Review: Precision medicine and driver mutations: Computational methods, functional assays and conformational principles for interpreting cancer drivers

Abstract

At the root of the so-called precision medicine or precision oncology, which is our focus here, is the hypothesis that cancer treatment would be considerably better if therapies were guided by a tumor's genomic alterations. This hypothesis has sparked major initiatives focusing on whole-genome and/or exome sequencing, creation of large databases, and developing tools for their statistical analyses-all aspiring to identify actionable alterations, and thus molecular targets, in a patient. At the center of the massive amount of collected sequence data is their interpretations that largely rest on statistical analysis and phenotypic observations. Statistics is vital, because it guides identification of cancer-driving alterations. However, statistics of mutations do not identify a change in protein conformation; therefore, it may not define sufficiently accurate actionable mutations, neglecting those that are rare. Among the many thematic overviews of precision oncology, this review innovates by further comprehensively including precision pharmacology, and within this framework, articulating its protein structural landscape and consequences to cellular signaling pathways. It provides the underlying physicochemical basis, thereby also opening the door to a broader community.

Fig 1. Examples of precision pharmacology.

(A) Crystal structure of HER2 extracellular domain in complex with trastuzumab (Herceptin) Fab (PDB code: 1N8Z). Trastuzumab is a monoclonal antibody targeting HER2 receptor positive for metastatic breast cancer. (B) Molecular structures of mercaptopurine (Purinethol), gefitinib (Iressa), and erlotinib (Tarceva). Mercaptopurine is specifically used to treat acute lymphocytic leukemia. Gefitinib is an EGFR inhibitor used to treat breast, lung, and other cancers. Erlotinib is also an EGFR inhibitor used to treat NSCLC, pancreatic cancer, and several other types of cancer. In the structure, C, N, and O atoms are denoted as white, blue, and red spheres, respectively. Hydrogen atom is represented as an edge of stick. Other heavy atoms, S, Cl, and Fe atoms, are marked on the sphere. Molecular topologies with the coordinates are generated by using Avogadro software [412]. (C) Crystal structures of EGFR extracellular domain in complex with cetuximab (Erbitux) Fab (PDB code: 1YY9) and (D) EGFR receptor domain 3 in complex with panitumumab (Vecitibix) Fab (PDB code: 5SX4). Both cetuximab and panitumumab are monoclonal antibodies targeting EGFR. EGFR, epidermal growth factor receptor; Fab, antigen-binding fragment; HER2, human epidermal growth factor receptor 2; NSCLC, non-small cell lung cancer; PDB, protein data bank.

Fig 2. Examples of chemotherapy drugs.

Molecular structures of (A) imatinib, (B) vemurafenib, and (C) dabrafenib. Imatinib is used for chronic myelogenous leukemia and acute lymphocytic leukemia. Both vemurafenib and dabrafenib are drugs for the treatment of melanoma targeting the B-Raf^V600E mutant. In the structure, C, N, and O atoms are denoted as white, blue, and red spheres, respectively. Hydrogen atom is represented as an edge of stick. Other heavy atoms, S, Cl, and Fe atoms, are marked on the sphere. Molecular topologies with the coordinates are generated by using Avogadro software [412].

Fig 3. A flowchart representing the comprehensive framework to precision oncology.

Here, our focus is the process of the interpretation of the driver mutation using the databases in cancer biology, cancer pathways, and the distribution of the ensembles of protein conformation. The next process to match the driver mutations against drug databases and known drug-target interactions is not discussed in this paper.

Fig 4. Examples of experimental strategies to explore the functional consequences of mutations altering tumorigenesis, disease progression, and drug responses.

These include (A) RPPA technology, (B) integrated proteogenomic analysis, (C) high-throughput gateway-compatible enhanced yeast two-hybrid, and (D) CRISPR-Cas9 genome editing. LC-MS/MS, liquid chromatography—tandem mass spectrometry; RPPA, reverse-phase protein assay.

Fig 5. An example shown for the dynamic landscape of the free energy surface representing the protein conformational ensembles.

Molecular events result in the population shift of the conformational ensembles, redistributing the populations of the states.

Fig 6. Kinase domain structures of protein kinases.

(A) Crystal structure of kinase domain of cAMP-dependent PKA Cα in the active state (PDB code: 4DH3). PKA is a member of the AGC kinase family. The kinase domain conformation is highly conserved among other protein kinases. Examples are shown for crystal structures of Akt1 (PDB code: 6CCY) and CAMKK2 (PDB code: 5UYJ). Akt is known as PKB and belongs to the serine/threonine-specific protein kinase family. CAMKK2 is a member of CaM kinase family. (B) Crystal structures of kinase domain of proto-oncogene tyrosine-protein kinase Src in the active (PDB code: 1YI6) and inactive (PDB code: 2SRC) states. Src is a member of the nonreceptor tyrosine kinase family. While C-spine is preserved, R-spine is significantly distorted in the inactive Src kinase domain due to outer movement of the αC-helix. AGC, kinase group AGC; C-spine, catalytic spine; CaM, Calmodulin; CAMKK2, Calcium/Calmodulin dependent protein kinase kinase 2; cAMP, cyclic AMP; PDB, protein data bank; PKA Cα, protein kinase catalytic subunit α; PKB, protein kinase B; R-spine, regulatory spine.

Fig 7. EGFR dimerization and activation.

(A) Model for ligand-induced homodimerization of EGFR. The inactive EGFR monomer forms a symmetric dimer in the inactive state. Ligand binding to the extracellular domain shifts the population to the active EGFR dimer. (B) EGF or ligand binding causes an extended conformation of the extracellular domain, promoting a conformational rearrangement through the transmembrane to juxtamembrane domains [263, 270]. This results in an asymmetric assembly of the kinase domains. In the cartoons, crystal structures of extracellular domain (PDB code: 5XWD), transmembrane domain (PDB code: 5LV6), and kinase domain (PDB code: 2GS7) were used to model the inactive monomer and symmetric dimer. The active asymmetric dimer model employs crystal structures of extracellular domain (PDB code: 3NJP), transmembrane/juxtamembrane domain (PDB code: 2M20), and kinase domain (PDB code: 2GS6). Spheres in the C-terminal tail represent tyrosine (Y) and phosphorylated tyrosine (pY) residues. The mutant kinase domain cartoon employs crystal structure of kinase domain with T790M/L858R mutant (PDB code: 5EDP). EGFR, epidermal growth factor receptor; PDB, protein data bank.

Fig 8. Ras activation and oncogenic mutations.

(A) KRas4B is activated by the son of sevenless 1 (SOS1) nucleotide exchange factor (GEF), while GAP inactivates KRas4B via the GTP → GDP hydrolysis. KRas4B oncogenic mutations at the active site Gly12, Gly13, and Gln61 aborts the hydrolysis reaction, keeping the Ras in a constitutively active GTP-bound state. (B) Of those with mutant Ras, KRas is the most highly mutated in cancer. In KRas mutation, Gly12 mutations are the most frequent. (C) The interactions of KRas4B with the anionic membrane composed of DOPC:DOPS (4:1 molar ratio). In the active GTP-bound state, the HVR is in contact with the membrane, but the catalytic domain is away, exposing the effector binding site. In contrast, autoinhibition persists in the inactive GDP-bound state with occluded catalytic domain conformation, yielding the effector binding site is inaccessible. In cartoons, the modeled KRas4B structures were adopted from our simulations [182, 285]. DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; GAP, GTPase-activating protein; GEF, Guanine nucleotide exchange factors; HVR, hyper variable region; SOS1, son of sevenless 1.

Fig 9. Akt1 domain structure.

(A) Akt1 is composed of PH (residues 5–108), kinase domain (residues 150–408), and regulatory domain (residues 409–480). The Gly-rich α-helix linker connects the PH and kinase domains. (B) Non-hotspot, passenger mutations can be functional as the driver mutation E17K. The PH domain mutants, L52R, C77F, and Q79K activate Akt1 as the E17K driver does, whereas D32Y, K39N, and P42T mutants at the interface between the PH and kinase domains reduce Akt1 in the inactive conformation. PH, pleckstrin homology.

Fig 10. A general scheme of cell-signaling pathway.

Representatives shown for the MAPK, PI3K/Akt, and Wnt pathways. In the MAPK pathway, Ras gets activated in the presence of the EGFR signal and forms nanoclusters that promote Raf dimerization. Autophosphorylated, active Raf dimer phosphorylates and activatesMEK 1/2, and subsequently phosphorylates and activates ERK 1/2, leading to cell proliferation. In the PI3K/Akt pathway, both Ras and RTK activate PI3K, recruiting it to the plasma membrane, where PI3K phosphorylates PIP₂ to produce PIP₃. The tumor suppressor PTEN can reverse the PIP₃ production. PIP₃ recruits both PDK and Akt to the plasma membrane, where PDK and mTORC2 activate Akt, and active Akt phosphorylates the mTORC1, leading to cell growth. The canonical Wnt pathway involves an accumulation of β-catenin in the cytoplasm and translocates β-catenin to the nucleus where it binds to TCF/LEF transcription factors. β-catenin upregulates c-Myc. In the noncanonical Wnt pathway, CaM-bound CaMKII interfere with the canonical β-catenin/TCF/LEF signaling. CaM, Ca²⁺-calmodulin; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MAPK, Raf/MEK/ERK; MEK, MAPK kinase; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; PDK, phosphoinositide-dependent protein kinase; PIP₂, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-bisphosphate; PTEN, phosphatase and tensin homolog; RTK, receptor tyrosine kinase; TCF/LEF, T-cell factor/lymphoid enhancing factor.

Fig 11. Crystal structures of KRas4B^G12C and covalent inhibitors.

Cartoon (left panel) and surface (middle panel) representations of the crystal structure of KRas4B^G12C-GDP in complex with covalently linked inhibitors (right panel) of (A) compound 4, (B) compound 9, and (C) compound 16 (PDB codes: 4LV6, 4LYJ, and 4M22, respectively) [116]. In the protein structures, light green, blue, and orange colors denote the P-loop, Switch I, and Switch II regions, respectively. In the compound structures, C, N, and O atoms are denoted as white, blue, and red spheres, respectively. Hydrogen atom is represented as an edge of stick. Other heavy atoms, S, Cl, and I atoms are marked on the sphere.