Epigenetic drivers of retinoblastoma
Retinoblastoma is an aggressive pediatric cancer caused by bi-allelic inactivation of the RB1 gene. Each year 250-300 new cases are detected in the US and 7,000-8,000 worldwide. In the US, the 5-year survival rate is around 95%, but the chances of survival steeply decreases with delayed detection and poor healthcare system access, a common situation in less developed countries. Despite progress in clinical outcomes of retinoblastoma, eye enucleation (removal of the eye) still remains a frequent treatment for retinoblastoma. Inevitably, this ocular surgery greatly impacts the future quality of life of the patient.
The Benavente Lab research in this area focuses on understanding the drivers of the epigenetic changes observed in retinoblastoma after RB/E2F pathway inactivation in order to identify new potential targets for therapeutic intervention. Particularly, we are evaluating the role of chromatin remodelers HELLS and UHRF1 in retinal tumorigenesis. These two chromatin remodelers are involved in maintaining DNA methylation. HELLS recruits DNA methyltransferase 3 (DNMT3) regulating de-novo DNA methylation, while UHRF1 along with DNA methyltrasferase 1 (DNMT1) controls cell cycle-dependent DNA methylation. Increased HELLS and UHRF1 expression is not unique to retinoblastoma, but it also observed in several other cancers.
Our findings so far: We developed conditional knock-out mouse models to study the effects of Hells or Uhrf1 inactivation in the mouse developing retina. Our studies reveal both Hells and Uhrf1 as transcriptional targets of the RB/E2Fs pathway in retina. While HELLS and UHRF1 appear to be nonessential for retinal development, failure to repress Hells or Uhrf1 transcription during terminal retinal differentiation in the absence of the RB1 family significantly contributes to retinoblastoma development. Loss of Hells expression significantly decreases the incidence of retinoblastoma, and increases overall survival of retinoblastoma-bearing mice. Surprisingly, the absence of HELLS expression does not results in altered chromatin structure in the retina. Instead, results in reduced expression of E2F3 cell cycle target genes, with HELLS playing a role as transcriptional co-activator of E2F3 (Zocchi L., et al., Oncogenesis 2020). The significant increase in survival observed in both Hells and Uhrf1 mouse model mouse make these two chromatin remodelers interesting targets for the treatment of retinoblastoma. They are both nonessential for retinal development and their expression is absent in fully differentiated retina. These unique characteristics may open up an avenue for the ocular delivery of UHRF1 or HELLS inhibitors.
Role of UHRF1 in osteosarcoma and its relationship to RB1
Osteosarcoma is the most common primary cancer of bone and typically occurs in children and young adults. As a highly metastatic cancer, 15-20% of osteosarcoma patients are diagnosed after the cancer has already metastasized (typically to the lungs), which translates to 5-year survival rates of less than 40%. In comparison, patients without metastases have survival rates of 65-75%. Unfortunately, pulmonary metastases occur in nearly half of all osteosarcoma patients. Thus, there is a pressing clinical need to determine the factors responsible for metastasis in osteosarcoma to facilitate development of new therapeutic strategies. Loss of the tumor suppressor gene RB1 is associated with increased mortality, metastasis and poor therapeutic outcome in patients with osteosarcoma. However, the mechanism(s) through which RB1 loss leads to poor prognosis remains to be elucidated. We are testing a model for how loss of RB1 worsens clinical outcome that has the potential of becoming an alternative therapeutic approach to prevent metastasis in osteosarcoma patients. The main goal of our study is to determine the mechanism(s) through which UHRF1 overexpression in osteosarcoma contributes to tumor progression and determine how RB influences its activity. We are trying to understand the function of UHRF1 in osteosarcoma formation and metastasis and further investigate its potential as a therapeutic target.
Identification of the UHRF1 functional domain critical for tumorigenesis
UHRF1 overexpression is associated with poor prognosis in many cancers. Studies from our research group found that suppression of UHRF1 in retinoblastoma and osteosarcoma leads to diminished tumor growth increased survival in mouse models. From these data, we hypothesize that UHRF1 may be used as a therapeutic target for the treatment of retinoblastoma and osteosarcoma (and perhaps other cancers). UHRF1 is a multidomain protein that acts as a key epigenetic regulator by bridging DNA methylation and chromatin modification. It recognizes and binds hemimethylated DNA at replication forks via its YDG domain and recruits DNA methyltransferase 1 (DNMT1) to ensure faithful propagation of the DNA methylation patterns through DNA replication. In addition to its role in maintenance of DNA methylation, it also plays a key role in chromatin modification through its TUDOR-like regions and PHD-type zinc fingers. Furthermore, UHRF1 also has E3 ubiquitin-protein ligase activity. Given the complexity of UHRF1, we must first identify which domain of UHRF1 is paramount for tumorigenesis in order to design a small molecule inhibitor that can interfere with the tumorigenic properties of UHRF1.
Identifying novel therapeutic targets for the treatment of small cell lung cancer
Following our promising results in studies of the role of UHRF1 in retinoblastoma and osteosarcoma, we expanded our research to include small cell lung cancer (SCLC), another cancer that presents mutations in the RB1 gene. SCLC is a very aggressive disease with 5-year survival rates of just 6%. Our observations using genetically engineered mouse models (using Adeno-Cre in Tp53/Rb1 and Tp53/Rb1/Uhrf1 mice) indicate that UHRF1 also plays a significant role in tumor formation in this disease. We are currently exploring the mechanism(s) by which UHRF1 overexpression results in enhanced tumor progression.
Harnessing the ubiquitin system as a therapeutic approach in triple negative breast cancer
Breast cancer is the most common cancer among women, claiming more than 40,000 lives in the U.S. every year. Among the different breast cancer subtypes, triple negative breast cancer (TNBC) is the most aggressive, with few treatment options and the worst prognosis for patients. The five-year survival rate for TNBC is 65% for patients with regional metastasis and only 11% for patients with distant metastatic disease. Compared to other breast cancer subtypes, TNBC more commonly affects younger patients, and is more common among African American and Hispanic women. Despite the recent advances in TNBC treatment, there is still a desperate need to better understand how and why these tumors are so aggressive in order to identify new vulnerabilities that can leveraged to develop better treatments. From studies in other cancers with similar genetic backgrounds as TNBC, the Benavente Lab identified UHRF1 as a new protein with therapeutic potential in TNBC. UHRF1 function is critical for tumor metastasis. However, there are currently no known inhibitors to target UHRF1. Independently, the Emanuele Lab, our collaborators at the University of North Carolina, identified UHRF1 as protein that is naturally tightly controlled by a cellular machinery that functions to eliminate proteins within the cell. This observation creates an opportunity, as drugs that turn off the enzymes that regulate UHRF1 disposal have already been developed. We are taking advantage of our labs complementary expertise and fortuitous overlapping interests, to join forces in an effort to exploit this natural protein-eliminating systems. We will determine if USP7 inhibitors, which take advantage of the cell natural machinery to eliminate UHRF1 from cells, can be used as a therapeutic intervention to stop the proliferation and metastasis of TNBC using both cell and animal models of disease. In this pilot project, we will determine if UHRF1 is critical for metastasis in TNBC and follow up with studies establishing the feasibility of exploiting the protein-eliminating pathway to decrease UHRF1 levels in TNBC cells. At the same time, we will determine the effects of these drugs on normal cells, shedding light on the overall safety of a potential treatment.