Developing Novel Therapies for High Risk Pediatric Cancers

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The goal of this project is to identify potential therapeutic targets for PAX3-FOXO1 funsion gene driven Rhabdomyosarcoma (FP-RMS) and Neuroblastoma (NB). We employ various assays, including the Incutyte and iCELLigence System. Furthermore, we conduct CRISPR screens using genome-wide and epigenetic-focused libraries to uncover critical genetic and epigenetic factors involved in RMS pathogenesis. For the drug screening phase, we conduct extensive analyses of single agent and combination responses to a panel of over 2000 drugs. This panel, known as the Mechanism Interrogation Plate, has been developed by NCATS. The library includes FDA-approved compounds, some of which are already used in cancer therapy, as well as those currently in clinical trials (phase 1, 2, or 3. The targets or mechanisms of action are known for these compounds. Our strategy revolves around identifying the most promising targets, which will undergo further evaluation using patient-derived xenograft animal models. In collaboration with NCATS, we are also conducting targeted efforts to identify inhibitors of the PAX3-FOXO1 fusion gene, which plays a pivotal role in RMS pathogenesis. To accomplish this, we utilize a PAX3-FOXO1 activity reporter cell line that we developed in our laboratory. This reporter line utilizes the super-enhancer region within the ALK gene, cloned upstream of a minimal CMV promoter that drives a Green Fluorescent Protein (GFP)-Luciferase reporter. This setup allows us to efficiently monitor PAX3-FOXO1 activity and assess its responses to different treatments. Additionally, we have now developed a more direct and accurate approach by using assay readouts of tagged endogenous proteins. To achieve this, we decided to fuse the pro-luminescent HiBiT peptide to the endogenous PAX3-FOXO1 using CRISPR/Cas9-mediated knockin. The HiBiT peptide is a small, 11-amino acid peptide capable of producing a luminescence signal that is approximately 100-fold brighter than traditional firefly or Renilla luciferases. This high-affinity complementation with LgBiT, an 18 kDa subunit derived from the NanoLuc luciferase, enables detection with exceptional sensitivity. With the HiBiT signal readily detectable, we were able to monitor HiBiT-tagged PAX3-FOXO1 levels at the protein level upon treatment with the BRD4 inhibitors JQ1 and CPI-0610, which have demonstrated promising effects on FP-RMS xenograft growth suppression. The results from our previous studies have been quite promising, revealing that PAX3-FOXO1 reprograms the cis-regulatory epigenetic landscape by inducing de novo super-enhancers. This phenomenon occurs in collaboration with the bromodomain and extra-terminal domain protein family member BRD4, effectively locking the RMS cells in a myoblast-like state. These findings demonstrate the feasibility of using unbiased high-throughput screening approaches to identify small molecules that can effectively disrupt the PAX3-FOXO1 core regulatory circuitry. Of particular significance, we have observed that the transcriptional activity of PAX3-FOXO1 relies heavily on its physical interaction with BRD4. To this end, we utilized the BRD4 inhibitor JQ1, which effectively ablates this interaction and subsequently reduces PAX3-FOXO1 protein levels. This reduction correlates with the suppression of FP-RMS xenograft growth in mice, making it a promising avenue for potential therapeutic intervention. Beyond protein-protein interactions, we recognize that protein homeostasis, which encompasses processes such as synthesis, folding, trafficking, and degradation, plays a crucial role in cellular function. Consequently, we are exploring strategies to selectively target specific post-translational modifications that could lead to decreased stability or activity of PAX3-FOXO1, offering an attractive approach to developing novel FP-RMS therapies. Our research efforts extend beyond PAX3-FOXO1, as we recognize the significance of other potential therapeutic targets. In this regard, we have identified FGFR4 as a rational target for RMS due to its essential role in myogenic differentiation and muscle regeneration after injury. Moreover, FGFR4 is highly expressed in all RMS and has been identified as a diagnostic and prognostic biomarker. In addition to this, we have noted that approximately 10% of FN-RMS have activating mutations in FGFR4, and cells harboring these mutations are oncogene-addicted and sensitive to pharmacological inhibition by small molecules. These findings solidify FGFR4 as a key cell surface tyrosine kinase receptor for RMS biology, growth, and survival. Our focus on FGFR4 has led us to develop monoclonal antibodies and human scFv binders to target this receptor. We are diligently examining FGFR4 expression levels in normal human organs to mitigate potential organ toxicity concerns. Furthermore, we are actively pursuing the development of FGFR4, GPC2, and CD276 chimeric antigen receptors (CARs) as potential therapies for RMS and NB. For RMS, we have designed a second-generation lentiviral construct for the FGFR4 CAR that contains the CD8 transmembrane region, 41BB, CD3zeta intracellular domains, and a human EGFR extracellular domain. This design has proven effective in clinical trials, showing CAR T cell persistence in patients' peripheral blood for several months after therapy. Our anti-FGFR4 CART cells have shown promising results in vitro and in vivo. Our research endeavors also extend to the development of novel TCRs (T-cell receptors) as potential therapies for pediatric solid tumors. The successful development of potent immunotherapeutic biologics and cell-based therapies for aggressive pediatric cancers is a key objective of our ongoing efforts. In conclusion, our research efforts for NB and RMS are multidimensional and encompass various cutting-edge technologies and approaches. We aim to identify and validate promising therapeutic targets while ensuring the safety and efficacy of potential treatment options. Through extensive drug screening, molecular investigations, and collaboration with leading institutions, we strive to advance novel therapies that will make a significant impact on improving outcomes for patients with RMS and other aggressive pediatric cancers.