Funding Childhood Cancer Research

Targeting RNA Processing Defects of Ewing Sarcoma
Ewing sarcoma (EwS) is a disease driven by the fusion of two genes, EWSR1 and FLI1, creating EWSR1-FLI1. In normal cells, EWSR1 has the ability to bind RNA and plays a role in RNA processing. EwS cells have problems properly processing RNA which is thought to be due to the fusion gene interfering with the normal function of EWSR1. We recently discovered that EWSR1 controls the activity of the RNA producing machinery (RNA polymerase) and that in EwS cells this machinery is overactive, producing more RNA than in normal cells. We believe that EwS cells are unable to process the increased amount of RNA efficiently and therefore these cancer cells may be more sensitive than normal cells to agents that target RNA processing. We screened the whole genome, targeting each gene individually, and found that EwS are very sensitive to targeting RNA processing genes. This is a key finding that could lead to alternate targeted treatment options for this disease. We propose to further investigate the results, identify which RNA processes are particularly important for EwS and whether available pharmaceuticals targeting these processes can be used effectively to treat this disease. As with any strategy to specifically target cancer, the key is to identify how these cancers differ from the norm and then explore whether this difference can be therapeutically targeted. Clinically relevant RNA processing inhibitors currently exist and we will test them in preclinical models of EwS.

Predicting Post-Treatment Relapse in Pediatric AML Using Single-Cell Proteomics
Roughly 500 children are diagnosed with acute myeloid leukemia (AML) each year in the United States. While most children initially respond to standard chemotherapy, nearly 40% ultimately relapse, making AML the deadliest blood cancer in childhood. Despite significant advancements in the treatment of other kinds of acute pediatric leukemia—such as B-cell Progenitor Acute Lymphoblastic Leukemia (BCP-ALL)— in recent years, clinical outcomes in pediatric AML have remained poor for decades, with the majority of mortalities attributable to relapsed disease. To better characterize why some pediatric AML patients ultimately relapse while others do not, the proposed project will use state-of-the-art molecular profiling approaches to characterize millions of primary AML cells and their computational “alignment” with the different stages of healthy blood cell development. These data will be used to develop a method of predicting at diagnosis which patients have a high risk of relapse and which patients have a low risk of relapse. Successful completion of this research will identify the most prognostically important cell populations in pediatric AML as well as potential therapeutic targets in the management of this poorly-understood childhood illness.

Activation of NF-kB in Neuroblastoma Myeloid Cells
Malignant tumors develop means to evade the immune system. An exciting recent advance is the use of checkpoint inhibitors to reactivate T cells within the immune system, which has led to major responses in several cancers. Myeloid cells are a second key component of the immune system. Cancers attract myeloid cells and stimulate them to adopt tumor-promoting M2 features. Conversion of tumor myeloid cells to the anti-cancer M1 state would represent a new immunotherapy strategy. Myeloid cells that lack a protein called NF-?B p50 adopt M1 features and cannot be directed to become M2 by cancers. Several cancers grow slower in mice lacking p50, and when we treat mice with myeloid cells lacking p50, several types of tumors respond. This proposal seeks to use mouse models to validate and optimize p50-deficient myeloid cell therapy against neuroblastoma, a pediatric cancer that is often highly aggressive in patients over 18 months of age. Aim 1 will determine whether neuroblastoma grows slower in mice lacking p50. Aim 2 will determine whether infusion of myeloid cells lacking p50 slows neuroblastoma tumor growth. In both aims, we will also evaluate whether combining T cell checkpoint inhibitors or agents that modify DNA to stimulate the immune response with myeloid cells lacking p50 benefits therapy of neuroblastoma. Successful completion of these studies will encourage us to test the utility of myeloid cells lacking NF-kB p50 as a novel immunotherapy for patients with neuroblastoma.

Overcoming Innate Immune Evasion in Pediatric High Grade Glioma
Few therapies have been aimed at stimulating the myeloid arm of the immune system to attack tumors. As one of the earliest immunologic defense mechanisms of the developing fetus, the innate immune system, which begins developing during the first trimester of gestation, is a potent cell scavenger within children. We previously showed that anti-CD47 monoclonal antibody activates the innate immune system showing significant activity against high-grade glioma including pediatric glioblastoma and Diffuse intrinsic pontine glioma. However for phagocytosis to occur eat-me signals are required to be exposed on the outer surface of the cells. Depending on the initiating stimulus such as Irradiation, cancer cell death can be immunogenic as they lead to surface expression changes termed, Damage Associated Molecular Patterns which acts as eat me signals marking them for clearance by macrophages. Therefore, there is an overlap in the signals that induce anti-tumor activity in the standard of care therapy and anti-CD47 therapy. Here We hypothesize that irradiating pediatric high-grade glioma tumors, activates DAMP pathways, leading to increased pro-phagocytosis signals on tumor cell surface, making them more susceptible to macrophage-mediated phagocytosis by overcoming the immune evasion mechanisms. Performing these studies we will facilitate a new clinical paradigm.

Targeting Signaling Pathways that Confer Cytarabine Resistance in Pediatric AML
Nearly 40% of children with acute myeloid leukemia (AML) relapse, and many of them die of progressive AML. Current treatments still use the same old drugs, including cytarabine. Patients with detectable AML after one cycle of treatment have a very high risk of relapse. This residual leukemia is the focus of our proposal. Our goal is to identify and block the processes that allow some AML cells to survive treatment. Interactions between AML cells and the bone marrow environment support survival of a small number of AML cells, but scientists have not yet figured out how to overcome this problem. Studies with AML patients and with mice that received cytarabine have shown that residual AML cells are changed in ways that favor cytarabine resistance. Our experiments confirm that AML cells from mice treated with cytarabine have higher activity of a survival protein, STAT3, than AML cells from mice treated with placebo. We hypothesize that resistance processes are enhanced in AML cells that survive cytarabine, and blocking these processes reduces residual disease and prolongs survival. We will engineer changes in AML cells to interrupt the processes that promote cytarabine resistance, and compare the effectiveness of cytarabine in mice with engineered AML cells compared to normal AML cells. Also, we will test drugs that block resistance processes in mice with human AML. This research will lead to novel strategies to overcome cytarabine resistance so that more children will be cured.

L3MBTL3, A Therapeutic Target in Acute Myeloid Leukemia
Billions of white blood cells are formed every day in the bone marrow. Leukemia occurs when cell proliferation becomes uncontrolled. As part of a complex ensemble of regulators of blood cells, the “Notch signaling pathway” helps maintain the balanced generation and proliferation of white blood cells. Several labs observed that increasing Notch signaling in Acute Myeloid Leukemia (AML) cells impairs their proliferation and may thus provide therapeutic benefit for AML patients. What mechanisms contribute to switching Notch signaling off in blood cells? Could these mechanisms be targeted for the therapeutic benefit of leukemia patients? The Rual lab recently discovered that the L3MBTL3 gene is a repressor of Notch signaling. We hypothesize that the inhibition of L3MBTL3 in AML cancer cells and the associated “de-repression” of Notch signaling could provide therapeutic benefit in AML. With the support of the B+ Foundation, we propose to test this hypothesis by studying the extent to which inhibiting L3MBTL3 modulates cancer progression in mouse models of AML. Our study could offer critical mechanistic insights on the role of the L3MBTL3 in AML that could be harnessed in the future for the therapeutic benefit of AML patients.

Uncovering the Contribution of RNA Binding Proteins to Neuroblastoma Aggression
High-risk neuroblastoma is an extremely aggressive pediatric cancer of the developing sympathetic nervous system. Unfortunately, fewer than 50% of patients survive and survivors experience multiple treatment-related side effects, including decreased growth/development, bone damage, and an increased risk of developing other cancers. Thus, novel targets and therapies must be developed for this disease.

This study focuses on a poorly characterized class of proteins known as RNA binding proteins (RBPs). These proteins bind RNA, which contains the direct instructions to make proteins (proteins carry out many key functions in cells). We have now discovered that the RBP, Musashi 2 (MSI2), is highly expressed in neuroblastoma, causing uncontrollable growth. We will determine how MSI2 causes neuroblastoma to act aggressively, studying its function in cell line and animal models and determining other signaling networks with which MSI2 communicates. MSI2 constitutes a promising drug target and our studies will help build the initial foundation for potentially drugging this protein directly or indirectly. In addition, there are approximately 1550 other RBPs and we will use a “high-throughput” technique that will allow us, for the first time, to determine how RBPs contribute to neuroblastoma aggression. We believe that our focused studies on MSI2 as well as our more global studies on the 1550 other RBPs will nominate new therapeutic targets for this aggressive disease.

A Novel Approach to Target ACVR1 as a Treatment to Pediatric Cancer
Brain cancers are the leading cause of cancer-related deaths in childhood. Each year about 300 children in the US are diagnosed with Diffuse Intrinsic Pontine Gliomas (DIPGs), which accounts for 10% of childhood cancer patients. Due to the delicate location of these tumors, surgical resection is not practical, and these patients also do not respond well to current cancer therapies. The survival rate for DIPG patients is less than 20% two years after the initial diagnosis. Therefore, there is a pressing need to develop new, effective therapeutics for DIPGs. Recently, several independent whole-genome sequencing studies on the DIPG tumor tissues have found a new cancer-driver gene known as ACVR1. About 30% of DIPG patients harbor deleterious genetic mutations in their ACVR1 genes, which are believed to contribute to the cause of the disease. Here, the goal is to target ACVR1 in a series of combined computational and experimental studies. By using high-resolution structural data, state-of-the-art virtual screening tools, and experimental validation, two strategies will be adopted to alleviate the deleterious effects of ACVR1 mutations. First, the team will look for novel inhibitors that target ACVR1 mutants found in DIPG patients. Second, the team will look for small-molecule compounds that may restore the regulation of ACVR1. The outcome of the study will suggest possible therapies using repurposed FDA approved drugs for compassionate use or novel small-molecule compounds for clinical trials.

Molecular Mechanism of CAR Activation in Targeting B Cell Leukemia
Childhood cancer is the #1 disease killer of children in the United States. Among all the childhood cancers, Leukemias, cancers of bone marrow and blood, are the most common type. The development of chimeric antigen receptors (CARs) marked a new era for leukemia therapy. CAR-mediated therapy has been successfully adopted in treating pediatric acute lymphoblastic leukemia and other childhood leukemias. Meanwhile, significant challenges remain including cytokine release syndrome, neurotoxicity, and incomplete patient response. This raises the necessity to understand CAR function at the molecular level so that new tools for CAR therapy could be developed to improve its therapeutic effect. In this proposal, I aim to reveal how a tumor antigen could activate CAR to induce immune responses towards cancer. It will provide guidelines for selecting antigens for building CARs targeting more types of childhood cancers. Moreover, I will explore how phase separation, an emerging biophysical principle in organizing biomolecules, promotes CAR activity. This will establish phase separation as a new target for increasing the efficacy of CAR’s killing tumors.

Developing A Novel Therapeutic Strategy for Rhabdomyosarcoma
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Even with intensive treatment, outcome for patients with metastatic or recurrent disease remains poor. The 5-year survival rate of children with high-risk RMS is only 20% – 30%, with very little improvement over the last 30 years. Therefore, new therapy based on better understanding of this disease is urgently needed. In this proposal, our goal is to develop an effective therapeutic strategy for RMS.

To achieve this aim, my colleagues and I recently combined a new computational approach (iExCN algorithm) and an experimental tool (CRISPR/Cas9-based screen) to identify cancer disease genes in RMS. We have successfully identified 29 RMS disease genes whose expression is driven by copy-number variations and have validated more than half of them. We then applied another computational approach (rFBA algorithm) to predict synergistic drug combinations targeting two RMS disease genes. We found the drug combination targeting both EZH2 and CDK4/6 is promising and our preliminary data support it. Here, we are going to further test the synergistic drug combination in preclinical models, and study the pharmacodynamics of the drug combination. The drug combination we are studying is either FDA-approved drug or drug currently in single-agent clinical trials in children. Therefore, upon completion of this work, we will be able to rapidly advance this promising combination to clinical trials for patients with RMS.