Principal Investigator: Jeffrey Magee, MD, PhD

Description: In collaboration with the McDonnell Pediatric Cancer Research Center (MPCRC), we have launched a funding initiative called “Amplification Awards” to ensure that highly meritorious pediatric cancer-related research is sustained to completion in the context of NIH funding cuts and rising research costs. The overarching premise is to supplement research projects that currently have NIH funding yet remain underfunded. These projects have already undergone rigorous scientific review and been deemed exceptional. However, they are at risk for not meeting their full potential – in terms of new knowledge and new therapies – because of budget cuts and rising overall costs.

While the MPCRC and the Division of Pediatric Hematology Oncology remain committed to funding new investigators and pilot research, as we have for a long time, the Amplification Awards meet an emerging need, especially for our talented mid-career faculty. They will ensure that the promise of successful pilot efforts goes fulfilled.

We have established a two-step review process under the auspices of the annual MPCRC funding cycles. The first stage involves a letter of intent to ensure eligibility. The awards are meant to amplify research that directly relates to childhood cancer and has successfully garnered NIH funding (or has received a clearly fundable score). The second stage involves review of progress to date on the funded aims. Since the proposals already have been deemed scientifically meritorious by the NIH, the internal review emphasizes accomplishment and relevance to pediatric cancer treatment. The MPCRC scientific advisory board oversees the review process. This year we anticipate funding 5 eligible investigators within the Division of Pediatric Hematology and Oncology.

Principal Investigator: Nicole Brossier, MD, PhD

Description: This project is funded in collaboration with Siteman Cancer Center. Children with the pediatric brain tumor predisposition syndrome Neurofibromatosis Type 1 (NF1) are at risk for low-grade gliomas in the optic pathway (optic pathway gliomas; OPGs). However, there is significant clinical heterogeneity in the presentation of these tumors, and we currently cannot predict which children will develop OPGs or how severe their clinical manifestations will become. Consistent with epidemiologic studies showing that maternal obesity is associated with higher rates of pediatric glioma formation, obesogenic (high-fat, high-sugar; HFHS) diet exposure has recently been shown to accelerate Nf1-OPG formation and increase tumor penetrance, in part through effects of maternal diet on the Nf1-OPG cell of origin. While neither the dietary component mediating this response nor its downstream effectors have been elucidated, Ob-exposed Nf1-OPG mice display significantly higher levels of EGF, a blood-brain penetrant, neuroactive growth factor and potent mitogen. The EGF receptor (EGFR) is expressed in the optic nerve, and preliminary data shows that EGF stimulates the proliferation of optic pathway glioma stem cells in vitro. EGFR signaling is critical for the development of other types of diet-driven tumors, including colon cancer and breast cancer, which can be driven by exposure to a high-fat (HF) diet without added sugar. Prior studies additionally show that maternal high fat (HF) diet exposure increases the proliferation of progenitors in the embryonic hypothalamus, where the Nf1-OPG cell of origin resides, similar to maternal HFHS diet exposure. HF diet and elevated EGFR signaling have also been shown to induce resistance to chemotherapeutic agents, including the Nf1-OPG therapeutic agent carboplatin. Based on these provocative findings, this application addresses the hypothesis that high dietary fat intake will increase EGF levels to accelerate gliomagenesis and induce resistance to therapy in Nf1-OPG mice. Experiments will first be performed to determine how dietary composition (fat, sugar content) affects murine Nf1-OPG formation and therapy resistance in relationship to EGF levels (Aim 1). This will be paired with mechanistic studies to determine whether EGFR signaling is required for Ob-accelerated gliomagenesis (Aim 2). Together, these experiments will determine the relationship between obesogenic diet composition, EGF and the rate of glioma formation in NF1. They will also provide a critical foundation for future investigations focused on the mechanisms by which diet affects EGF signaling to accelerate gliomagenesis in NF1.

Principal Investigators: Abby Green MD, PhD and Jeff Bednarski, MD, PhD

Description: The long-term goal of this project is to define the mutagenic processes that promote leukemic transformation of B cells in children. Precursor B cell acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood. The ETV6-RUNX1 translocation is present in 25% of childhood ALL, making it the most prevalent genomic aberration among childhood leukemias. While many patients with ETV6-RUNX1-translocated ALL have favorable outcomes, the incidence of late relapse is substantial (15-20%) necessitating mechanistic understanding of disease drivers to develop improved therapeutic strategies.

The ETV6-RUNX1 translocation is an inefficient oncogene and requires additional genomic alterations to promote leukemia development. Prior studies suggest two potential sources of mutagenesis in pre-B cells with ETV6-RUNX translocations. First, RNA-sequencing of ETV6-RUNX-translocated leukemias demonstrated elevated expression of RAG endonuclease. RAG is responsible for generating purposeful DNA breaks during normal B cell development, but can cause off-target DNA damage during dysregulated states. Second, genome sequencing of ETV6-RUNX1-translocated ALLs has demonstrated an enrichment in specific mutational patterns consistent with the activity of the APOBEC3A cytosine deaminase. APOBEC3A is expressed in hematopoietic cells and restricts virus infection by mutating pathogen genomes, but when acting aberrantly can induce widespread mutations through the cellular genome. We propose that ETV6- RUNX1 translocations alter B cells in a manner that enables accumulation of mutations. We hypothesize that RAG and APOBEC3A generate genomic aberrations that drive leukemogenesis in B cells with ETV6-RUNX1 translocations. To execute the proposed studies, we have generated a novel mouse model which enables co-expression of APOBEC3A and/or RAG in hematopoietic cells with ETV6-RUNX1 translocations. The overall objective of this proposal is to determine the mechanism of leukemic transformation in B cells with ETV6-RUNX1 translocations. We hypothesize that ETV6-RUNX1 disrupts gene expression to establish an aberrant B cell state that primes cells for mutagenesis and leukemic transformation. The Aims of the project are to: 1) determine how ETV6-RUNX1 translocations alter development of pre-B cells, and 2) define how APOBEC3A promotes malignant transformation of pre-B cells.

Principal Investigator: Emily Phillips, MD

Description: Although 70% of pediatric patients with acute myeloid leukemia (AML) achieve cure, survival upon relapse is poor, with less than 50% of patients achieving a cure. Memory-like NK (ML NK) cells are NK cells upon which exposure to IL12, IL15, and IL18 induces a differentiation process in which these now ML NK cells demonstrate increased metabolic fitness and increased activation and killing of malignant cells. ML NK cells have demonstrated efficacy against pediatric AML in a phase I trial pediatric of patients with relapsed AML undergoing adoptive transfer of donor lymphocytes and ML NK cells following allogeneic stem cell transplant. Although promising, most patients do not achieve long-term survival.

The aim of the study is to improve the recognition of AML and function of ML NK cells through targeting CD33 via CD33-IL15-CD 33 trispecific killer engager (CD33 TriKE), and targeting CD123 via CD123 chimeric antigen receptor (CAR), and to determine if dual targeting of CD33 and CD123 have synergistic effect on ML NK cell responses to AML.

Design/Method: ML NK cells were co-cultured with various CD33+ and/or CD123+ AML cell lines (MOLM13, THP1, HL60, OCI-AML3). ML NK activation (degranulation, interferon gamma) was evaluated in a 6- hour functional assay. AML killing was assessed in a 4-hour flow-based killing assay. Longer-term killing potential was assessed in 20-24 hour luciferase-based assay. Transgenic IL-15 producing NSG mice were injected with THP-1 and dosed with ML NK cells, CD123 CAR-expressing ML NK cells, or ML NK cells plus CD33 TriKE every other day for 9 doses.

Preliminary Results: ML NK cells co-incubated with tumor and CD33 TriKE exhibit increased markers of activation compared to ML NK cells co-incubated with AML (MOLM13: CD107a p<.0001, IFNg p=.0056; THP1: p<.0001, IFNg p=.0015). ML NK cells treated with CD33 TriKE exhibit increased killing of AML cell lines compared to ML NK cells alone at 4 hours (absolute IC50 MOLM13 2.002 vs 4.423; THP1: 566.1 vs 2.297). There is near elimination of AML after 20 hours at E:T ratios of 10:1. In a single donor experiment, mice treated with ML NK cells plus CD33 TriKE exhibit increased tumor control and prolonged survival compared to mice receiving only ML NK cells. ML NK cells expressing CD123 CAR exhibit increased markers of activation and trend towards increased killing. Conclusion: ML NK cells targeted to CD33 via CD33 TriKE and ML NK cells targeted to CD123 via CD123 CAR exhibit increased activation and increased killing potential.

Principal Investigators: Brooke Sadler, PhD and Jorge Di Paola, MD

Description: Platelet biogenesis (a process known as thrombopoiesis) involves a sequence of complex cellular events in mature bone marrow megakaryocytes (MKs) culminating with the generation of proplatelet extensions that release platelets into the circulation. Approximately 10¹¹ platelets must be produced daily to maintain normal concentrations of 150-400 x 10⁹ platelets per liter of human blood. Platelets are essential not only for providing adequate hemostasis but also for being implicated in other health related processes such as inflammation, wound healing and vascular integrity.^3,4 Thrombocytopenia (defined as a platelet count < 150 x 10⁹/L) is caused by either decreased production from a variety of medical conditions such as cancer, chemotherapy and radiation therapy and aplastic anemia. Thrombocytopenia is a major clinical complication that can lead to severe hemorrhage and even death. It is estimated that more than 2 million platelet units are transfused yearly in the United States. Therefore, a better understanding of the processes that govern MK differentiation and platelet production will potentially lead to an increased ability to manipulate MKs, generate functional platelets in vitro, target therapies for congenital and chemotherapy related thrombocytopenia, and ultimately improve clinical outcomes. Our laboratory has made seminal discoveries in the genetic regulation of platelet production including the discovery of genes that cause low platelet and cancer predisposition such as NBEAL2 and ETV6. In this proposal, we will study 250 individuals from Northern India in which 15-20% of people have  platelet counts of < 100 X 10⁹/L) without significant bleeding. We will perform whole genome sequencing (WGS) and bioinformatic analyses in 250 samples, that will hopefully determine the genetic regulators that drive this thrombocytopenic phenotype and shed light on actionable targets for therapeutics.