Nancy Berliner - US grants

Abstract Hemoglobinopathies, such as sickle cell disease (SCD) and thalassemia, are among the greatest public health concerns in the world. Although new therapeutic modalities, such as gene therapy, are currently being tested, there is a pressing need for pharmacologic approaches to treat general patient populations. Our long-term goal is to develop a compound(s) that induces fetal-type globin (HbF) production by targeting the transcriptional complex regulating globin switching. The objective of this application is to determine molecular mechanisms underlying the LRF-NuRD-mediated ?-globin silencing and identify a mean(s) to target them. Our central hypothesis is that the LRF-containing NuRD complex is a potential target for HbF reactivation therapy. The rationale for the proposed research is that understanding the LRF/NuRD-mediated globin regulation will provide greater understanding of the transcriptional complex regulating ?-globin repression and facilitate development of novel therapeutic strategies for HbF induction therapy. Guided by strong preliminary data, we expect to achieve our objective by pursuing the three specific aims: 1) to determine the molecular basis for ?- globin reactivation in the absence of the LRF/NuRD; 2) to identify a domain(s) of CHD3/4 necessary for ?- globin silencing; and 3) to determine functional significance of LRF/CHD3 interaction in controlling ?-globin silencing. In Aim1, we will employ ChIP-seq and ATAC-seq foot-printing to determine how the NuRD- associated pathways silences ?-globin expression in adult erythroid cells and how ?-globin is induced upon LRF depletion. In Aim2, we will identify a minimal domain(s) of CHD3/4 responsible for ?-globin silencing. To do so, we will perform a functional domain mapping using CRISPR-Cas9 gene mutagenesis. In Aim3, we will determine functional significance of the LRF/CHD3 interaction in controlling ?-globin silencing. Our published work and preliminary data strongly suggest that the NuRD-associated pathways, in which LRF and BCL11A are involved, represent the near entirety of the ?globin-switch?. We expect that the combined approaches proposed here will elucidate the role of LRF and the NuRD complex in ?-globin silencing and facilitate development of novel strategies for HbF reactivation therapy for hemoglobinopathies.

Project Summary Basic and translational research in hematology has been at the cutting edge of recent advances in our understanding of the molecular pathophysiology of disease for decades. This T32 grant, for which we request renewal, was submitted 5 years ago to replace the former Harvard Medical School Training Program in Molecular Hematology, which had a distinguished 25-year track record in training graduate students and postdoctoral fellows in the study of blood and its disorders, with many graduates of the program going on to highly successful academic careers. The past 30 years have seen many changes in the landscape of hematology-oncology in the Harvard Medical area, with the merging of the Hematology program at the Brigham and Women?s Hospital with the oncology programs at the Massachusetts General Hospital and the Dana-Farber to form a combined Hematology-Oncology fellowship. This has had many positive effects on the opportunities for clinical and research training, complemented by a proliferation of training programs. At the same time, an explosion of new technologies has ushered in an era in biomedical research that offers exciting opportunities for major breakthroughs in our understanding of human disease, offering hope for new clinical paradigms that can transform the treatment of many hematologic disorders. However, the loss of a dedicated Hematology fellowship has made us acutely aware of the importance of nurturing physician scientists dedicated to the study of hematology. Indeed, the disappearance of free-standing hematology fellowships nationwide and the shrinking numbers of Hematology trainees signals an urgent need to encourage future academic hematologists. This program places high priority on training physician-scientists focused on hematology, and we have been gratified to see an increasing number of clinical fellows who choose to do research as trainees on this grant. To date, we have accommodated all the MD and MD/PhD fellows with a research focus on Hematology, although we have a selection process in place should we have to limit availability because of an increase in interested fellows. This process has already been used in the first two years of the grant to select PhD fellows to fill available slots, although all subsequent slots have been taken by physician scientists. The primary site of training is the preceptor?s laboratory, but each trainee is expected to participate in relevant seminars and courses within the HMS community. Each trainee also assembles a training committee that monitors research progress with annual formal presentations. We strongly encourage the investigation of benign hematologic disorders in the areas of red cell disorders, iron metabolism, hemostasis and thrombosis, and neutrophil disorders, as well as hematologic malignancies such as leukemia, myelodysplasia, and myeloproliferative neoplasms. This strict focus on Hematology distinguishes this grant from other Harvard-related training grants, fulfilling a need that is not answered by any other training grant within adult medicine. We request renewal of this application with its 8 current training positions.!