Johns Hopkins University-School of Medicine Archives

2019

Scholar Grant

Nicolas Llosa

Baltimore, MD

Title: Exploiting the Immunosuppressive Niche of Rhabdomyosarcomas for New Immunotherapies.

Executive Summary:
Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Rhabdomyosarcoma usually begins in muscles that are attached to bones and that help the body move, but it can also begin in many places in the body including internal organs. Unfortunately, cure rates have stagnated since the 1990s; >70% of children diagnosed with the so-called “fusion-positive” rhabdomyosarcoma or metastatic disease will die from their cancer even with the most advanced multimodal therapies. Our research focuses on understanding how the immune system interacts with rhabdomyosarcoma cancer cells. Our goal is to develop immunotherapies for childhood rhabdomyosarcoma. Immunotherapy is a type of cancer treatment designed to boost the body’s natural defenses to fight the cancer and is one of the most promising current approaches for treating tumors. This novel strategy uses materials made in a laboratory to improve or restore the function of the body’s immune system with the ultimate goal of stopping the growth of tumors. Our plan is to use this approach to fight rhabdomyosarcomas and, in that way, transform (vs. optimize) the care for this group of hard to treat cancers and improve the survival of patients.

The key in predicting response to immunotherapy lies within understanding how the tumor interacts with the immune system. In an actively thriving tumor, the scale is tipped towards an environment that suppresses the immune response allowing for tumors to escape immune elimination. The numerous factors and players that maintain this balance are found in the tumor microenvironment (TME). The objective of our research is to investigate the TME of rhabdomyosarcomas, in hopes of understanding mechanisms by which RMS escapes immune detection and elimination. This information is key to the development of successful treatment regimens that can allow us to harness the patient’s immune system to help rid the body of cancer.

Our ultimate goal is to harness the immune system as a major therapeutic strategy for the treatment of RMS. It is clear that current immune-oncology approaches are ineffective in rhabdomyosarcoma (RMS), but the reasons why are extraordinarily poorly understood. In this project, we will examine the geography, nature and function of RMS tumor associated macrophages (TAMs) infiltration and how they spatially interact with T cells via multiplex immunohistochemistry staining followed by digital image analysis, gene expression profiling of specific tumor regions and multiparameter flow cytometry studies. We hypothesize that the presence of a higher concentration of TAMs in RMS impacts the trafficking and functionality of immune cells hampering proper antitumor immune responses.

Our systematic interrogation of the immunological microenvironment of RMS and circulating immune indicators which serve as a window into the TME will be critically important to identify relevant biomarkers for developing combinatorial immunotherapies.

2019

Young Investigator

Michael Koldobskiy

Baltimore, MD

Grant: $200,000 Young Instigator & $300,000 Hope Scholar

Executive Summary:
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive brainstem tumor of childhood. No effective treatment for DIPG has been developed to date, making it a universally fatal diagnosis. We are studying ways in which DIPG cells rely on “epigenetic” modifications, or chemical marks that modify the expression of genes without a change in the genetic sequence itself. Variability of such epigenetic marks allows DIPG cells flexibility in turning genes on and off, and may account for cellular heterogeneity and therapeutic resistance. By dissecting the mechanisms of epigenetic modification in this disease, we aim to identify selective vulnerabilities that serve as targets for intervention. In particular, we are evaluating the use of existing, FDA-approved drugs to target DNA methylation alterations for the treatment of this devastating tumor.

2018

Scholar Grant

Christine Pratilas

Baltimore, MD

Genomic and biochemical characterization of RAS dependency in tumors characterized by loss of NF1.

• Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive soft tissue sarcomas that develop most commonly in patients with the genetic syndrome neurofibromatosis (NF1). Despite the use of chemotherapy, radiation, and surgery, almost half of patients with MPNST will experience treatment failure and succumb to their disease.
• Clinical trials have tested both targeted and non-targeted agents in children and young adults with MPNST; however, no second-line therapy has yet to demonstrate an improvement in survival in patients who have had recurrence.
• One of the major drivers of cancer cell growth in MPNST is loss of NF1 gene function. NF1 is a tumor suppressor which functions to limit the activity of RAS (which is, itself, a major cancer-causing oncogene); thus, loss of NF1 leads to excessive activation of RAS-regulated pathways.
• Drugs that target RAS signaling pathways have been developed for other diseases, but do not work as well in MPNST for several reasons:

1. Since loss of NF1 is present in the benign form of tumors that leads to MPNST, it cannot be sufficient to promote development of cancer, so we know that additional “hits” must be present – either genomic, epigenetic, or adaptive.
2. The genes that are identified as additional drivers in MPNST are not targetable. It may be that they contribute to cancer growth by interacting with RAS, but we don’t understand these interactions well enough.
3. It is not clear which pathways regulated by RAS are most important.
4. Cancer cells adapt to inhibiting a pathway, i.e., targeting one pathway often leads to increase in activity of another, in ways that allow the cancer to escape from treatment.
5. When MPNST models do respond to inhibition of RAS signaling pathways, they can, over time, acquire resistance.

• In summary, studying adaptive changes and mechanisms of resistance can give clues to understand the molecular response to targeted therapy, and thereby develop approaches to prevent resistance and achieve more complete and durable responses. Our proposal aims to address these issues in search of rationally designed and effective therapies for patients with MPNST that has recurred after failure of first-line treatment.

2018

Young Investigator

Cara Rabik

Baltimore, MD

Epigenetic changes induced in acute myeloid leukemia harboring WT1 (Wilms Tumor 1) gene mutations

Leukemia is the most common childhood cancer, and the subtype of childhood leukemia called acute myeloid leukemia (AML) is challenging to treat, with survival rates stagnant at 50% despite intensification of therapies. AML can be caused by different genetic mutations, and my work focuses on mutations in one specific gene. WT1 mutations are found in 10% of AML patients, and they lead to poorer outcomes. The normal WT1 protein is important in normal blood cell differentiation from stem cells to mature white blood cells. WT1 binds DNA, stimulating expression of genes important for differentiation. WT1 does this by catalyzing the removal of methyl groups from the DNA, which allows for gene expression. Genes can be silenced with the addition of methyl groups, which is called epigenetics. Mutated WT1 cannot bind DNA; therefore, the DNA remains methylated and silenced, unable to express genes important for normal blood cell development. Drugs called hypomethylating agents, which include 5-azacytidine and decitabine, act independently of WT1 to demethylate DNA, which may be useful in treating patients with mutations in WT1 or other members of its pathway. I hypothesize that WT1 mutations result in excess DNA methylation of its target genes, silencing them, and that WT1-mutated AML may be sensitive to treatment with hypomethylating agents. My work aims to:

– Validate and prioritize WT1’s target genes to determine which ones are most important for normal blood cell development.
– Use genome editing techniques, such as CRISPR/Cas9, to impair expression of specific target genes to determine whether they are necessary in blood cell differentiation.
– Use hypomethylating agents in cell lines with wild-type, mutant, and no WT1 expression to test for re-expression of WT1 target genes.
– Use cells from patients who have AML with wild-type and mutant WT1 to measure the relative levels of target gene expression.
– Inject WT1 mutant AML cells into mice to express human leukemia in the mice followed by treatment with hypomethylating agents to test for disease response.

I anticipate that 5-azacytidine will result in restoration of expression of WT1 target genes and lead to increased sensitivity of these leukemia cells to standard chemotherapy. The next step after these experiments will be to initiate a clinical trial for patients with relapsed AML who have mutations in the WT1 gene using this treatment. If this goes well, then we will initiate a clinical trial for newly diagnosed AML patients with WT1 gene mutations incorporating hypomethylating agents into the treatment of these patients from their initial diagnosis in an effort to prevent relapse and increase cure rates.