Andrei Thomas-Tikhonenko - US grants

[unreadable] DESCRIPTION (provided by applicant): The ability of various infections to suppress neoplastic growth is well-documented. We have previously demonstrated that tumor suppression during acute toxoplasmosis does not involve cytotoxic functions of the immune system and readily occurs in immunocompromised mice. Instead, it relies on systemic inhibition of angiogenesis by circulating factors, most likely interferons. To determine whether AIDS-related Burkitt lymphoma would succumb to infection-mediated suppression, we have established a new mouse model for this disease. It is based on overexpression of the c-Myc oncoprotein in p53-null bone marrow progenitors. Using this model, we have found that growth of B-lymphomas during acute toxoplasmosis was completely abolished. In this proposal, we will study mechanisms whereby type I and II interferons suppress lymphomagenesis. We will use STATI-null mice in which both type I and type II interferon pathways are inactivated, and determine whether in these animals angiogenesis during infection is restored. We will also determine whether growth of neoplastic B-cells is directly inhibited by interferons. To this end, we will cross STATI- and p53-null mice and generate B-lymphomas that are deficient in STATI expression. They will be implanted into Toxoplasma gondii-infected STATl -null mice. Since in this system both host and tumor cells are refractory to interferons, we expect that B-lymphomagenesis would be completely restored. This would suggest that interferons play a dual role in lymphoma surveillance during infection: direct and aniogenesis-mediated. We will then determine whether exposure to T.gondii antigens (STAg) would lead to the induction of interferons and suppression of angiogenesis and lymphomagenesis. We will also perform tumor load studies in STAg-treated scid-beige mice, to demonstrate that anti-neoplastic properties of STAg do not rely on cell-mediated cytotoxic immunity. This anticipated result will establish that STAg or similar protozoan or bacterial antigens could be developed into new therapeutic modalities for AIDS-related Burkitt lymphoma.

Pax5 is a key regulator of B-cell differentiation, necessary for commitment to the B-lymphoid lineage. Its absence allows pro-B-cells to readily adopt other fates. Conversely, failure to silence Pax5 in pre-B-cells is thought to contribute to neoplastic transformation. For example, Pax5 is over-expressed in lymphoplasma-cytic lymphomas due to a chromosomal translocation juxtaposing pax5 and IgH loci. In other types of non-Hodgkin disease (diffuse large cell and Burkitt lymphomas), Pax5 is often co-expressed along with Myc, a confirmed oncogene. However, the role of Pax5 as an oncogene in its own right is yet to be established. To this end, a model system has been developed wherein p53-null bone marrow cells are transduced in vivo with retroviruses expressing Myc. In syngeneic hosts, these cells give rise to aggressive, Pax5-positive B-cell lymphomas. However, some cell lines derived therefrom spontaneously silence Pax5 and convert to a myeloid phenotype, with frequent loss of tumorigenicity. Thus, physiological levels of Pax5 are crucial for the B-lymphoid phenotype of Myc-induced neoplasms. Moreover, supraphysiological levels of Pax5, achieved via retroviral transduction, were found to greatly enhance Myc-induced tumor cell growth. In the current proposal, the retroviral transduction system will be used to elucidate the exact role of Pax5 in lymphomagenesis. Three important issues will be addressed. 1. The role of Pax5 in restricting Myc-induced hematopoietic neoplasms to B-lymphomas. The hypothesis is that Myc-transformed cells are multipotent. To remain committed to the lymphoid lineage and thwart myeloid differentiation, they require Pax5 which serves as an antagonist of the pro-myeloid transcription factor PU.1. 2. The transforming potential of Pax5 in bone marrow cells. The hypothesis is that at least in the absence of p53, Pax5 can initiate neoplastic growth and supplant Myc in tumor sustenance. 3. The consequences of Pax5 silencing for B-lymphomagenesis. The hypothesis is that inactivation of Pax5 in neoplastic pre-B cells will result in loss or attenuation of tumorigenicity. These experimental results may identify Pax5 as a valid target for gene-specific therapeutic interventions that would benefit patients with a variety of B-cell lymphomas.

DESCRIPTION (provided by applicant): The goal of this project is to investigate the molecular mechanisms by which the Myc oncoprotein enhances tumor neovascularization (the ingrowth of blood vessels into the nascent neoplasm). We have developed an experimental system wherein overexpression of this oncoprotein in murine colon carcinoma cells results in the hypervascular phenotype. We determined that this occurs without increased production of vascular endothelial growth factor (VEGF). Instead, Myc down-regulates the potent endogenous inhibitor of angiogenesis thrombospondin-1 and several other members of the TSR superfamily. Thrombospondin-1 is down-regulated primarily at the level of mRNA turnover, suggesting the involvement of microRNAs (miRNAs) which are known to mediate mRNA degradation. Indeed, Myc upregulates one of the miRNA clusters (miR17-92) whose predicted targets include thrombospondin-1 and other TSR proteins. miR17-92 knockdown with antisense 2'-O-methyl oligoribonucleotide partly restores Tsp1 and CTGF expression. Conversely, transduction of Ras-only cells with a miR17-92-encoding retrovirus reduces Tsp1 and CTGF levels. These key findings were also reproduced in HCT116 human colon carcinoma cells. Notably, miR17-92-transduced murine cells form larger, better-perfused tumors. Thus, we have formulated the following overall hypothesis: The contribution of Myc to tumor neovascularization is based on down-regulation of thrombospondin-1 and related proteins via a post-transcriptional mechanism involving the miR17-92 microRNA cluster. To corroborate this hypothesis, we propose to fulfill the following Specific Aims: 1. To dissect the molecular mechanisms underlying downregulation of TSR proteins by the miR17-92 cluster. 2. To establish the significance of Myc-dependent TSR protein down-regulation for neoplastic growth of Myc/Ras colonocytes and human colon cancer xenografts. 3. To elucidate the effects of transient Myc down-regulation on tumor vascularity and overall growth. After having fulfilled these Aims, we will be able to commence large-scale pre-clinical studies further validating miR17-92 and Myc as non-cell-autonomous therapeutic targets in vivo. RELEVANCE TO PUBLIC HEALTH: One of the promising anti-cancer drugs currently undergoing clinical trials is ABT-510. This drug mimics the effects of the natural tumor suppressor thrombospondin. Our research will help determine what types of colorectal cancer might be particularly sensitive to ABT-510 and allow for targeted patient enrollment. Additionally, our studies identify certain microRNAs as potential targets for anti-angiogenic therapies.

DESCRIPTION (provided by applicant): For patients with high-risk neuroblastoma (NB) survival remains below 40%, and survivors suffer from life-long treatment-related disabilities. This necessitates the development of new targeted therapies. Small-molecule inhibitors of insulin-like growth factor 1 receptor (IGF1R) exhibit antitumor effects on NB cells in vitro and in vivo. Yet given the apparent lack of somatic mutations in the IGF1R gene, it remains to be determined what subset of NB patients would benefit from this approach. We hypothesized that IGF1R expression is controlled by alterations in the 3'UTR which would affect binding by microRNAs. In our Preliminary Experiments, we first focused on the genetic variant rs3833015, which is a small 2-nt deletion found in ~50% of NB patients. The positive effects of this 3'UTR variant on IGF1R expression were established using luciferase sensor essays, immunoblotting of commonly available NB cell lines, and transcriptome profiling of 28 primary neuroblastomas using Gene Set Enrichment Analysis. Furthermore, rs3833015-overexpressed genes were also strongly enrichment for members of the IGF1R/MAPK pathway, which plays a well-established role not only in cell proliferation but also in cell survival in the face of chemotherapy. In paralel, during the course of unbiased expression-quantitative trait loci (eQTL) studies, we identified another variant, rs3743251, which creates a new miR-binding site and correlates with decreased IGF1R mRNA levels. Our current overall hypothesis is that rs3833015 and rs374251 change IGF1 signaling (up and down, respectively), resulting in altered resistance to conventional chemotherapeutic agents and small-molecule IGF1R inhibitors. We will pursue this hypothesis in the following two Aims. 1. To determine the mechanisms of IGF1R deregulation by rs3833015 and rs3743251 and to identify new SNPs linked to IGF1R over/under-expression. 2. To investigate the contribution of IGF1R variants to neuroblastoma sensitivity to small-molecule IGF1R inhibitors and chemotherapeutic agents. To this end, we will correlate IGF1R 3'UTR genotypes with survival of NB patients using Kaplan-Meier analysis and compare and contrast responses of rs3833015 and rs374251 tumors to conventional therapeutics (cyclophosphamide and topotecan) and IGF1R inhibitors. The main impact of this study is that it would allow prospective stratification of patients enrolled in clinical trials of IGF1R inhibitors nd more robust responses to these drugs, ushering in the era of miR pharmacogenomics.

DESCRIPTION (provided by applicant): The c-Myc proto-oncogene is a key factor in both B-cell homeostasis and B-lymphomagenesis. Its transforming potential is based largely on its ability to drive cell proliferation. At the same time, Myc can also induce apoptosis, either throug induction of p53 or in a p53-independent manner. Yet the goal of channeling the pro-apoptotic activity of Myc toward anti-cancer therapies has so far proven elusive, primarily because methods to transiently increased Myc levels didn't exist. Our recent research showed that boosting Myc expression elevates p53 and increases sensitivity to bortezomib. One limitation of that study was the reliance on the p53 pathway, which is frequently lost in human B-lymphomas. To determine how Myc contributes to p53-independent apoptosis in a bona fide therapeutic setting (CHOP therapy), we now used cells isolated from bone marrows of p53ERTAM knock-in mice and subsequently transduced with a Myc-expressing retrovirus. This inducible system allowed us to distinguish between p53-dependent (cells treated with tamoxifen) and independent (no tamoxifen given) cell deaths. Additionally, very recent data from our lab show that GSK-3¿ is actively involved in the regulation of Myc protein degradation in B-cells, and that inhibition of GSK-3¿ with CHIR99021 sharply increases Myc levels. Using these reagents, we observed that pharmacological stabilization of Myc with GSK-3¿ inhibitors strongly enhanced p53-independent doxorubicin-induced apoptosis. Most importantly, even in p53-mutated Ramos Burkitt's lymphoma cells, Myc stabilization resulted in increased responses to doxorubicin. These results fully support our innovative hypothesis that transient up-regulation of Myc could be a viable adjuvant therapy for Myc-driven tumors even with p53 loss or MDM2 amplification. We will pursue this hypothesis in the following two aims. 1) To investigate Myc-dependent and - independent events that drive p53-independent apoptosis in response to doxorubicin+GSK3¿ inhibitors. Specifically, we will determine whether genetic or pharmacological inhibition of Myc abolishes pro- apoptotic effects of CHIR99021 or whether other GSK-3¿ targets such as BCL2L12 contribute to chemosensitization. 2) To investigate how GSK3¿ inhibition affects Burkitt's lymphoma response to doxorubicin in acute and chronic treatment models. On the strength of our in vitro data, we will test if GSK-3¿ inhibition potentiates doxorubicin-based chemotherapy against murine syngeneic grafts and human xenografts, resulting in a more robust apoptotic response (acute model) and prolonged event- free survival (chronic model). Upon completion of this work we will have a better understanding of the apoptotic pathways regulated by GSK3¿ and its targets such as Myc and BCL2L12. Also, we will validate GSK3¿ inhibitors as adjuvant therapeutics to treat Myc-driven B cell lymphomas with mutant p53 or MDM2 amplification.

? DESCRIPTION (provided by applicant): Despite serving as the initial testing ground for cancer genetics and more recently - genomics, colorectal cancer (CRC) remains a deadly disease. One reason for the lack of major breakthroughs is that focusing on individual signaling pathways is not enough to understand pathogenesis and progression of this disease. The MYC oncogene is a case in point. It is involved in a dizzying number of functional interactions, few of which have been fully understood or sufficiently validated. The overarching goal of this grant is to identify MYC pathway interactions that can be targeted therapeutically. In CRC, MYC is frequently overexpressed due to activating mutations in the WNT pathway. Binding of WNT ligands to the co-receptors FZD and LRP prevents APC tumor suppressor-mediated degradation of ß-catenin. Stabilized ß-catenin translocates into the nucleus, where it forms a complex with the TCF4 transcription factor and drives MYC expression. However, what non-mutational events aid WNT-dependent Myc activation remains largely unknown. Nor has MYC function in highly genetically complex CRC been firmly established. We recently demonstrated that MYC confers upon CRC cells a hypervascular phenotype. This occurs through down-regulation of thrombospondin-1 (Tsp1) and related anti-angiogenic factors, many of which are normally sustained by transforming growth factor ß. The TGFß pathway, too, is frequently inactivated in CRC, but how it is regulated by non-mutational means remained to be seen. In the past several years we discovered that far from being a passive downstream effector of WNT and TGFß signaling, Myc has the potential to profoundly influence both pathways. This is due in large part to the ability o Myc to up-regulate the miR-17~92 microRNA cluster (a.k.a. oncomir-1). Of relevance to this proposal, in CRC miR-17-92 directly targets and down-regulates Tsp1, but with even broader impact - several key components of the TGFß pathway. Another relevant Myc | miR-17~92 target is DKK3, an inhibitory LRP ligand and WNT signaling suppressor. These discoveries have led to the following overall hypothesis: WNT, Myc, and miR-17~92 form a previously unrecognized positive feedback loop, which suppresses TGFß signaling and promotes tumor neovascularization. In this proposal, we aim to: 1. Define a role of DKK3 in sustaining Myc expression; 2. Determine the role of Wnt | Myc | miR-17~92 in overcoming angiogenesis suppression by TGFß; 3. Validate TGFß pathway mutations as determinants of resistance to drugs targeting the WNT pathway. After fulfilling the three Aims of this proposal we will have re-drawn the wiring diagrams of human CRC, identified novel targets for therapeutic anti-angiogenesis, and harnessed the power of cancer genomics to predict responses to new drugs targeting WNT and TGFß pathways.

PROJECT SUMMARY/ABSTRACT Successful immunotherapies for childhood cancers typically target lineage-, rather than cancer-specific markers, B-cell specific CD19 being the prime example. These successes culminated in the recent FDA approval of bi-specific T-cell engagers and chimeric antigen receptor (CAR)-armed T-cells for B-cell acute lymphoblastic leukemia (B-ALL). However, relapses frequently occur in patients treated with CD19- directed immunotherapies, often due to epitope loss. While strategies based on dual antigen targeting are beginning to emerge, they are still based on targeting canonical B-cell markers, with the unavoidable side effect of total ablation of normal B-cells. The apparent paucity of tumor-specific targets in pediatric cancers (including leukemias) is likely to limit future immunotherapies. We hypothesize that alternative splicing could be both a mechanism of epitope loss and a rich source of a neo-antigens in B-ALL. Indeed, using computational and biochemical approaches, we have identified hundreds of local splicing variations (LSVs), mapping to transmembrane proteins (e.g., CD19 and CD22) with prominent extracellular domains (ectodomains). In fact, in our prior work we described a mechanism of acquired resistance to CART-19 based on selective loss of the CD19 ectodomain, primarily through exon 2 skipping. The two large Aims of this U01 are as follows. Aim 1: To identify programs and determinants of altered splicing of B-ALL cell surface antigens. Our overarching goal is to construct a dedicated ?splicing code? for leukemic B-cells. Such a code will predict cis-acting genetic variants as well as trans-acting factors involved in alternative splicing and allow us to identify all alternatively spliced ectodomains. Then in Aim 2, we will investigate the effects of alternative splicing on B-ALL immunotherapy. Using CD22 as just one example, we will determine how truncated protein isoforms confer resistance to CD22-targeting immunotherapeutics, including antibody- drug conjugates such as inotuzumab ozogamicin, which was recently approved by FDA to treat relapsed or refractory B-ALL. We will also raise antibodies against peptides spanning novel exon junctions, generate antibody-drug conjugates, and test their efficacy against B-ALL cell lines and patient-derived xenografts. In summary, this leukemia-based U01 will create new computational and conceptual frameworks, which would be highly synergistic with Pediatric Immunotherapy Discovery & Development Network (PI-DDN) overall goals, including identification of antigenic epitopes that are uniquely expressed on childhood cancers and of cancer cell-intrinsic mechanisms of immune evasion.