Nivolumab, Pomalidomide, and Elotuzumab Combination Regimens for Treatment of Relapsed and Refractory Multiple Myeloma: Results from the Phase 3 CheckMate 602 Study.

BACKGROUND: Preclinical studies suggest that combining nivolumab, a programmed death-1 (PD-1) immune checkpoint inhibitor, with pomalidomide/dexamethasone (Pd) with or without elotuzumab, an antisignaling lymphocytic activation molecule F7 monoclonal antibody, may improve multiple myeloma (MM) treatment efficacy. PATIENTS AND METHODS: The phase 3 CheckMate 602 study (NCT02726581) assessed the efficacy and safety of nivolumab plus pomalidomide/dexamethasone (NPd) and NPd plus elotuzumab (NE-Pd). Eligible patients (aged ≥ 18 years) had measurable MM after ≥ 2 prior lines of therapy, that included an immunomodulatory drug (IMiD) and proteasome inhibitor (PI), each for ≥ 2 consecutive cycles, alone or combined, and were refractory to their last line of therapy. Patients were randomized 3:3:1 to receive NPd, Pd, or NE-Pd. The primary endpoint was progression-free survival (PFS); overall response rate (ORR) was a key secondary endpoint. RESULTS: At a median follow-up of 16.8 months, PFS was similar between treatment arms (Pd, 7.3 months [95% CI, 6.5-8.4]; NPd, 8.4 months [95% CI, 5.8-12.1]; NE-Pd, 6.3 months [95% CI, 2.4-11.1]). ORR was similar in the Pd (55%), NPd (48%), and NE-Pd (42%) arms. Nivolumab-containing arms were associated with a less favorable safety profile versus Pd, including a higher rate of thrombocytopenia (NPd, 25.0%; NE-Pd, 16.7%; Pd, 15.7%), any-grade immune-mediated adverse events (NPd, 13.9%; NE-Pd, 16.7%; Pd, 2.9%), and adverse events leading to discontinuation (NPd, 25.0%; NE-Pd, 33.3%; Pd, 18.6%). No new safety signals were identified. CONCLUSION: CheckMate 602 did not demonstrate clinical benefit of nivolumab (+/- elotuzumab) plus Pd versus Pd for patients with relapsed/refractory MM (RRMM).

High Dimensional Immune Profiling of Smoldering Multiple Myeloma Distinguishes Distinct Tumor Microenvironments.

BACKGROUND: Multiple myeloma (MM) is a malignancy of plasma cells that arises from premalignant Monoclonal Gammopathy of Undetermined Significance (MGUS) and often progresses through an asymptomatic Smoldering (SMM) phase. Understanding the interactions between abnormal clonal plasma cells and the tumor microenvironment (TME) in the early disease states (MGUS, SMM) may inform risk assessment and therapy. PATIENTS AND METHODS: We performed high dimensional immunologic analysis of bone marrow specimens from 73 subjects with SMM by mass cytometry and T cell receptor sequencing of CD138-depleted bone marrow (BM) mononuclear cells, and proteomics and seromic profiling of BM plasma. Analysis of individual assay data identified self-organizing subgroups of SMM patients. We then applied novel bioinformatic methods to integrate data from pairs, trios, and quartets of assays. RESULTS: Mass cytometry, TCRSeq and proteomics identified three taxa (sing. taxon) of subjects that shared common characteristics across all three assays. Differential levels of BM plasma pleiotropin (PTN) and BM T cells and their productive clonality emerged as strong distinguishing factors among these taxa. CONCLUSION: These results suggest that the continuum from MGUS to MM does not consist of a single pathway in the TME, and that complex interactions between myeloma cells and the TME may ultimately determine progression and inform clinical management.

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

MAGE-A inhibit apoptosis and promote proliferation in multiple myeloma through regulation of BIM and p21Cip1.

The type I Melanoma Antigen Gene (MAGE) A3 is a functional target associated with survival and proliferation in multiple myeloma (MM). To investigate the mechanisms of these oncogenic functions, we performed gene expression profiling (GEP) of p53 wild-type human myeloma cell lines (HMCL) after MAGE-A knockdown, which identified a set of 201 differentially expressed genes (DEG) associated with apoptosis, DNA repair, and cell cycle regulation. MAGE knockdown increased protein levels of pro-apoptotic BIM and of the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1. Depletion of MAGE-A in HMCL increased sensitivity to the alkylating agent melphalan but not to proteasome inhibition. High MAGEA3 was associated with the MYC and Cell Cycling clusters defined by a network model of GEP data from the CoMMpass database of newly diagnosed, untreated MM patients. Comparative analysis of CoMMpass subjects based on high or low MAGEA3 expression revealed a set of 6748 DEG that also had significant functional associations with cell cycle and DNA replication pathways, similar to that observed in HMCL. High MAGEA3 expression correlated with shorter overall survival after melphalan chemotherapy and autologous stem cell transplantation (ASCT). These results demonstrate that MAGE-A3 regulates Bim and p21Cip1 transcription and protein expression, inhibits apoptosis, and promotes proliferation.

Mutation-derived Neoantigen-specific T-cell Responses in Multiple Myeloma.

PURPOSE: Somatic mutations in cancer cells can give rise to novel protein sequences that can be presented by antigen-presenting cells as neoantigens to the host immune system. Tumor neoantigens represent excellent targets for immunotherapy, due to their specific expression in cancer tissue. Despite the widespread use of immunomodulatory drugs and immunotherapies that recharge T and NK cells, there has been no direct evidence that neoantigen-specific T-cell responses are elicited in multiple myeloma. EXPERIMENTAL DESIGN: Using next-generation sequencing data we describe the landscape of neo-antigens in 184 patients with multiple myeloma and successfully validate neoantigen-specific T cells in patients with multiple myeloma and support the feasibility of neoantigen-based therapeutic vaccines for use in cancers with intermediate mutational loads such as multiple myeloma. RESULTS: In this study, we demonstrate an increase in neoantigen load in relapsed patients with multiple myeloma as compared with newly diagnosed patients with multiple myeloma. Moreover, we identify shared neoantigens across multiple patients in three multiple myeloma oncogenic driver genes (KRAS, NRAS, and IRF4). Next, we validate neoantigen T-cell response and clonal expansion in correlation with clinical response in relapsed patients with multiple myeloma. This is the first study to experimentally validate the immunogenicity of predicted neoantigens from next-generation sequencing in relapsed patients with multiple myeloma. CONCLUSIONS: Our findings demonstrate that somatic mutations in multiple myeloma can be immunogenic and induce neoantigen-specific T-cell activation that is associated with antitumor activity in vitro and clinical response in vivo. Our results provide the foundation for using neoantigen targeting strategies such as peptide vaccines in future trials for patients with multiple myeloma.

SOX11 augments BCR signaling to drive MCL-like tumor development.

Mantle cell lymphoma (MCL) is characterized by increased B-cell receptor (BCR) signaling, and BTK inhibition is an effective therapeutic intervention in MCL patients. The mechanisms leading to increased BCR signaling in MCL are poorly understood, as mutations in upstream regulators of BCR signaling such as CD79A, commonly observed in other lymphomas, are rare in MCL. The transcription factor SOX11 is overexpressed in the majority (78% to 93%) of MCL patients and is considered an MCL-specific oncogene. So far, attempts to understand SOX11 function in vivo have been hampered by the lack of appropriate animal models, because germline deletion of SOX11 is embryonically lethal. We have developed a transgenic mouse model (Eµ-SOX11-EGFP) in the C57BL/6 background expressing murine SOX11 and EGFP under the control of a B-cell-specific IgH-Eµ enhancer. The overexpression of SOX11 exclusively in B cells exhibits oligoclonal B-cell hyperplasia in the spleen, bone marrow, and peripheral blood, with an immunophenotype (CD5+CD19+CD23-) identical to human MCL. Furthermore, phosphocytometric time-of-flight analysis of the splenocytes from these mice shows hyperactivation of pBTK and other molecules in the BCR signaling pathway, and serial bone marrow transplant from transgenic donors produces lethality with decreasing latency. We report here that overexpression of SOX11 in B cells promotes BCR signaling and a disease phenotype that mimics human MCL.

Precision Medicine for Relapsed Multiple Myeloma on the Basis of an Integrative Multiomics Approach.

PURPOSE: Multiple myeloma (MM) is a malignancy of plasma cells, with a median survival of 6 years. Despite recent therapeutic advancements, relapse remains mostly inevitable, and the disease is fatal in the majority of patients. A major challenge in the treatment of patients with relapsed MM is the timely identification of treatment options in a personalized manner. Current approaches in precision oncology aim at matching specific DNA mutations to drugs, but incorporation of genome-wide RNA profiles has not yet been clinically assessed. METHODS: We have developed a novel computational platform for precision medicine of relapsed and/or refractory MM on the basis of DNA and RNA sequencing. Our approach expands on the traditional DNA-based approaches by integrating somatic mutations and copy number alterations with RNA-based drug repurposing and pathway analysis. We tested our approach in a pilot precision medicine clinical trial with 64 patients with relapsed and/or refractory MM. RESULTS: We generated treatment recommendations in 63 of 64 patients. Twenty-six patients had treatment implemented, and 21 were assessable. Of these, 11 received a drug that was based on RNA findings, eight received a drug that was based on DNA, and two received a drug that was based on both RNA and DNA. Sixteen of the 21 evaluable patients had a clinical response (ie, reduction of disease marker ≥ 25%), giving a clinical benefit rate of 76% and an overall response rate of 66%, with five patients having ongoing responses at the end of the trial. The median duration of response was 131 days. CONCLUSION: Our results show that a comprehensive sequencing approach can identify viable options in patients with relapsed and/or refractory myeloma, and they represent proof of principle of how RNA sequencing can contribute beyond DNA mutation analysis to the development of a reliable drug recommendation tool.

Stage-Specific Human Induced Pluripotent Stem Cells Map the Progression of Myeloid Transformation to Transplantable Leukemia.

Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions.

A phase 2 study of panobinostat with lenalidomide and weekly dexamethasone in myeloma.

Phase 3 studies combining histone deacetylase inhibitors with bortezomib were hampered by gastrointestinal (GI) intolerance, which was not observed when combined with immunomodulatory drugs. This study is a single-center phase 2 study of panobinostat with lenalidomide and dexamethasone (FRD). Twenty-seven relapsed multiple myeloma patients were enrolled. Twenty-two patients (81%) were lenalidomide refractory and 9 (33%), 14 (52%), and 7 (26%) were refractory to pomalidomide, bortezomib, and carfilzomib, respectively. High-risk molecular findings were present in 17 (63%) patients. Responses included 2 complete responses (CRs), 4 very good partial responses (VGPRs), 5 partial responses (PRs), and 9 minimal responses (MRs) for an overall response rate of 41%, clinical benefit rate of 74%, and a disease control rate of 96%. The median progression-free survival (PFS) was 7.1 months. In the 22 lenalidomide-refractory patients, there were 1 CR, 4 VGPRs, 3 PRs, and 7 MRs, with a median PFS of 6.5 months. Median overall survival was not reached. Grade 3/4 toxicities were primarily hematologic. Gene expression profiling of enrollment tumor samples revealed a set of 1989 genes associated with short (<90 days) PFS to therapy. MAGEA1 RNA and protein expression were correlated with short PFS, and laboratory studies demonstrated a role for MAGE-A in resistance to panobinostat-induced cell death. FRD demonstrates durable responses, even in high-risk, lenalidomide-refractory patients, indicating the essential role of panobinostat in attaining responses. MAGEA1 expression may represent a functional biomarker for resistance to panobinostat. In contrast to PANORAMA 1, there were no significant GI toxicities and primarily expected hematologic toxicities. This trial was registered at www.clinicaltrials.gov as #NCT00742027.

Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma.

Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma.

Gli1-Mediated Regulation of Sox2 Facilitates Self-Renewal of Stem-Like Cells and Confers Resistance to EGFR Inhibitors in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients have very low survival rates because the current therapeutic strategies are not fully effective. Although EGFR tyrosine kinase inhibitors are effective for NSCLC patients harboring EGFR mutations, patients invariably develop resistance to these agents. Alterations in multiple signaling cascades have been associated with the development of resistance to EGFR inhibitors. Sonic Hedgehog and associated Gli transcription factors play a major role in embryonic development and have recently been found to be reactivated in NSCLC, and elevated Gli1 levels correlate with poor prognosis. The Hedgehog pathway has been implicated in the functions of cancer stem cells, although the underlying molecular mechanisms are not clear. In this context, we demonstrate that Gli1 is a strong regulator of embryonic stem cell transcription factor Sox2. Depletion of Gli1 or inhibition of the Hedgehog signaling significantly abrogated the self-renewal of stem-like side-population cells from NSCLCs as well as vascular mimicry of such cells. Gli1 was found to transcriptionally regulate Sox2 through its promoter region, and Gli1 could be detected on the Sox2 promoter. Inhibition of Hedgehog signaling appeared to work cooperatively with EGFR inhibitors in markedly reducing the viability of NSCLC cells as well as the self-renewal of stem-like cells. Thus, our study demonstrates a cooperative functioning of the EGFR signaling and Hedgehog pathways in governing the stem-like functions of NSCLC cancer stem cells and presents a novel therapeutic strategy to combat NSCLC harboring EGFR mutations.

YAP1 Regulates OCT4 Activity and SOX2 Expression to Facilitate Self-Renewal and Vascular Mimicry of Stem-Like Cells.

Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that stem-like cells contribute to the genesis and progression of NSCLC. Our results show that the transcriptional coactivator yes-associated protein 1 (YAP1), which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and ability to form angiogenic tubules. Inhibition of YAP1 by a small molecule or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of stem-like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor, Sox2. YAP1 could transcriptionally induce Sox2 through a physical interaction with Oct4; Sox2 induction occurred independent of TEAD2 transcription factor, which is the predominant mediator of YAP1 functions. The binding of Oct4 to YAP1 could be detected in cell lines as well as tumor tissues; the interaction was elevated in NSCLC samples compared to normal tissue as seen by proximity ligation assays. YAP1 bound to Oct4 through the WW domain, and a peptide corresponding to this region could disrupt the interaction. Delivery of the WW domain peptide to stem-like cells disrupted the interaction and abrogated Sox2 expression, self-renewal, and vascular mimicry. Depleting YAP1 reduced the expression of multiple epithelial-mesenchymal transition genes and prevented the growth and metastasis of tumor xenografts in mice; overexpression of Sox2 in YAP1 null cells rescued these functions. These results demonstrate a novel regulation of stem-like functions by YAP1, through the modulation of Sox2 expression.

Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ ß-arrestin-1 dependent manner in NSCLC.

Lung cancer remains the leading cause of cancer-related deaths worldwide. ß-arrestin-1 (ARRB1), a scaffolding protein involved in the desensitization of signals arising from activated G-protein-coupled receptors (GPCRs), has been shown to play a role in invasion and proliferation of cancer cells, including nicotine-induced proliferation of human non-small cell lung cancers (NSCLCs). In this study, we identified genes that are differentially regulated by nicotine in an ARRB1/ß-arrestin-1 dependent manner in NSCLC cells by microarray analysis. Among the identified genes, SCF (Stem cell factor) strongly differentiated smokers from non-smokers in the Director's Challenge Set expression data and its high expression correlated with poor prognosis. SCF, a major cytokine is the ligand for the c-Kit proto-oncogene and was found to be over expressed in human lung adenocarcinomas, but not squamous cell carcinomas. Data presented here show that transcription factor E2F1 can induce SCF expression at the transcriptional level and depletion of E2F1 or ARRB1/ß-arrestin-1 could not promote self-renewal of SP cells. These studies suggest that nicotine might be promoting NSCLC growth and metastasis by inducing the secretion of SCF, and raise the possibility that targeting signalling cascades that activate E2F1 might be an effective way to combat NSCLC.

Nicotine-mediated invasion and migration of non-small cell lung carcinoma cells by modulating STMN3 and GSPT1 genes in an ID1-dependent manner.

BACKGROUND: Inhibitor of DNA binding/Differentiation 1 (ID1) is a helix loop helix transcription factor that lacks the basic DNA binding domain. Over-expression of ID1 has been correlated with a variety of human cancers; our earlier studies had shown that reported ID1 is induced by nicotine or EGF stimulation of non-small cell lung cancer (NSCLC) cells and its down regulation abrogates cell proliferation, invasion and migration. Here we made attempts to identify downstream targets of ID1 that mediate these effects. METHODS: A microarray analysis was done on two different NSCLC cell lines (A549 and H1650) that were transfected with a siRNA to ID1 or a control, non-targeting siRNA. Cells were stimulated with nicotine and genes that were differentially expressed upon nicotine stimulation and ID1 depletion were analyzed to identify potential downstream targets of ID1. The prospective role of the identified genes was validated by RT-PCR. Additional functional assays were conducted to assess the role of these genes in nicotine induced proliferation, invasion and migration. Experiments were also conducted to elucidate the role of ID1, which does not bind to DNA directly, affects the expression of these genes at transcriptional level. RESULTS: A microarray analysis showed multiple genes are affected by the depletion of ID1; we focused on two of them: Stathmin-like3 (STMN3), a microtubule destabilizing protein, and GSPT1, a protein involved in translation termination; these proteins were induced by both nicotine and EGF in an ID1 dependent fashion. Overexpression of ID1 in two different cell lines induced STMN3 and GSPT1 at the transcriptional level, while depletion of ID1 reduced their expression. STMN3 and GSPT1 were found to facilitate the proliferation, invasion and migration of NSCLC cells in response to nAChR activation. Attempts made to assess how ID1, which is a transcriptional repressor, induces these genes showed that ID1 down regulates the expression of two transcriptional co-repressors, NRSF and ZBP89, involved in the repression of these genes. CONCLUSIONS: Collectively, our data suggests that nicotine and EGF induce genes such as STMN3 and GSPT1 to promote the proliferation, invasion and migration of NSCLC, thus enhancing their tumorigenic properties. These studies thus reveal a central role for ID1 and its downstream targets in facilitating lung cancer progression.

A novel five gene signature derived from stem-like side population cells predicts overall and recurrence-free survival in NSCLC.

Gene expression profiling has been used to characterize prognosis in various cancers. Earlier studies had shown that side population cells isolated from Non-Small Cell Lung Cancer (NSCLC) cell lines exhibit cancer stem cell properties. In this study we apply a systems biology approach to gene expression profiling data from cancer stem like cells isolated from lung cancer cell lines to identify novel gene signatures that could predict prognosis. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stem like properties. Differentially expressed genes that were over or under-expressed at least two fold commonly in all 4 cell lines were identified. We found 354 were upregulated and 126 were downregulated in SP cells compared to MP cells; of these, 89 up and 62 downregulated genes (average 2 fold changes) were used for Principle Component Analysis (PCA) and MetaCore pathway analysis. The pathway analysis demonstrated representation of 4 up regulated genes (TOP2A, AURKB, BRRN1, CDK1) in chromosome condensation pathway and 1 down regulated gene FUS in chromosomal translocation. Microarray data was validated using qRT-PCR on the 5 selected genes and all showed robust correlation between microarray and qRT-PCR. Further, we analyzed two independent gene expression datasets that included 360 lung adenocarcinoma patients from NCI Director's Challenge Set for overall survival and 63 samples from Sungkyunkwan University (SKKU) for recurrence free survival. Kaplan-Meier and log-rank test analysis predicted poor survival of patients in both data sets. Our results suggest that genes involved in chromosome condensation are likely related with stem-like properties and might predict survival in lung adenocarcinoma. Our findings highlight a gene signature for effective identification of lung adenocarcinoma patients with poor prognosis and designing more aggressive therapies for such patients.

Targeting multiple targets in Pseudomonas aeruginosa PAO1 using flux balance analysis of a reconstructed genome-scale metabolic network.

Constraint-based flux balance analysis (FBA) is a powerful tool for predicting target genes that can be engineered by analyzing the redistribution of metabolic fluxes on specific gene modifications. Specifically, the effects of metabolic gene deletions on flux distribution can be examined by forcing the fluxes of different reactions catalyzed by the corresponding gene product to zero. However, the target enzyme needs to be essential for survival of the organism to ensure that efficient chemical inhibition results in cell stasis or death. Here, we investigate the essentiality of enzymes in iMO1056 metabolic model of nosocomial pathogen Pseudomonas aeruginosa by performing in silico enzyme deletions using FBA. We identified 116/113 essential enzymes in rich medium in P. aeruginosa. These were then compared with human metabolic model to identify nonhomologous enzymes that could be possible drug targets. Here, we present a refined list of 41 novel potential targets for P. aeruginosa. These targets were then matched with the enzymes belonging to 97 correlated clusters through which we propose the concept of "one target per cluster." Our approach relates to the "single drug multiple target (SDMT)" concept and has potential in efficient drug target discovery.

Cloning and targeted disruption of two lipopolysaccharide biosynthesis genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by site-directed mutagenesis.

The emergence of antibiotic resistance in bacterial pathogens poses a great challenge to public health and emphasizes the need for new antimicrobial targets. The recent development of microbial genomics and the availability of genome sequences allows for the identification of essential genes which could be novel and potential targets for antibacterial drugs. However, these predicted targets need experimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the pathogen Pseudomonas aeruginosa PAO1 using insertion duplication. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP-glucose (heptosyl) LPS α-1,3-glucosyltransferase were selected as putative target candidates for the gene disruption experiments using plasmid insertion mutagenesis to determine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome resulted in lack of recovery of antibiotic-resistant colonies suggesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa.

A Comparative Study of Metabolic Network Topology between a Pathogenic and a Non-Pathogenic Bacterium for Potential Drug Target Identification.

Metabolic network provides a unified platform to integrate all the biological information on genes, proteins, metabolites, drugs and drug targets for a comprehensive system level study of the relationship between metabolism and disease. In recent times, drug-target identification by in silico methods has emerged causing a phenomenal achievement in the field of drug discovery. This paper focuses on describing how microbial drug target identification can be carried out using bioinformatic tools. Specifically, it highlights the use of metabolic 'choke point' and 'load point' analyses to understand the local and global properties of metabolic networks in Pseudomonas aeruginosa and allow us to identify potential drug targets. We also list out top 10 choke point enzymes based on the load point values and the number of shortest paths. A non-pathogenic bacterial strain Pseudomonas putida KT2440 and a related pathogenic bacteria P.aeruginosa PA01 was selected for the network anlaysis. A comparative study of the metabolic networks of these two microbes highlights the analogies and differences between their respective pathways. System analysis of metabolic networks will help us in identifying new drug targets which in turn will generate more in-depth understanding of the mechanism of diseases and thus provide better guidance for drug discovery.

A combined computational-experimental analyses of selected metabolic enzymes in Pseudomonas species.

Comparative genomic analysis has revolutionized our ability to predict the metabolic subsystems that occur in newly sequenced genomes, and to explore the functional roles of the set of genes within each subsystem. These computational predictions can considerably reduce the volume of experimental studies required to assess basic metabolic properties of multiple bacterial species. However, experimental validations are still required to resolve the apparent inconsistencies in the predictions by multiple resources. Here, we present combined computational-experimental analyses on eight completely sequenced Pseudomonas species. Comparative pathway analyses reveal that several pathways within the Pseudomonas species show high plasticity and versatility. Potential bypasses in 11 metabolic pathways were identified. We further confirmed the presence of the enzyme O-acetyl homoserine (thiol) lyase (EC: 2.5.1.49) in P. syringae pv. tomato that revealed inconsistent annotations in KEGG and in the recently published SYSTOMONAS database. These analyses connect and integrate systematic data generation, computational data interpretation, and experimental validation and represent a synergistic and powerful means for conducting biological research.

Differential genome analyses of metabolic enzymes in Pseudomonas aeruginosa for drug target identification.

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We used a differential pathway analyses approach (based on KEGG data) to identify essential genes from Pseudomonas aeruginosa. Our approach identified 214 unique enzymes in P. aeruginosa that may be potential drug targets and can be considered for rational drug design. About 40% of these putative targets have been reported as essential by transposon mutagenesis data elsewhere. Homology model for one of the proteins (LpxC) is presented as a case study and can be explored for in silico docking with suitable inhibitors. This approach is a step towards facilitating the search for new antibiotics.