Project 1: Kinase Fusion Protein DNAJB1-PRKACA as a driver for fibrolamellar hepatocellular carcinoma Background and Significance: Fibrolamellar hepatocellular carcinoma (FLHCC) is a liver cancer that predominantly affects adolescents and young adults. FLHCC does not respond well to chemotherapy and other effective therapeutic options are very limited. Given the low survival rate and lack of available treatment options, there is a pressing need for new diagnostic and therapeutic approaches to FLHCC. Inhibition of the tumor driver has the best chance of offering a curative treatment for FLHCC. The results of whole genome sequencing and transcriptome sequencing of FLHCC tumors show a single, consistent genetic alteration in the FLHCC tumor cells: a deletion of 400 kB between the first exon of the heat shock protein DNAJB1 and the first exon of the catalytic subunit of cAMP dependent Protein Kinase A (PKA), PRKACA, on one copy of chromosome 19. This deletion produces a chimeric gene that leads to a chimera of the J-domain of DNAJB1 with PRKACA. This chimeric DNAJB1-PRKACA protein has now been recognized as the driver of FLHCC. Inhibition of the DNAJB1-PRKACA chimeric tumor driver offers tremendous potential to treat FLHCC. In its inactive state in cells, PKA exists as a holoenzyme composed of two PRKACA catalytic subunits and one regulatory (R) subunit homodimer. There are four R-subunit isoforms (RIa, RIb, RIIa and RIIb), which define a primary mechanism for achieving PKA signaling specificity. The fusion chimera DNAJB1-PRKACA can also bind thus be inhibited by R-subunits. RIa is the only upregulated R isoform in FLHCC cancer cells. To understand how PKA signaling might be disrupted by the DNAJB1-PRKACA chimera in FLHCC, it is essential to know the architecture, as well as the conformational dynamics, of the chimeric and wildtype (wt) holoenzymes.
Specific Aims : We are studying the structure and dynamics of the DNAJB1-PRKACA chimera and how it is regulated with the long-term goal of developing precision medicines against this fatal pediatric cancer.
The specific aims of this project are: 1A) To determine the structures of the chimeric RIa2: DNAJB1-PRKACA2 and wt RIa2: PRKACA2 holoenzymes. In parallel, we will carry out biophysical and biochemical characterizations of PRKACA, DNAJB1-PRKACA and corresponding RIa holoenzymes to establish structure-function relationship; 1B) To develop therapeutics that directly target the DNAJB1-PRKACA fusion oncoprotein. Project 2: Structure and function studies of Leucine Rich Repeat Kinase 2 (LRRK2) and its homologue LRRK1 Background and Significance: Discovered in 2004, LRRK2 is the leading genetic contributor to familial Parkinson's disease (PD) and currently one of the most promising therapeutic targets for drug design in PD. LRRK2 is a large multi-domain protein containing two putative catalytic domains: a kinase domain and a Ras of Complex (ROC) GTPase domain. The most common mutation in PD patients is LRRK2 G2019S. G2019 is present in the kinase domain and its substitution to serine increases LRRK2 kinase activity. Mutations in LRRK2 are also associated with increased risk of cancer. Interestingly, G2019S mutation carriers exhibit increased risk of hormone-related cancers, including prostate cancer in men and breast and ovarian cancers in women. My long-term goal in this project is to understand how dysfunction of LRRK2 leads to the onset of PD as well as other diseases, including cancer. However, little is known about the LRRK2 structure and even less is known about the dynamic conformational changes that are associated with LRRK2 activation. LRRK2 and its closest homologue LRRK1belong to the Tyrosine Kinase Like (TKL) family of protein kinases, and they are closely related to the RAF kinases. Similarly, little is known about LRRK1. Curiously, in mammals only LRRK2 and LRRK1 possess a domain architecture with two enzymatic activities (a GTPase and a kinase) within the same molecule. Many important mechanisms remain to be elucidated, including how the GTPase cycle and kinase cycle of LRRK2 are regulated, and how GTPase and kinase activities contribute to the overall functional output of LRRK2. The present understanding of LRRK2 is severely handicapped by the lack of structural information, as only the ROC and WD40 domain structures have been determined. There is no insight into the global relationship of the domains and the functional interactions of these domains. The major objective of this project is to break this barrier using integrative structural and biochemical approaches and to reveal the molecular mechanisms of LRRK function and pathology. This project will generate results that allow us to capture high-resolution structures as well as a range of conformational states of LRRK proteins and create new hypotheses for how to intervene with therapeutic strategies.
Specific Aims :
We aim to advance the understanding of LRRK2 and LRRK1 domain structures, LRRK conformations in different states, and how these conformations are altered by pathological PD and cancer mutations through interdisciplinary structural and biochemical approaches.
The specific aims of this project are to: 2A) To establish an efficient system to purify full-length and stable LRRK proteins; 2B) To determine the full-length LRRK2 structure; 2C) To determine the full-length LRRK1 structure; 2D) To explore the GTPase and kinase regulation cycle, dynamic conformational states of the LRRK proteins and the LRRK2 mis-regulation in PD and cancer. Project 3: Structure and functional studies of RAF kinases in the RTK/RAS/RAF signaling pathway Background and Significance: The family of RAF kinases (ARAF, BRAF and CRAF) constitute core components of the RTK-RAS-RAF-MEK-ERK signaling pathway, which plays a major role in cell growth, differentiation and survival. Deregulation of this pathway commonly occurs in cancer, frequently due to mutations in RTK, RAS or BRAF resulting in constitutive activation of the pathway. BRAF mutations are present in approximately 8% of human tumors. One of the goals of this Project is to discern how cancer-associated mutations lead to dysfunction. For example, BRAF is mutated in 50% of melanomas (50%) with the V600E mutation being the most common; as discussed below, we are studying this mutation and comparing the mutated protein to wildtype. In quiescent cells, the wt RAF kinases exist as autoinhibited monomers in cytosol and this state can be further stabilized by 14-3-3 association. Direct interaction with Ras recruits the cytosolic RAF kinases to the plasma membrane and disrupts their autoinhibited state. Ras binding also induces RAF dimer formation and activation, which is a required step to activate the downstream MEK kinase. The RAF kinases can dimerize with any of the other RAF family members. Although the factors that determine the dimerization preferences are poorly understood, BRAF/CRAF heterodimers predominate in Ras-dependent signaling. The complex structural and biochemical mechanisms of RAF kinase signaling account both for the effectiveness of RAF inhibitors as potential therapeutics and for the various mechanisms of tumor resistance to them. However, at present, the mechanistic basis for RAF monomer autoinhibition and active dimer formation is not known.
Specific Aims :
We aim to elucidate the mechanistic basis of full-length active and inactive BRAF kinase complexes by applying interdisciplinary structural and biochemical approaches.
The specific aims are: 3A) To determine the structure of a full-length autoinhibited monomeric BRAF kinase; 3B) To explore the biochemical and structural changes that occur during BRAF activation and dimer formation; 3C) To explore how the inhibited and activated states of BRAF are altered by pathological mutations such as the V600E mutant.
