Analysis of global changes in gene expression induced by human polynucleotide phosphorylase (hPNPase(old-35)).

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPase(old-35)), we performed gene expression analysis of HeLa cells in which hPNPase(old-35) was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPase(old-35) was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPase(old-35) expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPase(old-35) expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPase(old-35) infected HO-1 melanoma cells and HeLa cells overexpressing hPNPase(old-35) under the control of a doxycycline-inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPase(old-35) is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation.

Evolutionary dynamics of Polynucelotide phosphorylases.

Polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3'→5' phosphate-dependent exoribonucease belonging to the PDX family of proteins. It consists of two catalytic RNase PH domains (PNP1 and PNP2), an α-helical domain and two RNA-binding domains. The PNP1 and PNP2 domains share substantial sequence and structural homology with RNase PH (RPH), which is another PDX family member found in all the three major kingdoms of life, suggesting that these three domains originated from a common ancestor. Phylogenetic analysis (based on the PNPase/RNase PH sequence information for 43 vertebrate taxa) shows that PNP2 and RPH are sister taxa which arose through duplication of the ancestral PNP1 domain. Also, all three domains (PNP1, PNP2 and RPH), along with the KH and S1 domains have undergone significant and directional sequence change, as determined by branch and site-specific dN/dS analyses. In general, codons that show dN/dS ratios that are significantly greater than 1.0 are outside the ordered regions (α-helices and ß-sheets) of these protein domains. In addition, sites that have been selected for mutagenesis in these proteins lie embedded in regions where there is a preponderance of codons with dN/dS values that are not significantly different from 0.0. Overall, this report is an attempt to further our understanding of the evolutionary history of these three protein domains, and define the evolutionary events that led to their refinement in the vertebrate lineage leading to mammals.

Human polynucleotide phosphorylase selectively and preferentially degrades microRNA-221 in human melanoma cells.

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase(old-35)), a type I IFN-inducible 3'-5' exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase(old-35) identified miRNAs differentially and specifically regulated by hPNPase(old-35). One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27(kip1), displayed robust down-regulation with ensuing up-regulation of p27(kip1) by expression of hPNPase(old-35), which also occurred in multiple human melanoma cells upon IFN-beta treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase(old-35). Inhibition of hPNPase(old-35) by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-beta-mediated growth inhibition, accentuating the importance of hPNPase(old-35) induction and miR-221 down-regulation in mediating IFN-beta action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase(old-35) might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma.

Distinct Inflammatory Macrophage Populations Sequentially Infiltrate Bone-to-Tendon Interface Tissue After Anterior Cruciate Ligament (ACL) Reconstruction Surgery in Mice.

Macrophages are important for repair of injured tissues, but their role in healing after surgical repair of musculoskeletal tissues is not well understood. We used single-cell RNA sequencing (RNA-seq), flow cytometry, and transcriptomics to characterize functional phenotypes of macrophages in a mouse anterior cruciate ligament reconstruction (ACLR) model that involves bone injury followed by a healing phase of bone and fibrovascular interface tissue formation that results in bone-to-tendon attachment. We identified a novel "surgery-induced" highly inflammatory CD9+ IL1+ macrophage population that expresses neutrophil-related genes, peaks 1 day after surgery, and slowly resolves while transitioning to a more homeostatic phenotype. In contrast, CX3CR1+ CCR2+ macrophages accumulated more slowly and unexpectedly expressed an interferon signature, which can suppress bone formation. Deletion of Ccr2 resulted in an increased amount of bone in the surgical bone tunnel at the tendon interface, suggestive of improved healing. The "surgery-induced macrophages" identify a new cell type in the early phase of inflammation related to bone injury, which in other tissues is dominated by blood-derived neutrophils. The complex patterns of macrophage and inflammatory pathway activation after ACLR set the stage for developing therapeutic strategies to target specific cell populations and inflammatory pathways to improve surgical outcomes. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Immune Response to Persistent Staphyloccocus Aureus Periprosthetic Joint Infection in a Mouse Tibial Implant Model.

Staphyloccocus aureus is one of the major pathogens in orthopedic periprosthetic joint infection (PJI), a devastating complication of total joint arthroplasty that often results in chronic and persistent infections that are refractory to antibiotics and require surgical interventions. Biofilm formation has been extensively investigated as a reason for persistent infection. The cellular composition, activation status, cytokine profile, and role of the immune response during persistent S. aureus PJI are incompletely understood. In this study, we used histology, multiparametric flow cytometry, and gene expression analysis to characterize the immune response in a clinically relevant orthopedic PJI model. We tested the hypothesis that persistent S. aureus infection induces feedback mechanisms that suppress immune cell activation, thereby affecting the course of infection. Surprisingly, persistent infection was characterized by strikingly high cytokine gene expression indicative of robust activation of multiple components of innate and adaptive immunity, along with ongoing severe neutrophil-dominated inflammation, in infected joint and bone tissues. Activation and expansion of draining lymph nodes and a bone marrow stress granulopoiesis reaction were also maintained during late phase infection. In parallel, feedback mechanisms involving T-cell inhibitory receptors and exhaustion markers, suppressive cytokines, and regulatory T cells were activated and associated with decreased T-cell proliferation and tissue infiltration during the persistent phase of infection. These results identify the cellular and molecular components of the mouse immune response to persistent S. aureus PJI and indicate that neutrophil infiltration, inflammatory cytokine responses, and ongoing lymph node and bone marrow reactions are insufficient to clear infection and that immune effector mechanisms are suppressed by feedback inhibitory pathways. These immune-suppressive mechanisms are associated with diminished T-cell proliferation and tissue infiltration and can be targeted as part of adjuvant immunotherapeutic strategies in combination with debridement of biofilm, antibiotics, and other therapeutic modalities to promote eradication of infection. © 2021 American Society for Bone and Mineral Research (ASBMR).

Immune and repair responses in joint tissues and lymph nodes after knee arthroplasty surgery in mice.

The importance of a local tissue immune response in healing injured tissues such as skin and lung is well established. Little is known about whether sterile wounds elicit lymph node (LN) responses and inflammatory responses after injury of musculoskeletal tissues that are mechanically loaded during the repair response. We investigated LN and tissue immune responses in a tibial implant model of joint replacement surgery where wounded tissue is subjected to movement and mechanical loading postoperatively. Draining inguinal and iliac LNs expanded postoperatively, including increases in regulatory T cells and activation of a subset of T cells. Thus, tissue injury was actively sensed in secondary lymphoid organs, with the potential to activate adaptive immunity. Joint tissues exhibited three temporally distinct immune response components, including a novel interferon (IFN) response with activation of signal transducer and activator of transcription (STAT) and interferon regulatory factor (IRF) pathways. Fibrovascular tissue formation was not associated with a macrophage type 2 (M2) reparative immune response, but instead with delayed induction of interleukin-1 family (IL-1ß, IL-33, IL-36), IL-17, and prostaglandin pathway genes concomitant with transforming growth factor (TGF)-ß and growth factor signaling, fibroblast activation, and tissue formation. Tissue remodeling was associated with activity of the HOX antisense intergenic RNA (HOTAIR) pathway. These results provide insights into immune responses and regulation of tissue healing after knee arthroplasty that potentially can be used to develop therapeutic strategies to improve healing, prevent arthrofibrosis, and improve surgical outcomes. © 2021 American Society for Bone and Mineral Research (ASBMR).

HBEGF+ macrophages in rheumatoid arthritis induce fibroblast invasiveness.

Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+ inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor-dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF+ inflammatory macrophages; however, in some cases, medication redirected them into a state that is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions.

Dissection and function of autoimmunity-associated TNFAIP3 (A20) gene enhancers in humanized mouse models.

Enhancers regulate gene expression and have been linked with disease pathogenesis. Little is known about enhancers that regulate human disease-associated genes in primary cells relevant for pathogenesis. Here we use BAC transgenics and genome editing to dissect, in vivo and in primary immune cells, enhancers that regulate human TNFAIP3, which encodes A20 and is linked with autoimmune diseases. A20 expression is dependent on a topologically associating subdomain (sub-TAD) that harbors four enhancers, while another >20 enhancers in the A20 locus are redundant. This sub-TAD contains cell- and activation-specific enhancers, including an enhancer (termed TT>A) harboring a proposed causal SLE-associated SNV. Deletion of the sub-TAD or the TT>A enhancer results in enhanced inflammatory responses, autoantibody production, and inflammatory arthritis, thus establishing functional importance in vivo and linking enhancers with a specific disease phenotype. These findings provide insights into enhancers that regulate human A20 expression to prevent inflammatory pathology and autoimmunity.

Tumor Necrosis Factor dynamically regulates the mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes.

During rheumatoid arthritis (RA), Tumor Necrosis Factor (TNF) activates fibroblast-like synoviocytes (FLS) inducing in a temporal order a constellation of genes, which perpetuate synovial inflammation. Although the molecular mechanisms regulating TNF-induced transcription are well characterized, little is known about the impact of mRNA stability on gene expression and the impact of TNF on decay rates of mRNA transcripts in FLS. To address these issues we performed RNA sequencing and genome-wide analysis of the mRNA stabilome in RA FLS. We found that TNF induces a biphasic gene expression program: initially, the inducible transcriptome consists primarily of unstable transcripts but progressively switches and becomes dominated by very stable transcripts. This temporal switch is due to: a) TNF-induced prolonged stabilization of previously unstable transcripts that enables progressive transcript accumulation over days and b) sustained expression and late induction of very stable transcripts. TNF-induced mRNA stabilization in RA FLS occurs during the late phase of TNF response, is MAPK-dependent, and involves several genes with pathogenic potential such as IL6, CXCL1, CXCL3, CXCL8/IL8, CCL2, and PTGS2. These results provide the first insights into genome-wide regulation of mRNA stability in RA FLS and highlight the potential contribution of dynamic regulation of the mRNA stabilome by TNF to chronic synovitis.

Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense.

One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. Mycobacterium tuberculosis (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro vs. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of rpoB>aroK>ppk>rpoC>ilvB. RpoB was used as the cidality control. In-vitro and in-vivo studies feature aroK (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute infection phase for the high fidelity translation, the compound efficacy may also be evaluated in the low pH, in addition to the standard replication condition.

Examination of Epigenetic and other Molecular Factors Associated with mda-9/Syntenin Dysregulation in Cancer Through Integrated Analyses of Public Genomic Datasets.

mda-9/Syntenin (melanoma differentiation-associated gene 9) is a PDZ domain containing, cancer invasion-related protein. In this study, we employed multiple integrated bioinformatic approaches to identify the probable epigenetic factors, molecular pathways, and functionalities associated with mda-9 dysregulation during cancer progression. Analyses of publicly available genomic data (e.g., expression, copy number, methylation) from TCGA, GEO, ENCODE, and Human Protein Atlas projects led to the following observations: (a) mda-9 expression correlates with both copy number and methylation level of an intronic CpG site (cg1719774) located downstream of the CpG island, (b) cg1719774 methylation is a likely prognostic marker in glioma, (c) among 22 cancer types, melanoma exhibits the highest mda-9 level, and lowest level of methylation at cg1719774, (d) cg1719774 hypomethylation is also associated with histone modifications (at the mda-9 locus) indicative of more active transcription, (e) using Gene Set Enrichment Analysis (GSEA), and the Virtual Gene Overexpression or Repression (VIGOR) analytical scheme, we were able to predict mda-9's association with extracellular matrix organization (e.g., MMPs, collagen, integrins), IGFBP2 and NF-κB signaling pathways, phospholipid metabolism, cytokines (e.g., interleukins), CTLA-4, and components of complement cascade pathways. Indeed, previous publications have shown that many of the aforementioned genes and pathways are associated with mda-9's functionality.

Human polynucleotide phosphorylase (hPNPaseold-35): should I eat you or not--that is the question?

RNA degradation plays a fundamental role in maintaining cellular homeostasis whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3'-5' exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel "overlapping pathway screening" approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. The objective of this review is to provide an up-to-date perspective of the recent discoveries made relating to hPNPaseold-35 and the impact they continue to have on our comprehension of its expanding and diverse array of functions.

Identification of genes potentially regulated by human polynucleotide phosphorylase (hPNPase old-35) using melanoma as a model.

Human Polynucleotide Phosphorylase (hPNPase(old-35) or PNPT1) is an evolutionarily conserved 3'→ 5' exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPase(old-35) in cellular physiology, we knocked-down and overexpressed hPNPase(old-35) in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPase(old-35) resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPase(old-35) caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPase(old-35) knockdown and overexpression datasets allowed us to identify 77 potential "direct" and 61 potential "indirect" targets of hPNPase(old-35) which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPase(old-35) expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.

Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells.

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda- 7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca (2+). Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda- 7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.

SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling.

The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p = 0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced overexpression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p = 0.0015-0.030) and in vivo (p = 0.016) cellular growth, invasion (p = 0.029-0.049), and colony formation (p = 0.023-0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser(473)), STAT3 (Tyr(705)), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated ß-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.

Raf kinase inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melanoma.

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor, RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored a hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested cross-talk between these two proteins. Furthermore, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Finally, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth, and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.

MDA-9/syntenin: a positive gatekeeper of melanoma metastasis.

Melanoma differentiation associated gene-9 (MDA-9), synonymous with syntenin, is an adapter protein that provides a central role in regulating cell-cell and cell-matrix adhesion. MDA-9/syntenin transduces signals from the cell-surface to the interior through its interaction with a plethora of additional proteins and actively participates in intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies demarcate a seminal role of MDA-9/syntenin in cancer metastasis. In the context of melanoma, MDA-9/syntenin functions as a positive regulator of melanoma progression and metastasis through interactions with c-Src and promotes the formation of an active FAK/c-Src signaling complex leading to NF-k B and matrix metalloproteinase (MMP) activation. The present review provides a current perspective of our understanding of the important features of MDA-9/syntenin and its significant role in tumor cell metastasis with special focus on molecular mechanism of action.

Selected approaches for rational drug design and high throughput screening to identify anti-cancer molecules.

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specificpromoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

Developing an effective gene therapy for prostate cancer: New technologies with potential to translate from the laboratory into the clinic.

Prostate cancer is the second leading cause of cancer-related deaths in men in the U.S. At present, no single or combination therapy has shown efficacy in decreasing disease progression in patients with metastatic disease. A potentially viable approach for treating late-stage prostate cancer is gene therapy. Adenoviruses (Ad) are the most commonly used mode of gene delivery, but progress using this vector has been hampered by concerns over the safety and practicality of viruses including conditionally replicating Ads (CRAds), particularly for intravenous delivery, and the inefficiency of non-viral transfection techniques. Major challenges for effective gene therapy using Ads are the limited infectivity of regular Ad serotype 5 (Ad5) and the inability to specifically deliver the therapeutic directly into diseased tissue without trapping in the liver or elimination by the immune system. The shortcoming in using Ad5 is mostly attributed to a reduction in Coxsackie-adenovirus receptors (CAR) on the surface of cancer cells, which can be mitigated by generating tropism-modified Ads permitting CAR-independent infection of tumor cells. The limitations of systemic gene delivery can now be overcome by using a novel targeted-delivery approach such as ultrasound (US) contrast agents (microbubbles) to deliver effective therapeutic reagents, Ads, or recombinant proteins, combined with ultrasound-targeted microbubble destruction (UTMD), to develop a site-specific therapy in immune competent transgenic mouse models. These unique strategies for enhancing the efficacy of gene therapy provide a direct path to translation from the laboratory into the clinic for developing an effective gene therapy of prostate cancer.