Ann Richmond - US grants

In most self-renewing tissues, one control point in the regulation of cell growth is availability of hormones and/or growth factors produced by other tissues. One method by which cancer cells may achieve autonomous growth is through production of autostimulatory growth factors. We have found that Hs0294 human melanoma cell line produces at least two growth stimulatory activities: a monolayer mitogen (MGSA) and a class I transforming growth factor (TGF-alpha). The monolayer mitogen is at least partially responsible for the growth of Hs0294 cells in serum-free medium. The melanoma growth stimulatory activity (MGSA) produced by this mitogen is autostimulatory in that it produces an increased incorporation of [[unreadable]3[unreadable]H]-thymidine into DNA and an increase in cell number in the Hs0294 cells which produce it. MGSA is stable to heat (100~C/10 min) and acid (IN acetic acid), is sensitive to dithiothretol and trypsin, and has no associated proteolytic activity. MGSA can be purified by molecular sieve chromatography, followed by RP-HPLC and preparative electrophoresis. In RP-HPLC the major peak of MGSA elutes from a mu-Bondapak C[unreadable]18[unreadable] column at 35+3% acetonitrile and is separated from the TGF-alpha activity eluting at 30+4% acetonitrile which does not compete with [[unreadable]125[unreadable]I]-EGF for binding to EGF receptors. When RP-HPLC purified MGSA is subjected to preparative electrophoresis under non-reducing conditions, the major bioactivity is recovered from a 16 kilodalton moiety. Monoclonal antibodies have been raised to a heterogenous preparation of MGSA and antibodies produced by five of the hybridoma clones will inhibit the growth of Hs0294 cells. An enzyme-linked immunoabsorbent assay developed with one of the antibodies, FB2AH7, shows peaks of immunoreactive protein coincident with the major MGSA peak. Immunoaffinity columns prepared with F82AH7 antibody bind the 16 kilodalton MGSA moiety. Our studies will now be directed toward: (1)\characterizing the MGSA receptor; (2)\determining the cellular specificity of production and response to MGSA; and (3)\determination of effects of MGSA and antibodies to MGSA on the growth of human melanoma tumors growing in nude mice. (J)

The investigations focus on the characterization of an endogenous growth factor produced by malignant melanoma cells and the description of the early expression of this growth factor in premalignant nevi. An autostimulatory growth factor for melanocytes has been isolated and partially characterized. This melanoma growth stimulatory activity (MGSA) resides in a family of antigenically related, acid and heat stable polypeptides which appear to be different from other previously described growth factors. MGSA is not detectable in chromosomally normal, inactive nevi, but nevi exhibiting chromosomal abnormalities are MGSA positive. MGSA can be purified from acetic acid extracts of lyophilized culture medium conditioned by the Hs0294 human melanoma cell line. When this extract is subjected to molecular sieve chromatography, RP-HPLC and preparative gel electrophoresis, low (greater than 14-16Kd) and high (24-26Kd) molecular weight forms of MGSA can be isolated. MGSA is separable from the 125I-EGF competing Class I TGF activity which is also produced by this cell line. MGSA can also be purified by immunoaffinity chromatography using a monoclonal antibody to MGSA, followed by gel filtration HPLS. When immunoprecipitates from 35S-methionine labeled extracts of Hs0294 cells were subjected to reducing SDS-PAGE followed by autoradiography, the major labeled bands had a Mr of greater than 20Kd. Efforts will now be directed toward: (1) determining the relationship between below the low and high molecular weight forms of MGSA and developing an invitro translation system to determine the nature of the precursor form of MGSA; (2) developing a radioreceptor assay for MGSA; (MGSA will be purified by the methods described above, iodinated using Bolton-Hunter reagent and binding assays will be developed using Hs0294 and NRK cells) (3) characterizing the MGSA receptor in isolated plasma membrane preparations using 125I-MGSA and the bifunctional cross-linking reagent, disuccinimidyl suberate; (4) comparing the cellular distribution of bioactive and immunoreactive MGSA and the distribution of MGSA receptors in normal nevi, malignant melanomas, and non-malignant and non-melanoma malignant controls. Nevus, melanoma and other tumor cells will be cultured in vitro, MGSA production will be determined by immunohistochemical and bioactivity assays, and MGSA receptor distribution will be evaluated using the MGSA-radioreceptor assay. Comparisons will be made regarding MGSA binding versus biological response to MGSA in these cultures.

Cytokine networking plays an important role in the regulation of cell growth, differentiation wound healing, and the inflammatory process. For example, TNFalpha and IL-1 bind to specific cellular receptors and stimulate the secretion of a number of secondary cytokines. One such family of secondary cytokines are the chemokines which play a major role in chemotaxis of neutrophils, lymphocytes, and monocytes and also effect growth and cell functions of non-hematopoietic cells. MGSA/GRO and IL-8 are members of the chemokine-alpha group and these proteins are highly chemotactic for neutrophils, and to a lesser extent for lymphocytes and monocytes. The neutrophil receptors for IL-8 have been characterized as 7-transmembrane 6-protein coupled receptors. Two receptors have been cloned: one binds IL-8 with high affinity but not MGSA/GRO. The other binds MGSA/GRO and IL-8 with high affinity. In addition to hematopoietic cells, melanocytes and keratinocytes respond to MGSA and/or IL-8 with enhanced growth. The proposed study is based upon the hypothesis that there are a number of receptors which bind MGSA/GRO proteins and that epithelial cells and mesenchymal cells respond to these ligands differently than neutrophils, basophils, lymphocytes and monocytes, as a result of different receptors, G-protein coupling, or signal transduction pathways. In work described in this proposal we plan to l) use expression cloning, degenerate PCR, and/or low stringency cDNA library screening to identify mouse receptor homologs and to determine whether there are additional human receptors for the MGSA/GRO chemokines and to compare the affinity of the alpha, beta, gamma isoforms for these receptors; 2) determine which serine residues are phosphorylated in response to MGSA and whether these phosphorylation events enhance or inhibit signaling through the receptor and/or biological response; 3) characterize the MGSA signal transduction pathway in non-lymphoid mesenchymal and epithelial cells as compared to neutrophils. 4) determine the effects of antagonism of MGSA receptor function in vivo using a variety of techniques to eliminate receptor function. These studies will utilize cloning strategies, site- directed mutagenesis and deletion mutagenesis, classical signal transduction methodology, purification/characterization of G-proteins, eucaryotic transfection and over-expression strategies, as well as transgenic models. The ultimate goal of the studies will be to develop a mechanism for antagonizing the MGSA receptor so that we may learn more about the role of this group of chemokines in development, growth regulation, wound healing and inflammation.

The MGSA/GRO gene was initially described as an autocrine growth factor for melanoma cells and as a growth related early response gene in fibroblasts. Subsequently, a family of these cytokines was identified including MGSA/GRO alpha, beta, gamma, and delta. the homology of the cDNAs for these four MGSA/GRO genes range from 82-93%. Many tumor cells over-express MGSA/GRO and there is evidence for tissue specific over- expression of the various subtypes of MGSA/GRO. The degradation rate of the various sub-forms appear to differ in tissues. The mRNA levels for these genes is increased when cells are treated with TPA, LPS, TNFalpha, IL-1, EGF, cycloheximide, serum, thrombin, and a number of other factors. MGSA/GRO mRNA levels are suppressed by TGFbeta in some cells. The regulation of mRNA levels by these agents appears to be the result of transcriptional effects as well as increased/decreased mRNA stability. The Hs294T malignant melanoma cell line over-expresses MGSA/GROalpha but not MGSA/GRObeta, gamma, or delta. Mutations of the NF-kappaB element markedly reduces basal transcription of a 5' MGSA/GRO CAT construct in melanoma tumor cells. In order to determine the mechanism for the elevated expression of MGSA/GRO, we will compare the basal and cytokine regulated expression of MGSA/GRO genes in melanoma to that in normal melanocytes. The goal of this study is to test the hypothesis that altered expression of MGSA/GROalpha in melanoma cells results from a combination of alterations in the level of NF-kappaB transactivation and altered mRNA stabilization. Specifically, we plan: (1) to compare the basal levels of transcription of MGSA/GRO alpha, beta, gamma, delta genes in normal and transformed melanocytes; (2) to compare the transcriptional regulation of the MGSA/GRO alpha, beta, gamma, delta genes by the cytokines IL-1, TNFalpha, TGFbeta, and by cycloheximide in these cell types, and to characterize alterations in NF-kappaB regulation of basal transcription in transformed melanocytes as compared to non-transformed melanocytes; (3) to examine the role for basal and cytokine effected MGSA/GRO in mRNA stabilization in normal melanocytes as compared to malignant melanoma cells, and (4) to examine the expression of MGSA/GRO alpha, beta, gamma, delta genes in non-malignant and malignant melanocytic lesions by in situ hybridization.

The overall objective of the Molecular Biology Core (C) is to facilitate training and utilization of molecular biology techniques by investigators in the Vanderbilt SDRC. The facilities and expertise of this Core will be available to investigators needing assistance with quality control or analysis of Critical molecular biology data needing independent verification. The Molecular Biology Core will find wide use among the SDRC investigators and those engaged in Pilot & Feasibility studies. This Core will provide several services to participants in the SDRC. 1. Instruction/Consultation in General Molecular Biology Methods - Core facilities and services will include not only the equipment necessary to perform specific methods, but also consultation to determine the level of expertise and skill required by the investigator to successfully complete the experiments proposed. If necessary, analysis of the experimental design, alternative techniques and data analysis will be provided and appropriate quality control analysis provided. 2. Molecular Kitchen - Services/consultations not already provided at Vanderbilt through other training centers or Cores will be provided by making vectors/probes for specific projects of SDRC members. 3. Instruction/Consultation in Specific Molecular Biology Methods - Since not all investigators have access to or the necessary expertise, the Core will provide specific help with Plasmid Construction, Verification and Transformation, Primer Extension Analysis, Cloning, Expression Systems, PCR Amplification, Transfections, Nuclear Run On Analysis, CAT assays, DNA Sequencing and Computerized Analysis of DNA/RNA/Protein Sequences. The coordination, training and consultation service for general and specific molecular biology techniques are the major benefits of the Molecular Biology Core.

DESCRIPTION: (adapted from the investigator's abstract) Chemotactic cytokines, known as chemokines, modulate the inflammatory response, wound healing, tumorigenesis, angiogenesis, and pathogen entry into cells. Chemokine subfamilies are denoted based upon the structural presentation of the first two cysteine residues as CXC or CC. Chemokines exert their effects by binding to seven-transmembrane-G protein-coupled receptors and transducing intracellular signals which include activation of serine and tyrosine kinases and mobilization of calcium. Chemokine receptors (CXCR or CCR, depending upon the subfamily of chemokines the receptors bind) are present on leukocytes, endothelial cells, epithelial cells, melanocytes, macrophages, and dendritic cells. Disregulation of chemokine/ chemokine receptor expression is associated with a number of disorders: chronic inflammatory diseases such as rheumatoid arthritis, ARDS, and psoriasis, sepsis, angiogenesis associated with tumorigenesis, squamous cell carcinoma and melanoma. So that they might better understand the mechanism by which CXC chemokines regulate the immune response, wound healing and cell growth, they have partially characterized signal transduction through the CXCR2 receptor for CXC chemokines and the events involved in desensitization and sequestration of CXCR2. To further explore the hypothesis that CXC chemokines acting through CXCR2 regulate important events of cell migration, cell growth, and wound healing, they propose to more completely characterize the signal transduction pathway through CXCR2 and explore in vitro and in vivo models involving expression of gain and loss of function CXCR2 receptors in keratinocytes. There are three aims: 1) to develop in vitro and in vivo models to examine the biological consequence of gain or loss of function of CXCR2 receptors in the epidermis during wound healing; 2) to characterize the events which occur in response to ligand binding to CXCR2 which facilitate homologous/heterologous cross desensitization and CXCR2 sequestration; and 3) to characterize the role of tyrosine phosphorylation of pyk2, src, and p130CAS in the cell motility responses mediated through CXCR2. Characterization of the events of signaling and receptor sequestration and desensitization will facilitate rationale drug design for disorders where sustained sensitivity to chemokines is essential for therapeutic efficacy.

The purpose of the molecular biology core will be facilitate training and utilization of molecular biology techniques among investigators in the Vanderbilt SDRC. The facilities and expertise of this Core will be available for all investigators who need assistance with quality control or analysis of critical molecular biology data needing independent verification. Core facilities will include not only the molecular equipment necessary to perform each of these techniques, but also an experienced staff for consultation on experimental design, demonstration of crucial techniques which might be new to investigators, and consultation on data interpretation. Specifically, we will assist and coordinate efforts to detect, quantitate and isolate novel regulatory and structural skin associated genes; coordinate and assist in the characterization of noel genes; assist in the functional analysis of novel genes: provide consultation and training for SDRC investigators and the pilot and feasibility projects; and maintain commonly used skin specific reagents, such as a library of RNA from human and mouse skin tissues; normal, diseased, and genetically altered. A core service for the design and cloning of new vectors/expression constructs, preparation of plasmid DNA, subcloning, and DNA sequence verification will be available. We will also provide consultation and help with nuclear run on analyses, in situ hybridization, and RNAse protection assays. Computerized analysis of sequence data will be available through the Genebank programs, BLAST, and the Vanderbilt Center VAX. The provision of these services should facilitate studies into the etiology of skin diseases and encourage clinical and basic scientists to utilize the clinical material and animal models available at Vanderbilt to further dermatological research.

DESCRIPTION (provided by applicant): A key event in the development of malignant melanoma is the disregulation of NF-kappa B which results in constitutive production of inflammatory mediators and chemokines and escape from apoptosis. NF-kB is regulated by an inhibitory protein-IkB which sequesters the NF-kappaBp65/p50 complex in the cytoplasm, preventing its translocation into the nucleus. When this IkappaB protein is phosphorylated by the IkappaB kinase (IKK), it subsequently becomes ubiquitinated and degraded by the 26S proteasome. We have demonstrated that human melanoma cell lines exhibit constitutive activation of IKK, enhanced phosphorylation and degradation of IkappaB, and elevated levels of activated the NF-kappaB p65/p50 complex in the nucleus. This results in enhanced NF-kappaB mediated gene transcription, resulting in constitutive expression of chemotactic cytokines (chemokines), VEGF, IL-6, and other regulatory factors that augment the growth of the tumor cells, promote tumor angiogenesis and metastasis. Our hypothesis is that during tumor progression the disregulation of NF-kappaB in melanocytes and/or stromal cells leads to enhanced production of inflammatory mediators and melanocyte tumor progression. Moreover, we propose that blocking NF-kappaB will interfere with tumor growth and/or metastasis. Our preliminary data suggest that blocking NF-kappaB in human melanoma cells growing on nude mice targets the cells for apoptosis and inhibits tumor growth. To further clarify the potential of inhibition of NF-kB for melanoma therapy in an immunocompetent setting, we propose the following specific objectives: la) To determine the effects of knocking down IKKa and IKKb on the growth and metastasis of melanoma tumors in vitro and in vivo; Ib) To determine the effect of expressing a constitutively active IKKb in melanocytes on incidence of melanoma and the effect of melanocyte specific expression of a super repressor form of IkappaB on resistance to tumor induction by oncogenic Ras; 2) To determine the effects of the IKKb specific inhibitor, BMS-345541, alone and in combination with the DNA alkylating agent, temozolamide, on the growth and metastasis of melanoma tumors and the immune response to the tumor in immunocompetent mice; 3) To determine the mechanism for the induction of apoptosis in melanoma tumor cells by inhibiting IKKb. Significance: Melanoma is one of the fastest growing tumor types in the US and aging persons are increasingly affected due to sun and chemical exposure. Surgical intervention prior to the time of tumor invasion is the key factor in reduction in fatality for this form of cancer. For metastatic disease, immunotherapy, often combined with chemotherapy, is one of the most promising approaches. Our data indicate that targeting NF-kappaB may target tumor cells for apoptosis, inhibit tumor angiogenesis, tumor growth and metastasis. However, we need to determine the effects of blocking NF-kappaB on the host response to the tumor and on the incidence of melanoma tumor formation. The studies designed here will help design better clinical trials using inhibitors like VELCADE or IKK inhibitors in combination with chemotherapy.

DESCRIPTION (provided by applicant): A key event in the development of melanoma is the mutation of key cell regulatory genes resulting in loss of tumor suppressors and endogenous expression of angiogenic and growth regulatory factors. The constitutive activation of nuclear factor-kappa beta (NF-kappaB) during tumorigenesis has been extensively documented in our lab, and a number of other laboratories. We have demonstrated that during melanoma tumor progression, IKKalpha-beta become constitutively activated, leading to nuclear activation of NF-kappaB, which in turn facilitates escape from apoptosis and immortalization of tumor cells. We have observed that coordinate with the activation of NF-kappaB is enhanced activation of the NF-kappaB inducing kinase, NIK. We hypothesize that NIK is activated by an upstream effector during tumor progression and these events lead to enhanced NF-kappaB mediated transcription and tumor progression. We propose to characterize the mechanism for the activation of NIK and NF-kappaB, and to develop protocols which explore new therapeutic intervention for melanoma based upon our findings. The specific aims of this proposal are 1) To determine the mechanism by which NIK is constitutively activated during melanoma tumor progression; 2) To develop approaches to ablate constitutive NIK and NF-kkappa activity and block tumor growth; 3) To determine the stage in melanoma tumor progression where constitutive activation of NIK occurs. The work described in this proposal should provide insight for the development of therapeutic reagents designed to intervene in the progression and growth of tumors showing constitutive NIK activation.

DESCRIPTION (provided by applicant): We propose to test the hypothesis that when ligand binds chemokine receptors, a "dynamic chemosynapse" forms, comprised of receptor and adaptor proteins that serve to enable polarization, activation, amplification, and oscillation of small GTPases and kinases involved in organization of the actin cytoskeleton, and chemotaxis. Moreover, we propose that interference of chemokine receptor association with these adaptors will ablate CXCR2- and CXCR4-mediated chemotaxis, intravasation, extravasation, and metastasis. There are three specific aims. I) To test the hypothesis that a major role of AP-2 in mediating CXCR2/CXCR4 chemotaxis and metastasis is to orchestrate polarization and amplification of intracellular signals. We will separately interfere with AP-2 mediated receptor trafficking versus polarization of intracellular signals to distinguish the importance of these two functions of AP-2 in CXCR2/CXCR4 mediated chemotaxis and metastasis. 2) To characterize the role of IQGAP1 and VASP in linking the CXCR2 and CXCR4 chemosynapse to the actin cytoskeleton. We will characterize the interacting domains of IQGAP1 and VASP with CXCR2 and CXCR4 and determine the effects of altering these interactions on the functional responses to chemokine (including extravasation, intravasation, metastasis) of leukocytes, endothelial cells and breast cancer cells. 3) To test the hypothesis that Src-family kinase activation at the CXCR2 and CXCR4 `chemosynapse'drives chemotaxis that is Dock2 dependent and largely PI3K independent. We will determine the role of the Src-p130Cas-CrkL-Dock2 pathway in PI3K independent CXC2/CXCR4 chemotaxis. We will study these processes in dHL-60, dU937, HMEC-1 CXCR2 expressing cells and in the breast cancer cell lines naturally expressing CXCR4 (highly invasive MDA-MB-231, DU4475, BT-549 and non-invasive MCF-7, MDA-MB- 453 and MCF-10A cells). Microfluidic devices and real time intravital microscopy using 2 photon imaging will be used to track chemotaxis in vitro and intravasation/extravasation /metastasis in vivo, respectively. Characterization of the functional significance of this interaction between chemokine receptors and adaptor proteins that comprise the chemosynapse will unveil important new therapeutic targets for the treatment of malignancies. Characterization of the mechanism for PI3K independent chemotaxis will provide valuable information for design of therapies for sepsis, arthritis, and metastasis. PUBLIC HEALTH RELEVANCE: This study aims to test the hypothesis that receptors involved in regulating the motility of immune cells and tumor cells must interact with a number of proteins in the cytoplasm of the cell to stimulate proper organization of the cytoskeleton. In this study we propose to disrupt these protein/protein interactions to develop a new way of blocking tumor cell metastasis and chronic inflammation. We also will interrupt specific intracellular signals initiated by these protein/protein interactions to point the way to new targets for drug therapy for chronic inflammation and metastasis.

DESCRIPTION (provided by applicant): Chronic inflammation is causally associated with the development of many cancers, often resulting in a "cytokine storm" that facilitates tumor progression. Therapy could be directed to appropriately "quiet the storm" while beneficial for the tumor, could result in negative effects on the host. The NF-?B family of transcription factors is required for the generation of the 'cytokine storm'. Malignant melanoma, the most deadly type of skin cancer, provides a uniquely valuable model for testing the balance between immunity and intrinsic resistance in tumor growth. We have demonstrated that inhibiting IKK2, the major kinase that activates the canonical NF-?B pathway, in melanoma tumor bearing mice results in reduced tumor growth. Moreover, targeted deletion of IKK2 in melanocytes prevents mutant Ras-mediated melanoma tumor formation in mice that have lost the tumor suppressors INK4a/ARF. Current therapies for disseminated melanoma are largely ineffective. Small molecule inhibitors of IKK2, by directly blocking the canonical NF-?B pathway, may be effective in the clinic for metastatic melanoma. Aurora Kinase (Aurk) inhibitors readily block cell cycle progression in tumor cells and also indirectly inhibit NF-kB, while inhibitors of NF-?B inhibit Aurk and target many types of tumor cells for apoptosis. Clinical trials are currently ongoing using small molecule inhibitors of IKK2 and AurK for other solid tumors and haematological malignancies. Melanoma might be highly responsive to inhibitors to these two kinases, but before initiating such studies, potentially harmful effects of inhibiting the NF-kB pathway on the host immune response require clarification. In this proposal we will test the hypothesis that the benefits of targeting IKK2 or AurkA will outweigh the risks for patients with aggressive metastatic melanoma and constitutive activation of these pathways. We also hypothesize that inhibiting NF-kB with IKK2 or AurKA inhibitors will boost the immune response to the tumor by shifting leukocyte profile in the tumor microenvironment from one that is pro-tumorigenic (M2, N2, Th2) to one that is anti-tumorigenic (M1/N1/Th1). Moreover, there are documented cases of tumors developing resistance to Aurk inhibitors and in these instances we hypothesize that resistance to therapy with Aurk or IKK inhibitors will be associated with mutations in Aurk or kinases in the IKK pathway. There are three specific aims: 1) To determine whether treatment with IKK2 or AurkA inhibitors results in loss of intrinsic immunity to "silent" tumors. 2) To characterize the effects of systemic inhibition of IKK2 versus AurkA on the leukocytes infiltrate and cytokine profile in the tumor microenvironment;3) To characterize the mechanism by which melanoma tumors sensitive to IKK2 or Aurk inhibitors become growth arrested then later resistant to these inhibitors. Insights from this pre-clinical work should allow appropriate design of clinical trials to test the efficacy of IKK2 and Aurk inhibitors for melanoma therapy. The ultimate goal of this work is to develop insight for improved individualized therapy for malignant melanoma patients to increase survival.

Strong ties have developed between cancer researchers at Meharry Medical College (MMC), Tennessee State University (TSU) and Vanderbilt-lngram Cancer Center (VICC) resulting in this formal Cancer Partnership. Our Partnership is dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities in the US. The U54 award is the bedrock of this collaboration and has moved our collaborative research more forcefully toward a relationship that is based on reciprocity and stronger mutual benefit. The overall objectives of this competing renewal application are: (1) to increase and stabilize the competitive cancer research capability of MMC and TSU;(2) to create stable, long-term collaborative relationships between MMC, TSU and the VICC in cancer research, research training, career development and cancer outreach;and (3) to improve the effectiveness of VICC research, training, career development, cancer education and cancer outreach activities specifically designed to benefit minority populations in the region served by VICC. Funds provided by this U54 will solidify our collaboration. During this phase of our Cancer Partnership, we continue to concentrate on amassing sufficient infrastructure for cancer research that will strengthen reciprocity between MMC, TSU and VICC. In addition to funding two full projects and two pilot studies involving collaborators from all three institutions, funds will be used to recruit investigators to TSU and VICC and to strengthen infrastructure in basic, epidemiologic and clinical cancer research through the support of core facilities providing expertise in biostatistics, clinical trial accruals, histopathology and cancer outreach. All activities involve coordinated, well-planned interactions between MMC, TSU and VICC and will be monitored by an Internal Advisory Committee composed of members from each institution and a Program Steering Committee composed of external, nationally-recognized cancer investigators and representation from the National Cancer Institute. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The institution's complementary strengths will help the Cancer Partnership establish effective cancer research and increase training opportunities at two minority-serving institutions enhance community- and population-based science research targeting minority cancer-related disparities and increase training of minority scientists at an NCI-funded Comprehensive Cancer Center.

Strong ties have developed between cancer researchers at Meharry Medical College (MMC), Tennessee State University (TSU) and Vanderbilt-lngram Cancer Center (VICC) resulting in this formal Cancer Partnership. Our Partnership is dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities in the US. The U54 award is the bedrock of this collaboration and has moved our collaborative research more forcefully toward a relationship that is based on reciprocity and stronger mutual benefit. The overall objectives of this competing renewal application are: (1) to increase and stabilize the competitive cancer research capability of MMC and TSU; (2) to create stable, long-term collaborative relationships between MMC, TSU and the VICC in cancer research, research training, career development and cancer outreach; and (3) to improve the effectiveness of VICC research, training, career development, cancer education and cancer outreach activities specifically designed to benefit minority populations in the region served by VICC. Funds provided by this U54 will solidify our collaboration. During this phase of our Cancer Partnership, we continue to concentrate on amassing sufficient infrastructure for cancer research that will strengthen reciprocity between MMC, TSU and VICC. In addition to funding two full projects and two pilot studies involving collaborators from all three institutions, funds will be used to recruit investigators to TSU and VICC and to strengthen infrastructure in basic, epidemiologic and clinical cancer research through the support of core facilities providing expertise in biostatistics, clinical trial accruals, histopathology and cancer outreach. All activities involve coordinated, well-planned interactions between MMC, TSU and VICC and will be monitored by an Internal Advisory Committee composed of members from each institution and a Program Steering Committee composed of external, nationally-recognized cancer investigators and representation from the National Cancer Institute. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The institution's complementary strengths will help the Cancer Partnership establish effective cancer research and increase training opportunities at two minority-serving institutions enhance community- and population-based science research targeting minority cancer-related disparities and increase training of minority scientists at an NCI-funded Comprehensive Cancer Center.

DESCRIPTION (provided by applicant): Gene expression profiling has indicated that the up-regulation of specific subsets of chemokines/cytokine/ growth factors is often linked to poorer prognosis for women with invasive mammary carcinoma. Chemokines are important for the recruitment of leukocytes to the tumor microenvironment (TME) which can affect invasion and metastasis. It has been proposed that specific chemokines released by circulating tumor cells signal to leukocytes in the pre-metastatic niche and entrain myeloid cells to exhibit an anti-tumorigenic phenotype. The entrainment process is thought to require activation of neutrophil NADPH oxidase to ultimately generate extracellular H2O2, which is toxic to tumor cells. This process is reported to be dependent upon activation of PI3 kinase. The goal of this research is evaluate mechanisms of entrainment and to characterize the role of inhibition of PI3 kinase in chemokine mediated entrainment of leukocytes. We hypothesize that perisurgical delivery of entraining chemokines to cancer patients may reduce recurrence and metastasis. Moreover, current PI3K inhibitor therapies for breast cancer patients might fail if they reverse this entrainment, counteract the effect of anti-tumor leukocytes, and thus promote metastasis. There are three specific aims for this study: 1) Using an immunocompetent mouse model we will determine whether manipulation of CCL2, CCL5, CXCL1, CXCL12 and/or TGF-beta levels will differentially effect the recruitment of anti- or pro-tumor leukocytes into the primary tumor and the pre-metastatic niche. Aim1B. We will determine whether blocking the receptors for each of these chemokines will facilitate or disrupt the metastatic capacity of mammary carcinoma; 2) Using immunocompetent mice, intravital imaging, and ex-vivo micro-bioreactors we will determine whether current therapeutics for breast cancer that inhibit PI3K are able to block or facilitate chemokine-mediated 'entrainment of myeloid cells in tumor bearing mice; We will deliver PI3K inhibitors to tumor bearing mice, isolate myeloid cells, and determine whether neutrophils respond to entraining chemokines with induction of H2O2 and have the capacity to kill tumor cells; 3) We will evaluate changes in the myeloid 'entrainment' status of cancer patients before and after chemotherapy and determine whether leukocytes can be entrained 'ex vivo'. Using a humanized mouse model and implanted patient tumors, we will determine whether drug induced alterations in myeloid entrainment that result in reduced tumor cell killing and/or enhanced metastasis correlates with time to progression in the patient. In humanized mice, we will also compare the effects of ex vivo treatment with chemokines or PI3K inhibitors on 'entrainment' properties of circulating myeloid cells of cancer patients prior to surgery, after surgery, or perisurgery to determine the appropriate time to deliver entraining chemokines or PI3K inhibitors.

DESCRIPTION (provided by applicant): Metastatic melanoma is one of the fastest growing tumor types in the US and also is one of the most challenging malignancies to treat once metastasis occurs. Though several new therapeutic approaches are available for melanoma patients, mean overall survival with these therapies remains in the 12 month range. Clearly, better treatments are needed and excellent targets for therapy are found within the pathways regulated by products of the CDKN2A gene which is mutated or deleted in 43% of melanomas. Two products of the CDKN2A gene are INK4A/p16 (an inhibitor of the cyclin-dependent kinases, CDK4/6) and ARF (a suppressor of the E3 ubiquitin ligase MDM2 that mediates ubiquitination and degradation of p53). When p16 binds to CDK4/6, it inhibits the CDK4/6 mediated hyper-phosphorylation of RB1, thus allowing RB to block entry into S- phase of the cell cycle. When p16 is deleted or mutated this regulation of cell cycle is compromised. Deletion or mutation of ARF results in the unbridled MDM2 mediated ubiquitination and degradation of p53, resulting in a loss of p53-mediated transcription of regulators of cell cycle and apoptosis. Since p53 is seldom mutated in melanoma, restoring its function is key for inducing apoptosis in tumor cells. Recently, drugs that target CDK4/6 and HDM2 have moved into clinical trials. We have preliminary data that combining a CDK4/6 inhibitor with an HDM2 antagonist induces marked inhibition of melanoma growth. This important finding, coupled with our recent success in preclinical models of melanoma with MDM2/HDM2 inhibitors combined with agents that block cell cycle and induce senescence, form the basis for this proposal. Hypothesis: We postulate that melanoma patient tumors that are RB1WT/ TP53WT will be inhibited by therapy that combines CDK4/6 inhibitors (CDK4/6i) with agents that disrupt MDM2/p53 interaction. There are three aims for this proposal: 1) To compare the senescence associated secretory program (SASP) in melanocytes of oncogene induced senescence (OIS) to that of therapy induced senescence pathways in vitro and in vivo and evaluate the effectiveness of MDM2 (MDM2i) and CDK4/6 inhibitors (CDK4/6i) on melanoma tumor growth, senescence, SASP, and apoptosis, and on recruitment of immune cells into the tumor microenvironment (TME) in immune competent mice with focus on BRAFWT/NRASWT or NRASmut melanoma; 2) To characterize the benefit of combining an HDM2 antagonist with CDK4/6 inhibitors for treatment of metastatic melanoma using patient derived xenografts (PDX) from BRAFWT/NRASWT and NRASmut tumors that are RBWT/TP53WT and to characterize gene mutation/expression signatures that will predict response to this therapy; 3) To characterize the effectiveness of CDK4/6 inhibitor combined with HDM2 inhibitor in the treatment of RBWT/TP53WT melanoma tumors that progressed on therapy. PDX models will be utilized and coordinated with biopsy from patient tumor at the time of progression from 3 ongoing clinical trials at Vanderbilt involving patients with tumors that are BRAFWT/NRASmut or BRAFWT/NRASWT.

SUMMARY Particular emphasis is devoted in the US to identify the determinants of, and eventually remedies, for the disproportionate share of the cancer burden borne by underrepresented minorities. Incidence rates for many forms of cancer are higher among blacks than whites. The disparity is compounded by lower rates of relative survival for almost all cancers, so that age-adjusted mortality rates are substantially higher among African Americans than whites for about two-thirds of all types of cancers. Our Cancer Partnership provides an exceptional environment to focus the efforts of diverse investigators working across many disciplines to address health disparities in NCI designated cancer centers and to develop research infrastructure, capacity and effectiveness of minority-serving institutions. Collectively, these efforts offer the best opportunity to overcome cancer disparities and optimize research resources and infrastructure. Our Partnership has matured and developed strong, successful collaborative interactions based on excellence, true reciprocity and mutual benefit to achieve this goal. Moreover, this Partnership is geographically placed in a region with the highest cancer incidence in the US, pointing to the enormous potential for improved outcomes through the proposed work. We are dedicated to enhancing the strengths and eliminating the weaknesses of our three institutions as we move toward our shared goal of eliminating cancer disparities. The overall objectives of this competing renewal application are to: (1) increase and stabilize the competitive cancer research capability of Meharry Medical College (MMC) and Tennessee State University (TSU); (2) create stable, long-term collaborative relationships between MMC, TSU and the Vanderbilt Ingram Cancer Center (VICC) in cancer research, research education, career development and cancer outreach; (3) expand access to clinical trials for minority populations served by Nashville General Hospital (NGH)/MMC, and (4) improve the effectiveness of VICC research, career development, education and outreach activities specifically designed to benefit minority populations served by VICC. All activities will be monitored by an Internal Advisory Committee and the Program Steering Committee with representation from the NCI. A sustained and comprehensive Cancer Partnership is of immense benefit to the three participating institutions as well as the mid-South region of the US. The common mission statement for our Cancer Partnership is to conduct rigorous basic, translational and clinical research directed towards the reduction of disparities in the incidence and treatment of cancer through a multidisciplinary collaborative approach to research, education and community engagement. As a triad we will amplify cancer research capabilities in the greater Nashville academic and healthcare community, improve and document outcomes in the diagnosis and treatment of cancer in minority and underserved communities, minimize or eliminate measurable disparities in the incidence of cancer among minority populations, and fortify collaborations among the three partner organizations.

Summary The main goal of our research education program is to increase the number and quality of minority researchers dedicated to making an impact on cancer research. We have a previously established research education program that supports summer cancer research for medical students at Meharry Medical College (MMC) and Vanderbilt; summer research for undergraduates at Tennessee State University (TSU); and provides support for graduate students at MMC and TSU. These programs have proven successful at progressing students into careers in cancer research. In this application our focus is to engage students early in a research path in order to augment recruitment and retention into graduate programs and careers in cancer research. With that in mind, we propose to formalize a Pathway to Discovery Program to recruit high school juniors and seniors into cancer research and continue this experience through graduate/medical school, with particular focus on underrepresented minorities (URM). Our Partnership is particularly suited to target African American or Black, Hispanic or Latino students at the undergraduate (TSU) and graduate student level (MMC and TSU). High school students will be recruited from two academic STEM magnet schools in the Nashville metropolitan school district with predominant URM student population profiles. In order to achieve our goal, we propose the following aims. In aim 1, we will implement a pathway program that provides early and continuous research education for high school and undergraduate students leveraging the cancer research opportunities at our Partnership Institutions. This will be accomplished with : 1) a high school cancer research experience (HSCRE) both in the summer and academic year with scholarship opportunities and multi-tier peer and faculty mentoring; 2) early undergraduate cancer research experience (EUCRE) in the summer and academic year providing the opportunity for engagement in projects supported through peer and faculty mentoring; and 3) a late undergraduate cancer research experience (LUCRE) in the summer and academic year to conduct mentored-independent research projects. In aim 2, we propose to recruit and support master and doctoral graduate students to engage in cancer research in order to diversify the cancer research workforce and develop research interests in the area of cancer disparities. Finally, in aim 3, we provide research educational opportunities to medical students to contribute to clinical population sciences and translational cancer research with focus on cancer disparities.

Treatment of metastatic melanoma with immune checkpoint inhibitors (ICI) has extended the life of many melanoma patients, but the vast majority of patient experience disease progression, prompting the need for alternate therapies. For ~50% of patients with BRAFmut tumors, treatment with BRAF and MEK inhibitors provide a good second-line treatment option. Unfortunately, there are few second-line options for the 25-30% of patients whose tumors harbor NRAS mutations. Since nearly 40% of all melanoma patients exhibit loss, mutation, or epigenetic silencing of the CDK4/6 regulator CDKN2A, we postulate that inhibition of CDK4/6 may induce response in NRASmut RBWT tumors with loss of CDKN2A. Because loss of CDKN2A also disrupts ARF, a suppressor of MDM2-mediated degradation of p53, it will be essential to also inhibit MDM2 to restore cell cycle control in p53WT melanoma. We have shown that NRASmut melanoma tumors with acquired resistance to ICI respond to co-treatment with a CDK4/6 inhibitor plus an MDM2, demonstrating both with reduced tumor growth and enhanced CD8+T cell recruitment into the tumor. These tumors contain a significant number of CXCR1,2 expressing myeloid-derived suppressor cells (MDSCs) that create an immune suppressive tumor microenvironment. Our preliminary data show that when CXCR2 is deleted in myeloid cells, MDSC recruitment to tumor is reduced and tumor growth is inhibited. Moreover, systemic delivery of a CXCR1,2 inhibitor reduced the growth of NRASmut melanoma in mice (p<0.02), and inducible deletion of CXCR2 in melanocytes blocks melanoma formation in the inducible BRAFV600E/PTEN-/- melanoma mouse model. These intriguing findings support prior studies indicating a role for CXCR2 inhibitors for treatment of melanoma. However, the mechanisms and generality of response to CXCR1,2 antagonism require further elucidation. Premise and Hypothesis: CXCR2 plays critical and pleotropic role in melanoma by promoting tumorigenesis and inducing an immunosuppressive tumor environment. Moreover, combined CDK4/6 and MDM2 inhibition significantly inhibits the growth of mouse and human NRASmut melanoma tumors. We hypothesize that co-inhibition of CXCR1,2, CDK4/6, and MDM2 in NRASmut melanoma with acquired resistance to ICI will inhibit tumor cell proliferation, induce tumor cell death, stimulate anti- tumor immunity, and potentially overcome the acquired resistance to ICI. We propose 3 specific aims. 1)To examine the ability of CDK4/6i plus MDM2i, combined with a CXCR1,2 antagonist treatment, or CXCR1,2 agonist alone, to enhance or restore ICI sensitivity for NRASmut melanoma tumors. 2) To determine the role of melanocyte-expressed CXCR2 in melanoma initiation. CXCR2 will be deleted coincident with induction of melanoma formation in mice and effects of this deletion on melanocyte apoptosis, senescence, differentiation and proliferation will be characterized. 3) To determine whether findings in mouse translate to human melanoma, changes in the tumor immune microenvironment (TIME) will be characterized over time in response to a CXCR1,2 antagonist (CXCR1,2i) with or without CDK4/6i+MDM2i followed by ICI therapy in humanized NRASmut patient-derived xenograft (PDX) models and human NRAS mut melanoma organoid co-cultures. These data will inform future clinical trials.

Metastatic breast cancer (BC) is a major health issue for women across the world. About 40,610 women and 460 men in the US are expected to die this year alone from BC. While there are numerous treatment options for hormone receptor positive (HR+) and HER2+ BC patients, the standard of care for triple negative BC (TNBC) patients largely relies on conventional chemotherapy and radiation. Improved options for treating metastatic BC represent a vast unmet medical need. Recently, several clinical trials investigated combined treatment with either HR pathway blockers or HER2 antagonists and PI3K inhibitors, since the PI3K pathway is constitutively activated or mutated in over 50% of BC patients. However, the results have shown only 2-4 months increase in progression free survival and there is extensive Grade 3 and 4 toxicity with the dosage schedules used. The discovery of immune checkpoint inhibitors (ICIs) is revolutionizing cancer therapy, but thus far BC patients are not showing strong responses to ICI therapy, due to low mutational load and minimal infiltration of CD4+ and CD8+ T cells (?cold?). Our hypothesis is that response to ICI therapy in immunologically ?cold? BCs can be enhanced by combining therapies that inhibit AKT with paclitaxel (PTX) to induce immunogenic tumor cell death and shift tumor-associated immune cells to an anti-tumor phenotype. To test this hypothesis, we will utilize immune competent mouse models, organoid/immune cell co-cultures, and humanized mouse models bearing patient-derived xenograft (PDX) to determine if PI3K pathway inhibitors, when enhance response to ICIs, and improve survival in mice. There are two specific aims. Aim 1. To develop the optimal strategy for reducing growth of TNBC through treatment with an AKT inhibitor, ipatasertib, combined with paclitaxel, and immune checkpoint inhibitors (ICIs) anti-CTLA4 + anti-PD1. Using immune competent mouse models, we will determine the functional significance of reprograming the tumor immune environment in mammary tumors in response to AKT inhibition in reference to response to ICIs at early, mid and late time points during therapy. Mechanisms of therapeutic response will be investigated based upon analysis of the following parameters: toxicity; tumor growth; metastasis; immune cell content (immunome); cytokine/chemokine expression profile in tumor, bone marrow, lung and blood samples; angiogenesis; and transcriptome in responding and non- responding tumors. State of the art technology will include multiplex immunohistochemistry (IHC), flow cytometry, CyTOF, reverse phase protein analysis (RPPA), RNA sequencing (RNAseq), DNA sequencing (DNAseq) and pathway analysis. Transcriptomic and immunome signatures predicting response to treatment in mice will be compared to RNAseq data from ongoing clinical trials available to us and published ?response signatures? 26. Aim 2: To determine the efficacy of treatment with AKT inhibitors combined with PTX and ICIs in two human TNBC models: 1) organoid co-cultures human TNBC plus fibroblasts, endothelial cells and patient immune cells: 2) humanized patient-derived xenografts (PDX) mouse models established from TNBC patients. Genetic/immunome signatures will be evaluated and compared to data from ongoing clinical trials.