investigation in clinical trials for subjects with FGFR gene amplifications, mutations and translocations. Here, we develop cell line models of acquired resistance to FGFR inhibition by exposure of cell lines harboring FGFR3 gene amplification and translocation to the selective FGFR inhibitor BGJ398 and multi-targeted FGFR inhibitor ponatinib. We show that the acquisition of resistance is rapid, reversible and characterized by an epithelial to mesenchymal transition (EMT) and a switch from dependency on FGFR3 to ERBB family members. Acquired resistance was associated with demonstrable changes in gene expression including increased production of ERBB2/3 ligands which were sufficient to drive resistance in the setting of FGFR3 dependency but not dependency on other FGFR family members. These data support the concept that activation of ERBB family members is sufficient to bypass dependency on FGFR3 and suggest that concurrent inhibition of these two pathways may be desirable when targeting FGFR3 dependent cancers.
Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Bar... more Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-kB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to con-stitutively activate NF-kB signalling, and we observed mutual exclusivity among tumours with somatic NF-kB pathway aberrations and LMP1-overexpression, suggesting that NF-kB activation is selected for by both somatic and viral events during NPC pathogenesis.
Substrates and inhibitors of chromatin-modifying enzymes are generated in intermediary metabolism... more Substrates and inhibitors of chromatin-modifying enzymes are generated in intermediary metabolism, so changes in nutrient availability and utilization can influence epigenetic regulation 1,2. Importantly, recent studies have indicated that the interplay between metabolism and epigenetics can serve as a programmed switch in cell states. For example, mouse embryonic stem cell differentiation is promoted by succinate-mediated inhibition of histone demethylases (HDMs) and TET DNA demethylases 3 , or by decreased S-adenosyl-methionine (SAM) levels leading to loss of histone H3K4 methylation 4. Moreover, aberrant metabolic activity can produce pathological effects by altering chromatin regulation. Most notably, mutations in the genes encoding the isocitrate dehydrogenase (IDH)1 and IDH2 enzymes lead to the generation of 2-hydroxyglutarate, which inhibits HDMs and TETs and thereby alters DNA and histone methylation—changes that have been implicated in overriding cell differentiation and promoting tumorigenesis 5. Whether this paradigm extends more generally to other oncogenic mutations remains unclear, and this question has implications for understanding cancer pathogenesis and developing improved treatments. Here, we demonstrate that dynamic exchange between metabolism and chromatin regulation contributes to pancreatic tumorigenesis driven by mutation of the LKB1 serine–threonine kinase. LKB1 is mutationally inactivated in a range of sporadic cancers, including pancreatic carcinomas 6–8. Additionally, germline mutations in LKB1 cause Peutz-Jeghers syndrome, which comprises gastrointesti-nal polyps and a high incidence of gastrointestinal tract carcinomas (for example, an approximately 100-fold increase in pancreatic cancer) 9,10. Cancers with LKB1 mutations tend to exhibit aggressive clinical features and different therapeutic sensitivity from cancers without these mutations 11–14. LKB1 directly activates a family of 14 kinases related to AMP-activated protein kinase (AMPK), many of which are coupled to nutrient sensing and broadly reprogram cell metabolism 15. Thus, metabolic rewiring is thought to be a driver of tumorigenesis after LKB1 loss. We now identify an LKB1-regulated program that links metabolic alterations to control of the epigenome and is involved in malignant growth. Our results provide evidence that coupled metabolic and epige-netic states have a more general role in cancer pathogenesis and suggest therapeutic strategies that could target these intersecting processes. Synergy between LKB1 and KRAS mutations LKB1 inactivation frequently coincides with mutations in the RAS–RAF pathway in human cancers and these genetic alterations cooperate to drive tumorigenesis in genetically engineered mouse models (GEMMs) 6,11,14,16. We examined the interactions between oncogenic KRAS G12D and deletion of LKB1 in adult pancreatic ducts using a tamoxifen-inducible GEMM (Extended Data Fig. 1a). The combined alterations resulted in pancreatic cancers by 20–25 weeks, whereas the individual mutations had no pathological effects at this age (Fig. 1a and Extended Data Fig. 1b). To investigate the mechanisms of tumorigenesis, we isolated primary pancreatic ductal epithelial cells from mice with conditional KRAS G12D and LKB1 alleles (n = 2 lines per genotype) and transduced them with adenoviruses expressing Cre and/or Flp recom-binase to generate KRAS G12D/+ , LKB1 −/− and KRAS G12D/+ ;LKB1 −/− cells (K, L and KL cells, respectively) as well as wild-type parental lines (Extended Data Fig. 1c). Only KL cells were tumorigenic following injection into severe combined immunodeficient (SCID) mice or growth in soft agar, and tumorigenicity was blocked by restoration of Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine–glycine– one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.
Given the success of targeted agents in specific populations it is expected that some degree of m... more Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted "panel" sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from wholegenome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate "drivers." Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.
The perceived inefficiency of pharmaceutical drug development has been widely discussed . Only 20... more The perceived inefficiency of pharmaceutical drug development has been widely discussed . Only 20 to 30 new chemical entities (NCEs: drugs not containing a previously approved active ingredient) are approved per year in the US , and each successful NCE requires an average of US$1.78 billion and 13.5 years from discovery to market . Although estimates of drug discovery costs vary (a recent study suggested that the minimum cost of developing an NCE is US$204 million [6]), it is important to note that these estimates do not yet account for drug failures. Given that only 11% of drugs investigated in clinical trials are eventually approved [3], the actual cost of drug development is much higher than the published estimates.
We describe cisRED, a database for conserved regulatory elements that are identified and ranked b... more We describe cisRED, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (www.cisred. org). The database and high-throughput predictive
We have developed a computational pipeline for the prediction of protein-small molecule interacti... more We have developed a computational pipeline for the prediction of protein-small molecule interactions and have applied it to the drug repositioning problem through a large-scale analysis of known drug targets and small molecule drugs. Our pipeline combines forward and inverse docking, the latter of which is a twist on the conventional docking procedure used in drug discovery: instead of docking many compounds against a specic target to look for potential inhibitors, one compound is docked against many proteins to search for potential targets. We collected an extensive set of 1,055 approved small molecule drugs and 1,548 drug target binding pockets (representing 78 unique human protein therapeutic targets) and performed a large-scale docking using ICM software to both validate our method and predict novel protein-drug interactions. For the 37 known protein-drug interactions in our data set that have a known structure complex, all docked conformations were within 2.0 A of the solved conformation, and 30 of these had a docking score passing the typical ICM score threshold. Out of the 237 known protein-drug interactions annotated by DrugBank, 74 passed the score threshold, and 52 showed the drug docking to another protein with a better docking score than to its known target. These protein targets are implicated in human diseases, so novel protein-drug interactions discovered represent potential novel indications for the drugs. Our results highlight the promising nature of the inverse docking method for identifying potential novel therapeutic uses for existing drugs.
Background Gastrointestinal carcinomas are genomically complex cancers that are lethal in the met... more Background Gastrointestinal carcinomas are genomically complex cancers that are lethal in the metastatic setting. Whole-genome and transcriptome sequencing allow for the simultaneous characterization of multiple oncogenic pathways.
Uploads
Papers by Yvonne Li