Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.

Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.

AAA ATPase protein-protein interactions as therapeutic targets in cancer.

AAA ATPases are a conserved group of enzymes that couple ATP hydrolysis to diverse activities critical for cellular homeostasis by targeted protein-protein interactions. Some of these interactions are potential therapeutic targets because of their role in cancers which rely on increased AAA ATPase activities for maintenance of genomic stability. Two well-characterized members of this family are p97/VCP and RUVBL ATPases where there is a growing understanding of their structure and function, as well as an emerging landscape of selective inhibitors. Here we highlight recent progress in this field, with particular emphasis on structural advances enabled by cryo-electron microscopy (cryo-EM).

Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.

FDA Approval Summary: Futibatinib for Unresectable Advanced or Metastatic, Chemotherapy Refractory Intrahepatic Cholangiocarcinoma with FGFR2 Fusions or Other Rearrangements.

On September 30, 2022, the FDA granted accelerated approval to futibatinib for the treatment of adult patients with previously treated, unresectable, locally advanced or metastatic intrahepatic cholangiocarcinoma (iCCA) with FGFR2 fusions or other rearrangements. Approval was based on Study TAS-120-101, a multicenter open-label, single-arm trial. Patients received futibatinib 20-mg orally once daily. The major efficacy outcome measures were overall response rate (ORR) and duration of response (DoR) as determined by an independent review committee (IRC) according to RECIST v1.1. ORR was 42% (95% confidence interval, 32%-52%). Median DoR was 9.7 months. Adverse reactions occurring in ≥30% patients were nail toxicity, musculoskeletal pain, constipation, diarrhea, fatigue, dry mouth, alopecia, stomatitis, and abdominal pain. The most common laboratory abnormalities (≥50%) were increased phosphate, increased creatinine, decreased hemoglobin, and increased glucose. Ocular toxicity (including dry eye, keratitis, and retinal epithelial detachment) and hyperphosphatemia are important risks of futibatinib, which are listed under Warnings and Precautions. This article summarizes the FDA's thought process and data supporting the approval of futibatinib.

Allosteric modulation of ryanodine receptor RyR1 by nucleotide derivatives.

The coordinated release of Ca2+ from the sarcoplasmic reticulum (SR) is critical for excitation-contraction coupling. This release is facilitated by ryanodine receptors (RyRs) that are embedded in the SR membrane. In skeletal muscle, activity of RyR1 is regulated by metabolites such as ATP, which upon binding increase channel open probability (Po). To obtain structural insights into the mechanism of RyR1 priming by ATP, we determined several cryo-EM structures of RyR1 bound individually to ATP-γ-S, ADP, AMP, adenosine, adenine, and cAMP. We demonstrate that adenine and adenosine bind RyR1, but AMP is the smallest ATP derivative capable of inducing long-range (>170 Å) structural rearrangements associated with channel activation, establishing a structural basis for key binding site interactions that are the threshold for triggering quaternary structural changes. Our finding that cAMP also induces these structural changes and results in increased channel opening suggests its potential role as an endogenous modulator of RyR1 conductance.

FDA Approval Summary: Amivantamab for the Treatment of Patients with Non-Small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations.

The FDA granted accelerated approval for amivantamab-vmjw (hereafter referred to as amivantamab), a bispecific antibody directed against EGFR and mesenchymal-epithelial transition receptor, on May 21, 2021, for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations whose disease has progressed on or after platinum-based chemotherapy. Approval was based on results of an ongoing, multicenter, nonrandomized, open-label, multicohort clinical trial (CHRYSALIS, NCT02609776), demonstrating a substantial overall response rate (ORR) and durable responses, with an ORR of 40% [95% confidence interval (CI): 29-51] and a median response duration of 11.1 months (95% CI: 6.9-not evaluable). Guardant360 CDx was contemporaneously approved as a companion diagnostic for this indication to identify EGFR exon 20 insertion mutations in plasma specimens. The most notable safety finding was the high incidence (66%) of infusion-related reactions, which is addressed in both the Dosage and Administration and Warnings and Precautions sections of the product label. Other common adverse reactions (occurring in ≥20% of patients) were rash, paronychia, musculoskeletal pain, dyspnea, nausea and vomiting, fatigue, edema, stomatitis, cough, and constipation. The approval of amivantamab was the first approval of a targeted therapy for patients with advanced NSCLC harboring EGFR exon 20 insertion mutations.

FDA Approval Summary: Lutetium Lu 177 Vipivotide Tetraxetan for Patients with Metastatic Castration-Resistant Prostate Cancer.

On March 23, 2022, the FDA approved Pluvicto (lutetium Lu 177 vipivotide tetraxetan, also known as 177Lu-PSMA-617) for the treatment of adult patients with prostate-specific membrane antigen (PSMA)-positive metastatic castration-resistant prostate cancer (mCRPC) who have been treated with androgen receptor pathway inhibition and taxane-based chemotherapy. The recommended 177Lu-PSMA-617 dose is 7.4 gigabecquerels (GBq; 200 mCi) intravenously every 6 weeks for up to six doses, or until disease progression or unacceptable toxicity. The FDA granted traditional approval based on VISION (NCT03511664), which was a randomized (2:1), multicenter, open-label trial that assessed the efficacy and safety of 177Lu-PSMA-617 plus best standard of care (BSoC; n = 551) or BSoC alone (n = 280) in men with progressive, PSMA-positive mCRPC. Patients were required to have received ≥1 androgen receptor pathway inhibitor, and one or two prior taxane-based chemotherapy regimens. There was a statistically significant and clinically meaningful improvement in overall survival (OS), with a median OS of 15.3 months in the 177Lu-PSMA-617 plus BSoC arm and 11.3 months in the BSoC arm, respectively (HR: 0.62; 95% confidence interval: 0.52-0.74; P < 0.001). The most common adverse reactions (≥20%) occurring at a higher incidence in patients receiving 177Lu-PSMA-617 were fatigue, dry mouth, nausea, anemia, decreased appetite, and constipation. The most common laboratory abnormalities that worsened from baseline in ≥30% of patients receiving 177Lu-PSMA-617 were decreased lymphocytes, decreased hemoglobin, decreased leukocytes, decreased platelets, decreased calcium, and decreased sodium. This article summarizes the FDA review of data supporting traditional approval of 177Lu-PSMA-617 for this indication.

FDA Approval Summary: Decitabine and Cedazuridine Tablets for Myelodysplastic Syndromes.

On July 7, 2020, the Food and Drug Administration approved Inqovi (Otsuka Pharmaceutical Co.), an oral fixed-dose combination tablet comprising 35 mg decitabine, a hypomethylating agent, and 100 mg cedazuridine, a cytidine deaminase inhibitor (abbreviated DEC-C) for treatment of adult patients with myelodysplastic syndromes (MDS). Evidence of effectiveness of DEC-C was established in phase III ASTX727-02 (N = 133) in adults with MDS. The study involved a two-sequence crossover comparing DEC-C and intravenous (IV) decitabine 20 mg/m2 once daily for the first 5 days of each 28-day cycle in the first 2 cycles. From cycle 3 onward, patients received DEC-C. Five-day cumulative area under the curve (5-d AUC) of decitabine for DEC-C was similar to that of IV decitabine, with geometric mean ratio 0.99 (90% confidence interval: 0.93-1.06). Clinical benefit was supported by study ASTX727-02 and the similarly designed phase II study ASTX727-01-B (n = 80), with complete remission (CR) of 21% and 18% and median duration of CR 7.5 and 8.7 months, respectively. Adverse reactions were consistent with IV decitabine. Postmarketing assessments were issued to address the effect of cedazuridine on QT prolongation, food effect, moderate and severe hepatic impairment, and severe renal impairment on the pharmacokinetics and safety of DEC-C.

A molecular mechanism for the generation of ligand-dependent differential outputs by the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

AAA+ ATPase p97/VCP mutants and inhibitor binding disrupt inter-domain coupling and subsequent allosteric activation.

The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), (2) the interprotomer interactions (p97A232E), or (3) the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function.

New insights into Raf regulation from structural analyses.

BRAF is a highly regulated protein kinase that controls cell fate in animal cells. Recent structural analyses have revealed how active and inactive forms of BRAF bind to dimers of the scaffold protein 14-3-3. Inactive BRAF binds to 14-3-3 as a monomer and is held in an inactive conformation by interactions with ATP and the substrate kinase MEK, a striking example of enzyme inhibition by substrate binding. A change in the phosphorylation state of BRAF shifts the stoichiometry of the BRAF:14-3-3 complex from 1:2 to 2:2, resulting in stabilization of the active dimeric form of the kinase. These new findings uncover unexpected features of the regulatory mechanisms underlying Raf biology and help explain the paradoxical activation of Raf by small-molecule inhibitors.

Visualizing Cancer.

Imaging has had a profound impact on our ability to understand and treat cancer. We invited some experts to discuss imaging approaches that can be used in various aspects of cancer research, from investigating the complexity and diversity of cancer cells and their environments to guiding clinical decision-making.

FDA Approval Summary: Ruxolitinib for Treatment of Steroid-Refractory Acute Graft-Versus-Host Disease.

On May 24, 2019, the Food and Drug Administration approved ruxolitinib for steroid-refractory acute graft-versus-host disease (SR-aGVHD) in adult and pediatric patients 12 years and older. Approval was based on Study INCB 18424-271 (REACH-1; NCT02953678), an open-label, single-arm, multicenter trial that included 49 patients with grades 2-4 SR-aGVHD occurring after allogeneic hematopoietic stem cell transplantation. Ruxolitinib was administered at 5 mg twice daily, with dose increases to 10 mg twice daily permitted after 3 days in the absence of toxicity. The Day-28 overall response rate was 57.1% (95% confidence interval [CI]: 42.2-71.2). The median duration of response was 0.5 months (95% CI: 0.3-2.7), and the median time from Day-28 response to either death or need for new therapy for acute GVHD was 5.7 months (95% CI: 2.2 to not estimable). Common adverse reactions included anemia, thrombocytopenia, neutropenia, infections, edema, bleeding, and elevated transaminases. Ruxolitinib is the first drug approved for treatment of SR-aGVHD. IMPLICATIONS FOR PRACTICE: Ruxolitinib is the first Food and Drug Administration-approved treatment for steroid-refractory acute graft-versus-host disease in adult and pediatric patients 12 years and older. Its approval provides a treatment option for the 60% of those patients who do not respond to steroid therapy.

The National Cryo-EM Facility at Frederick National Laboratory: Operational Procedures, Methods and Outputs.

The National Cryo-Electron Microscopy Facility (NCEF) at the National Cancer Institute was launched in May of 2017 to provide free and rapid access to high resolution cryo-EM data collection to United States researchers working on problems of broad general relevance to cancer biology. The decision about suitability of projects for data collection is made on a first-come, first-served basis by NCEF staff, and is based solely on the quality of the screening images provided without need for a scientific proposal. Here, we provide an overview of the operation of the facility, typical data collection procedures and some insights that have emerged from the structures reported from data collected at the facility.

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases.

Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical "active" conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.

Structure of the primed state of the ATPase domain of chromatin remodeling factor ISWI bound to the nucleosome.

ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at ∼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI.

FDA Approval Summary: Glasdegib for Newly Diagnosed Acute Myeloid Leukemia.

On November 21, 2018, the FDA approved glasdegib (Daurismo; Pfizer), a small-molecule Hedgehog inhibitor, in combination with low-dose cytarabine (LDAC) for treatment of newly diagnosed acute myeloid leukemia (AML) in adults ≥ 75 years or with comorbidities that preclude use of intensive induction chemotherapy. Evidence of clinical benefit came from Study BRIGHT AML 1003, a randomized trial comparing glasdegib+LDAC with LDAC alone for treatment of newly diagnosed AML in 115 patients either ≥ 75 years old or ≥ 55 years old with preexisting comorbidities. Efficacy was established by improved overall survival (OS) with the combination compared with LDAC alone (HR, 0.46; 95% confidence interval, 0.30-0.71; one-sided stratified log-rank P = 0.0002). Median OS was 8.3 months with the combination and 4.3 months with LDAC alone. Common adverse reactions included cytopenias, fatigue, hemorrhage, febrile neutropenia, musculoskeletal pain, nausea, edema, dyspnea, decreased appetite, dysgeusia, mucositis, constipation, and rash. The label includes a boxed warning for embryo-fetal toxicity and a warning for QT interval prolongation. There is a limitation of use for patients with moderate-to-severe hepatic and severe renal impairment; trials studying glasdegib in these patient populations are required as a condition of this approval.See related commentary by Fathi, p. 6015.

FDA Approval Summary: Midostaurin for the Treatment of Advanced Systemic Mastocytosis.

In April 2017, the U.S. Food and Drug Administration granted regular approval to midostaurin for the treatment of adult patients with aggressive systemic mastocytosis (ASM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), or mast cell leukemia (MCL). Approval was based on results from CPKC412D2201, a single-arm trial of midostaurin (100 mg orally twice daily) in previously treated or untreated patients. For the patients with ASM and SM-AHN, efficacy was established on the basis of confirmed complete remission (CR) plus incomplete remission (ICR) by modified Valent criteria with six cycles of midostaurin. There were no CRs reported; ICR was achieved by 6 of 16 patients (38%; 95% confidence interval [CI]: 15%-65%) with ASM and by 9 of 57 patients (16%; 95% CI: 7%-28%) with SM-AHN. Within the follow-up period, the median duration of response was not reached for the patients with ASM (range, 12.1+ to 36.8+ months) or with SM-AHN (range, 6.6+ to 52.1+ months). For the patients with MCL, efficacy was established on the basis of confirmed CR using modified 2013 International Working Group-Myeloproliferative Neoplasms Research and Treatment-European Competence Network on Mastocytosis criteria. Of 21 patients with MCL, 1 (5%) achieved a CR. Of 142 patients with SM evaluated for safety, 56% had dose modifications for toxicity, and 21% discontinued treatment due to a toxicity. Over 50% reported nausea, vomiting, or diarrhea, and ≥30% reported edema, musculoskeletal pain, fatigue, abdominal pain, or upper respiratory tract infection. New or worsening grade ≥3 lymphopenia, anemia, thrombocytopenia, or neutropenia developed in ≥20%. Although midostaurin is an active drug for treatment of advanced SM, it is not clear that the optimal dose has been identified. IMPLICATIONS FOR PRACTICE: Midostaurin is the only U.S. Food and Drug Administration-approved therapy for patients with systemic mastocytosis with associated hematological neoplasm and mast cell leukemia and is the only therapy approved for patients with aggressive systemic mastocytosis regardless of KIT D816V mutation status. Based on response rate and duration, midostaurin has meaningful clinical activity in these rare, life-threatening diseases.