Project 1:) The study of kinase fusion proteins as drivers of cancer. (i)DNAJB1-PRKACA in FLHCC FLHCC is a liver cancer that predominantly affects adolescents and young adults. Effective therapeutic options are very limited because FLHCC does not respond well to chemotherapy. 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 Protein Kinase A (PKA), PRKACA, on one copy of chromosome 19. This deletion produces a chimeric gene that leads to a chimera protein. This chimeric DNAJB1-PRKACA protein has now been recognized as the driver of FLHCC. My group studies the structure of this chimera and its regulatory mechanism with the long-term goal of developing precision medicines against this fatal pediatric cancer. The wild type (WT) PKA holoenzyme consists of two catalytic (PRKACA) subunits and a regulatory (R) subunit homodimer. There are four functionally non-redundant R-isoforms (RIa, RIb, RIIa and RIIb). RIa predominates in the fibrolamellar tumor. Additionally, inactivation of RIa is found in Carney complex disease and in the very few (1%) cases of FLHCC that do not have the DNAJB1-PRKACA fusion gene. Research objectives and progresses. A1) To characterize the structure and function of a R2:DNAJB1-PRKACA2 and WT R2:PRKACA2 holoenzymes. A2) To perform screening for inhibitors of DNAJB1-PRKACA and R2: DNAJB1-PRKACA2. I propose to perform screening on vast compound libraries for inhibitors of DNAJB1-PRKACALC and R2: DNAJB1-PRKACALC2 holoenzymes to identify putative inhibitors of either the catalytic pocket or an allosteric site. Likely binders will be tested for selective inhibitory activity via kinase assays. The identified small molecules should be able to selectively inhibit the chimera DNAJB1-PRKACA over the WT PRKACA or should selectively inhibit the activation of chimera R2: DNAJB1-PRKACA2 holoenzymes over the R2: PRKACA2 holoenzymes. In the past year, my group has performed structural and biochemical studies of DNAJB1-PRKACA and compared this protein with the wildtype WT PRKACA. My group has determined the crystal structures of the RIa2:DNAJB1-PRKACA2 and wildtype WT RIa2:PRKACA2 holoenzymes, showing different conformations with respect to other wildtype PKA holoenzymes. These biochemical and structural findings provide new strategies for therapeutic targeting. It will be of interest to elucidate how the holoenzymes communicate with their neighbors and substrates in macromolecular assemblies for achieving signaling specificity. Understanding in detail how DNAJB1-PRKACA signaling pathways drive disease will shed light on understanding its transformation to FLHCC and hopefully will lead to improved diagnosis and therapeutic treatment for this cancer. (ii) EML4-ALK Non-small cell lung cancer (NSCLC) is a major cause of death worldwide, with most of the patients being diagnosed with disease in advanced stage. This disease also has a key driver in the form of a kinase fusion protein. The fusion between echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) has been identified in a subset (7%) of NSCLCs, equivalent to over 70,000 patients diagnosed annually worldwide. EML4-ALK occurs most often in non-smokers with lung cancer and is oncogenic both in vitro and in vivo. All EML4-ALK fusions contain a coiled-coil domain within EML4 that mediates constitutive dimerization and activation of EML4-ALK. NSCLCs harboring rearrangements involving the ALK gene are sensitive to treatment with the ALK inhibitor drug crizotinib, which is an ATP analogue. However, the enormous success of traditional kinase ATP analogue inhibitors in the treatment of cancer is being rapidly overshadowed by the emergence of drug resistance. Drug-resistant mutations increase the kinase binding affinity for ATP, shifting them to the active conformation, and/or introducing new steric hindrance that interferes with inhibitor structural motifs outside the highly conserved ATP binding boundary. Overall, the traditionally designed, oversized kinase inhibitors are destined to develop many different resistance profiles in the clinic. Continuously introducing new inhibitors to overcome new evolving mutations is becoming increasingly hard to sustain. Targeting resistance-driven kinase active conformation with a small compact molecule that is completely located inside the ATP binding boundary offers tremendous potential to tackle the ever evolving mutation resistances. Research objectives. We will achieve the goal of generating novel strategies to target ALK through three research objectives. B1) Solve the structure of EML4-ALK. EML4-ALK fusion protein is an oligomer with a molecular weight over 200kD in total and is constitutively active. Our goal is to capture the active conformation of ALK using both x-ray crystallography and cryoEM approaches. B2) Explore EML4-ALK's interactions with small compact molecules that are completely located inside the ATP binding boundary. The top three EML4-ALK variants (V1, V2 and V3) will be tested for expression in different expression systems followed by purification. These purified WT and mutant proteins will be used in the apo form or with drugs for crystallization screening and cryoEM trials. In parallel, we will carry out biophysical and biochemical characterizations of the EML4-ALK to elucidate its oligomerization state, ATP and substrate binding, and activity in pathological states and further test structure-guided hypotheses by using an appropriate functional assay. We hope to combine structural and functional studies to reveal the molecular mechanism of dysfunction of ALK kinase complexes and to help develop new strategies for structure-based drug design. Project 2: Structures of Nucleosome and Nucleosome-protein complexes Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form various nucleosomes distinguished by their DNA sequences, histone variants and post-translational modifications. Despite their important functional roles, our knowledge of the structure of nucleosome with native genomic DNA is limited. Research objectives and progress:
We aim to study nucleosome and nucleosome-protein complexes with native DNA by cryo-EM with a long-term aim to understand the break and fusion mechanism of chromosomes. This project therefore relates to project 1 described above. In the past year, we have found that we can use a single chain variable fragment of an antibody (scFv), which was discovered in patients with systemic lupus erythematosus, to stabilize the Nuc-601 and obtained a Nuc-Ab complex Cryo-EM structure at 3A resolution, which is currently the highest cryoEM resolution structure of a nucleosome complex. We further demonstrate scFv enables us to determine the structure of the centromeric nucleosome with the alpha-satellite DNA by cryo-EM at atomic resolution, whereas the nucleosome completely dissociates in the absence of scFv. Our findings pave an exciting avenue for future structural studies of nucleosome or nucleosome-protein complexes with native DNA such as kinase DNA with potential breakpoints and we are poised to carry out these experiments. We now have two publications in preparation, on which I am the corresponding author.