Stanford University Lucille Packard Children's Hospital Archives

2019

Scholar Grant

Alice Bertaina

Palo Alto, CA

Title: Generating a novel humanized mouse model of Juvenile myelomonocytic leukemia (JMML) by knockout of hNF1 using CRISPR/Cas9 genome editin

Executive Summary:
Juvenile myelomonocytic leukemia (JMML) is an aggressive cancer that exclusively affects children, usually before age two. Children with JMML produce abnormal white blood cells, which overwhelm the bone marrow and other organs, causing them to fail. Without treatment, the five-year survival is only around 5%. The only curative treatment is hematopoietic stem cell transplant (HSCT), a transplant of stem cells from a healthy donor, however, HSCT offers, at best, a 50% survival rate. Thus, there is a desperate need for new treatment strategies which is only possible once we fully understand the mechanism of disease development. This is especially challenging because 1., there is low patient sample availability since JMML is a rare pediatric disease, and 2., there is currently no reliable humanized animal model as is the gold standard for studying other rare diseases.

Thus, the main goals of our project are:
– To ‘knock-out’ (turn-off) through CRISPR/Cas9 gene editing the gene hNF1 in human umbilical cord blood (CB) stem cells because it has previously been established that loss of hNF1 expression leads to JMML (Aim 1).
– To develop a novel humanized animal model of JMML by transplanting CB cells genetically modified such that the hNF1 gene is no longer expressed (Aim 2).

We will confirm the success of the gene knockout by evaluating the replenishing potential of the engineered cells through their growth in specialized media (nutritive semi-solid).

Thereafter, we will transplant these engineered cells into humanized mice (mouse carrying functioning human genes and cells) with the goal of recapitulating JMML. We will compare the disease appearing in mice with what is observed in humans to validate our model. We will look for definite JMML disease characteristics such as presence of leukemic cells in the blood and bone marrow of the mice, enlargement of spleen and liver.

This novel animal model will serve as an infinite JMML sample pool which will allow us to study disease development. Our ultimate goal using this model is to find effective approaches to identify new and efficient treatment options for this devastating childhood leukemia.

2019

Young Investigator

Sneha Ramakrishna

Palo Alto, CA

Executive Summary:
Patients with relapsed or refractory pediatric B-cell acute lymphoblastic leukemia (B-ALL) have very limited options that provide hope for cure. Chimeric antigen receptor (CAR) T cells harness the power of the immune system by teaching T cells to recognize specific targets on the surface of leukemia cells. Clinical trials have found that CARs directed toward either CD19 or CD22 are able to treat patients with relapsed B-cell ALL, but approximately half of these patients will eventually relapse. The three main causes of relapse are 1) loss of target expression, 2) decreased target expression, and 3) failure of CAR T cells to persist. To address loss of antigen expression, we are currently evaluating a CAR that can simultaneously target both CD19 and CD22. This dual-targeting approach may decrease relapse due to target loss, but is not likely able to overcome escape due to low target expression or CAR failure. We have identified a transcription factor, c-JUN, which, when over expressed, can improve CAR activity against low-antigen tumors, enhance CAR expansion, and improve CAR persistence in various single-antigen-targeting CAR constructs. Here, we propose to develop a next generation CAR incorporating c-JUN into the CD19-CD22 CAR. We hypothesize that incorporating c-JUN into the CD19-CD22 CAR construct will improve activity against low antigen expressing leukemia, and enhance CAR expansion and persistence, thereby improving durability of responses with this dual-targeting CAR. We will evaluate whether the addition of c-JUN improves CD19-CD22 CAR activity against low-antigen leukemia. We will also assess how c-JUN affects CD19-CD22 CAR expansion, persistence, and exhaustion in mouse models. Results of these studies will extend our understanding of how next generation CAR engineering can overcome three serious limitations to long-term CAR success in patients and will form the basis for clinical application of this “next generation” CAR T cell for pediatric B-ALL.

2018

Scholar Grant

Kathleen Sakamoto

Stanford, CA

The Role of RSK in the Pathogenesis of Pediatric AML and as a Target for Therapy

“Children with Acute Myeloid Leukemia (AML) have a 5-year survival of less than 60% despite intensive chemotherapy and stem cell transplantation. Treatment of AML is associated with significant morbidity and late effects, impacting the quality of life in children and adolescents who survive their disease. Furthermore, AML patients who relapse have few effective approaches to treat their disease. Therefore, it is important that we study the underlying pathogenesis of AML to identify novel targets for therapy.

We previously demonstrated that the transcription factor CREB is overexpressed in bone marrow cells from most pediatric and adult AML patients. CREB knockdown inhibits AML cell proliferation but not normal hematopoietic stem cell activity in vitro and in vivo. Tool compounds that inhibit CREB suppress AML progression in vivo and are nontoxic in mice. The major kinase phosphorylating CREB is the serine/threonine kinase RSK1 (Ribosomal S6 Kinase). We propose to study the role of RSK-CREB signaling axis and molecularly characterize the possibility of RSK as a target for AML therapy.

Project Aims
Aim 1: We will study the molecular effects of RSK inhibition and downregulation in AML cells in vitro.
Aim 2: We will characterize the set of genes regulated by RSK1 in AML and examine RSK1-mediated crosstalk between the NF-kB and CREB signaling pathways.

Outcomes
The project aims will result in a greater understanding of the role of RSK in the pathogenesis of pediatric AML. The proposed studies will lead to identification of RSK as a novel target for AML therapy.

Benefits to Pediatric Cancer
Understanding the role of RSK in AML could lead to novel approaches to treat pediatric AML.

Lay Language
The goals of this project are to:
1. Understand the molecular pathways involving RSK in the development of pediatric AML.
2. Study how blocking RSK activity could inhibit AML growth in cells. “

2017

Scholar Grant

Dr. Kara L. Davis

Palo Alto, CA

For pediatric patients with acute lymphoblastic leukemia, integration of early testing to look for leukemia cells which have survived the first weeks of chemotherapy has transformed the clinical system of risk prediction. Yet, despite knowing that having these chemotherapy-resistant minimal residual disease (MRD) cells places a patient at higher risk of future relapse, we have yet to determine what it is about these cells that permits them to be resistant to chemotherapy. Is it cell-intrinsic, that is features of the MRD cells themselves vs. a permissive immune environment where the patient’s own immune system cannot recognize these rogue remaining cells? Herein, we propose to take a novel approach to this problem based on single-cell studies of primary patient samples collected at diagnosis and then at standard timepoints for MRD detection. Applying a high-parameter, single-cell technology, mass cytometry, we will determine the phenotype and activated signaling networks which characterize these treatment-resistant cells to look for cell intrinsic features of resistance. However, utilizing the same primary samples, we will also characterize the immune state of each patient to determine the frequency and dynamics of immune cell subsets to decide if certain immune states are permissive to persistent disease (MRD) and to identify immune subsets which are present at highest frequency in those patients without detectable MRD.

The aims of this project are to:

Compare surface protein and signaling characteristics of minimal residual disease (MRD) cells from early on-therapy timepoints versus their matched diagnostic bone marrow in primary B-cell progenitor acute lymphoblastic leukemia samples.
Utilizing advanced computational analysis (machine learning), determine if there is a set of unifying signaling or surface protein features which characterize chemotherapy-resistant (MRD cells) across patients to identify potential druggable targets.
Characterize the recovery of the immune system during early chemotherapy and determine correlation between presence or absence of immune subsets and MRD positivity.

In childhood acute lymphoblastic leukemia, a major cause of mortality is relapse. By bringing novel single-cell technologies to this translational study in pediatric cancer we will identify key cell populations (both malignant and healthy) associated with poor clinical outcomes in order to identify therapeutic targets for future therapy with the goal of improving outcomes by preventing relapse altogether.

2017

Young Investigator

Dr. Robbie G. Majzner

Palo Alto, CA

Target Antigen Density as a Driving Factor in Chimeric Antigen Receptor (CAR) T-cell Efficacy

Chimeric Antigen Receptor (CAR) T cell therapy targeting CD19 has emerged as an exciting modality for the treatment of pediatric leukemia. This type of immunotherapy uses genetic engineering to redirect a patient’s immune cells to recognize and kill cancer cells that express high levels of CD19 on their surface. Thus far, attempts to use CART cells to treat non-hematologic malignancies in patients have not been as successful, and our preliminary data suggest that low expression of the target protein by the tumor cells impairs CAR-T cell efficacy. Our hypothesis is that the success of the CD19 CAR against leukemia is partially dependent on homogenously high expression of CD19 on leukemia cells. Understanding the contribution of antigen density to CART cell efficacy is essential to applying this technology to other tumor types.

The goals of this project are to:

Investigate how the amount of target protein on the tumor contributes to the success of CD19 CART cells
Determine what signals in a T cell are needed for an effective CAR T cell response
Use this knowledge to determine which patients are likely to respond to CD19 CAR therapy
Develop CARs that can recognize lower amounts of target protein which will allow us to use this technology to better treat other pediatric cancers

2014

Scholar Grant

Dr. Kathleen Sakamoto

Palo Alto, CA

Although the survival of children with acute lymphoblastic leukemia (ALL) has improved to 80-90% 5-year survival, it is becoming increasingly known that there are significant side effects and long-term complications of treatment. In addition, for the 10-20% of children who relapse, there are very few treatment options except for stem cell transplantation, which is associated with a high rate of complications and even death. Therefore, it is critical to identify new approaches to treat ALL that are more effective and less toxic. The goal of this project is to understand the role of a protein known as CREB. CREB is a transcription factor that is overexpressed in over 80% of patients with ALL. CREB controls genes that are important for leukemia cell growth and survival. Our laboratory has been studying the role of CREB in AML over the past 10 years, but very little is known about how CREB might cause pediatric ALL. Therefore, Hyundai Hope on Wheels will be supporting a new project and research focus of my laboratory. We have also identified a small molecule that blocks CREB function and can inhibit the growth of leukemia cells in culture and in mice. In this grant, we propose to 1) study the pathways that control CREB activity in ALL cells; 2) study genes that are regulated in ALL cells overproducing CREB; and 3) study the effects of CREB inhibitors on the growth and survival of ALL cells. These studies will enable us to identify new pathways that cause ALL and provide novel approaches to treat children with ALL.