Andrei V. Gudkov - US grants

Understanding the molecular basis of tumor metastasis should provide important insights into new strategies for the treatment of cancer. In contrast to the greater progress achieved in elucidating molecular genetic mechanisms of neoplastic transformation, the nature of the genes responsible for metastasis is far less well understood. Only a few genes directly or indirectly associated with metastatic behavior have been identified so far. This may be related to the possible recessive nature of many of the genetic alterations that lead to tumor cell survival and proliferation in a heterologous tissue environment. While dominant genes can be detected and characterized by gene transfer, identification of recessive mechanisms of metastasis may be achieved by the application of the recently developed general procedure for functional selection of recessive genes using genetic suppressor elements (GSEs) which induce dominant phenotypic changes by interfering with the function of genes from which they are derived. The methodology for GSE isolation has been developed on the model of human topoisomerase II (topo II). In that study expression selection of GSEs was carried out from a mixture of recombinant retroviruses containing a library of random fragment of topo II cDNA. A set of topo II-cDNA-derived fragments, encoding either antisense RNA sequences or short truncated proteins were selected for their ability to induce cellular resistance to topo II-interactive drugs by suppressing topo II expression or function. The GSE selection approach was then expanded to include all the expressed genes by preparing a large (3X10 independent clones) library from normalized (uniform abundance) cDNA of NIH 3T3 cells which should contain GSEs for most of the genes expressed in these cells. By using different selection strategies, we have isolated from this library several biologically active GSEs representing short fragments of known and unknown genes and inducing different selectable phenotypes (drug resistance or neoplastic transformation of NIH 3T3 cells). In the present project, we are planning to isolate from the same library GSEs capable of conferring the ability for spontaneous or experimental metastasis on non-metastasizing mouse cells, by in vivo selection of cells carrying such GSEs. The cloned GSEs will be used as probes for the isolation of the full-length cDNAs of their corresponding genes, whose function would be associated with preventing metastatic conversion. Cell phenotypes associated with suppression or overexpression of these genes in different rodent tumor cell lines will be characterized. The GSE approach should allow the identification and functional analysis of new metastasis suppressor genes.

DESCRIPTION The parent project for this FIRCA application proposes to identify new genes involved in cell growth control by their ability, when inactivated, to transform mouse fibroblasts. Genetic suppressor elements (GSE) are fragments of genes that encode either antisense RNA or truncated proteins acting as dominant negative mutants. The effects of these GSEs (derived from a library of randomly fragmented normalized cDNA) and resulting isolated genes can then be characterized by their effects on cultured cells. Finally, the investigators would clone the human homologs to analyze their association with cancer. The purpose of the FIRCA study is to employ the GSE approach of the parent grant and apply it to a specific gene, p53, to identify the domains of this gene that affect the specific phenotypes, anchorage dependence and response to dominant oncogenes. A proposed methodology in the parent grant, the two-hybrid system, will be applied to p53 to isolate and characterize p53 interactors. Using the GSE and two-hybrid approaches, the investigators hope to characterize new factors in the p53 pathway.

DESCRIPTION: Using technology based upon genetic suppressor elements (GSEs), which the applicant has played a large role in developing, Dr. Gudkov wishes to identify cellular factors affecting either expression or function of the p53 tumor suppressor gene. Regulation of transcription of p53 has not received a lot of scientific scrutiny since elevated levels of p53 protein- associated with its growth inhibitory and apoptotic roles is primarily due to conformational changes leading to an increased stability. Using a p53 responsive promoter-Lac Z reporter construct in transgenic mice Dr. Gudkov has shown tissue and developmental stage-specific expression of the Lac Z reporter. This expression correlates directly with p53 mRNA expression. He has further demonstrated that widespread apoptosis in early embryos following DNA damage is correlated directly with p53 mRNA expression and its translation. The applicant intends to determine the regulation of this mRNA expression. Various parameters will be investigated, including the possibility of positive and negative transcriptional factors and mRNA stability. cDNA expression libraries (full length cDNAs and GSEs) with be used to screen for clones affecting p53 mRNA expression. Using the GSE strategy that was used successfully to identify a growth suppressor, p33(ING1), the applicant will screen for clones that induce phenotypes associated with p53 inactivation. In this case GSEs are isolated from retroviral libraries of randomly fragmented cDNA sequences identified through two-hybrid interaction trap procedures using p53 and p33(ING1) proteins as baits. The third major aim will be to attempt to place the position and role of these candidate proteins in the p53 pathway, via overexpression and suppression studies. The expression patterns of the candidate proteins and their chromosome map positions are included in these aims.

Studies of tumor resistance and sensitivity to anti-cancer therapy is largely limited to the analysis of intrinsic mechanisms of cell response to treatment. However, tumor response also involves paracrine effects, including interactions of tumor cells with each other, with the stroma and with factors released by distant tissues. Here we propose a program of studies that is aimed at the determination of the role and mechanisms of cell-cell communication in drug and radiation treatment. It is based on two lines of preliminary observations. First, by using the genetic suppressor element (GSE) technology, ubiquitous kinesin heavy chain (uKHC) was identified as a drug-sensitivity gene, deregulation of which results in an unusual type of drug resistance. More recently, it was found that normal and tumor cells under conditions of stress secrete in a p53-dependent manner growth inhibitory factors causing negative paracrine regulation ("bystander effect"), affecting growth of neighboring cells. Preliminary data were generated showing that kinesin-mediated drug resistance is determined by modulation of the bystander effect, suggesting that kinesin participates in p53-dependent stress-induced secretion of growth inhibitors. Current program is devoted to characterization of the role of stress-induced bystander effect in tumor response to anticancer treatment, identification of factors determining this phenomenon and the analysis of molecular mechanisms and consequences of deregulation of p53- and kinesin-mediated secretion. Specific aims include: (i) the establishment of experimental approaches and model systems to study the role of stress- dependent bystander effect in vitro and in vivo, (ii) the analysis of involvement of the kinesin function in the p53-dependent bystander effect in drug- or radiation- treated cell populations, (iii) identification of cellular proteins involved in stress-induced kinesin-mediated bystander effect, and (iv) isolation and characterization of cellular factor(s) determining stress- induced bystander effect in tumor sensitivity and resistance to therapeutic treatments and in identification of molecular determinants of this form of therapy response.

DESCRIPTION (provided by applicant): Progress in curing prostate cancer could be greatly facilitated by identification of reliable and highly specific new serological markers for diagnostics and development of new therapeutic strategies and pharmaceuticals for effective elimination of prostate cancer cells. Both goals could be achieved through identification of new molecular targets for selective killing or selective detection of cancer cells of prostate origin. The present program is aimed at identification of prospective gene targets for both diagnostic and therapeutic applications using a combination of recently developed powerful functional gene cloning methodologies with cDNA array-based gene expression profiling and rationally designed experimental models. Diagnostic and therapeutic value of prospective candidates will be evaluated using specific inhibitors and antibodies. Aim 1 is devoted to identification of new candidate prostate tumor markers among proteins encoded by prostate-specific genes that are under negative control of tumor suppressor genes (PTEN, Rb, p53 and p27) followed by designing immunodiagnostic serological assay for prostate cancer. This approach is an extension of a recent work that linked tumor specificity of PSA expression with its negative regulation by p53. Aims 2 and 3 represent cells by small molecules. They are based on two new genetic methodologies recently developed by research the research team. Genetic elements specifically killing prostate carcinoma cells (with specific focus on hormone-independent tumors) will be isolated by functional screening of expression libraries of prostate-specific gene sequences collected based on a combination of experimental and bioinformatics-based criteria. Genes essential for viability of prostate cells (EVP-genes) will be defined and used as targets for selection of specific small molecules inhibiting growth of cells of prostate origin that will serve as prototype new drugs for prostate cancer treatment.

[unreadable] DESCRIPTION (provided by applicant): Identification of genes and pathways that contribute to tumorigenesis should lead to the defining of novel targets for therapeutic intervention and provide biomarkers for better diagnosis, staging, and risk assessment for individual cancer patients. Further progress in molecular genetics of cancer would greatly benefit from a reliable methodology of assigning gene functions based on phenotypic changes resulting from modulations in gene expression. Existing techniques of this kind are based on screening of genetically modified cells for genetic elements favoring cell growth under restrictive conditions (positive selection). Recently, a novel gene discovery methodology, named selection subtraction approach (SSA), was developed that allowed for a direct negative selection. Although proven useful for isolation of killing or growth suppressive genetic elements, SSA's capabilities are limited by the necessity to construct expression libraries. This proposal is focused on developing a new Insertional Selection Subtraction Approach (ISSA) that combines the advantages of SSA with the power of retroviral insertional mutagenesis and is based on a completely new vector system. The insertional mutagenesis arm is enhanced by the addition of a regulatable promoter, splice donor sequences, and the ability to trap polyadenylation signals. The second arm of ISSA involves "tagging" or "bar-coding" each mutant (SSA), thereby allowing to monitor the relative abundance of mutants within the population during selection by using "retrophage arrays," the key component of SSA. ISSA technique promises to become a universal functional screening tool, free from major drawbacks of its precursors. After "technical" testing of ISSA, its power will be determined by identification of genes involved in regulation of cell sensitivity to TNF, a well-characterized system that has been already well studied by numerous approaches, including functional selection. [unreadable] [unreadable]

This proposal deals with a new class of radioprotectors - natural products generated by microbes and tumor cells that act by activating internal cellular mechanisms of defense and inhibiting programmed cell death (apoptosis) as part of natural survival strategy in the host organism. Specifically, it is focused on development of the product originating from flagellin of Salmonella and latent form of human TGFbeta2, both acting as NF-kappaB-activating agents through interaction with specific cellular receptors (toll-like receptor 5 and TGFbeta receptors 1 and 2, respectively). During our preliminary work, we have completed structure-functional optimization of both factors and created products named, which demonstrated powerful anti-apoptotic activity. CBLB502 has been thoroughly characterized in vivo and showed outstanding efficacy against hematopoietic and gastro-intestinal radiation syndromes, thus validating our approach to radioprotection. So far we have been working with lethal radiation doses and treatment of acute consequences of radiation injury. The success of this program allows us now to address another radioprotective aspect of activity of our products - protection of the immune system - and to evaluate the perspectives of their use for this indication. The study involves: (i) thorough characterization of protective effect of both products on different compartments of immune system (adaptive, innate immunity and stromal components), (ii) evaluation of safety of CBLB502 and CBLB102 by estimation of their effect on radiation-induced carcinogenesis and (iii) testing their protective effect in non-human primates as an essential and final step before full-scale program of development of at least one of the tested compounds into radioprotecting drug.

DESCRIPTION (provided by applicant): The toxicity of ionizing radiation (IR) in mammals is largely due to damage to the most radiosensitive tissues, the gastrointestinal (GI) tract and the hematopoietic (HP) system. A number of approaches and compounds have been developed to protect or restore HP, including immune, function following IR exposure. In contrast, GI radiation syndrome remains poorly treatable resulting in significant mortality and reduced quality of life for survivors. In preliminary studies it was demonstrated that the bacterial flagellin protein, acting as an NF-?B activating agonist of Toll-like receptor 5 (TLR5), is a powerful radioprotectant and radiomitigator capable of improving survival when given either before or after lethal IR, respectively. Notably, the anti-radiation effects of flagellin are associated with protection of the GI tract as well as the HP system. A pharmacologically optimized TLR5 agonist with reduced immunogenicity (CBLB502) was generated by deleting portions of flagellin that are non-essential for TLR5 activation. CBLB502 is effective and non-toxic in mice and non-human primates and is being developed into a stable, self-administrable radiation antidote projected for biodefense applications. This proposal is focused on deciphering the molecular and cellular mechanisms of action of CBLB502. Aim 1 will involve identification of cellular mediators of CBLB502's effect on protection and healing of small intestine in irradiated mice with particular attention on the role of bone marrow-derived cells. Aim 2 will be devoted to analysis of molecular signaling pathways induced by CBLB502 that are critical for its radioprotection and radiomitigation properties. A combination of hypothesis-driven and unbiased functional screening approaches will be used to determine CBLB502 molecular targets. Finally, Aim 3 will concentrate on assessing the effects of CBLB502 on the short- and long-term pathological consequences of IR in other radiosensitive tissues, including liver, kidney and lung. The frequency and severity of fibrosis and cancer development will be assessed in a mouse model. Completion of this program will provide critical information about an emerging class of radiation antidotes that will allow improved understanding of their potential as human therapeutics targeting acute radiation syndrome. The potential scenarios for nuclear accidents and terrorist attacks in today's world imply mass radiation injury casualties affected by systemic exposure to radioactive materials. The toxicity of total body irradiation (TBI) is associated with induction of acute radiation syndrome (ARS) primarily involving damage to the highly radiosensitive gastrointestinal (GI) tract and hematopoietic (HP) system. While a number of approaches and compounds have been developed to protect or restore HP function following TBI, GI radiation syndrome remains poorly treatable, resulting in significant mortality and reduced quality of life for survivors. An ideal radiation countermeasure would protect both the GI and HP systems and would be capable of both radioprotection (prophylaxis) and mitigation of radiation injury. Moreover, biodefense applications require a drug that is easily self-administered, has a long shelf life and does not require special handling or storage conditions. We have recently discovered a new class of agents that satisfy all of the above requirements, most notably being effective against GI radiation syndrome as well as HP syndrome. Our lead compound, named CBLB502, is a powerful radioprotectant and mitigator of radiation injury suitable for intramuscular injections. It is a polypeptide derived from bacterial protein flagellin that acts as an agonist of Toll-like receptor 5 (TLR5), which is expressed in radiosensitive cells of the intestine and bone marrow progenitors and, upon binding with the ligand, activates several pro-survival pathways, mediated by NF-?B signaling. Here we propose a program to comprehensively characterize the molecular and cellular mechanisms underlying the anti-radiation activity of CBLB502, particularly focusing on GI radiation syndrome. We will also investigate the effect of CBLB502 on the pathological consequences of IR in other tissues, including radiation carcinogenicity. The proposed experiments will provide essential mechanistic insight into the previously observed radio-protective and -mitigative effects of CBLB502.

Summary The long term goal of this research is to utilize toll-like receptor 5 (TLR5) agonist based immunotherapy with immunomodulatory radiation treatment in the development of a safe and effective combinatorial approach to control liver metastatic disease. The objective of this project is to demonstrate improved therapeutic efficacy against liver metastatic disease using this novel treatment approach and to provide a mechanistic foundation to advance further development of the combined strategy. The hypothesis underlying this research is that TLR5 agonist mediated immune response in the normal liver microenvironment combined with the immunomodulatory effects of liver radiation treatment (RT) generate potent antitumor immunity to eliminate liver metastases. This theory is based on recent cumulative data indicating that the liver microenvironment possesses a unique character in which TLR5 agonists elicit a significant antitumor immune response mediated by TLR5 responsive hepatocytes and on pilot experiments illustrating that the immune stimulatory effects of ionizing radiation applied to the liver enhance and extend the therapeutic reach of TLR5 agonist immunotherapy against liver metastases. In this project, the hypothesis will be tested using the highly aggressive murine colorectal cancer and uveal melanoma liver metastasis models and the newly generated and pharmacologically optimized TLR5 agonist, entolimod. The research plan includes testing different combination therapy regimens and comparing the results of their application to that of liver radiation and TLR5 stimulation individually for antitumor efficacy against liver metastatic growth using the experimental colorectal cancer and spontaneous uveal melanoma liver metastasis models (Aim 1). Single dose and fractionated RT will be applied at different times (adjuvant and neoadjuvant) relative to systemic entolimod administration to determine the most efficacious regimens of the combined therapy. The role of the immunological changes in the hepatic microenvironment in the development of an efficacious antitumor immune response will be determined using phenotypic and functional assays (Aim 2). The results will further the development of a clinical protocol for this innovative approach. While the murine colorectal cancer and uveal melanoma liver metastasis models were chosen due to the significant medical need, clear translational path and encouraging preliminary results obtained with these models, the results will provide information relevant to other cancers with a high propensity to metastasize to the liver. Completion of this project will provide supporting pre-clinical efficacy data to facilitate advancement to clinical trials and a mechanistic foundation on which to base further investigation of the molecular mechanisms responsible for the antitumor activity of this innovative therapeutic approach that is critically needed against liver metastasis.