Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

Mediator structure and conformation change.

Mediator is a universal adaptor for transcription control. It serves as an interface between gene-specific activator or repressor proteins and the general RNA polymerase II (pol II) transcription machinery. Previous structural studies revealed a relatively small part of Mediator and none of the gene activator-binding regions. We have determined the cryo-EM structure of the Mediator at near-atomic resolution. The structure reveals almost all amino acid residues in ordered regions, including the major targets of activator proteins, the Tail module, and the Med1 subunit of the Middle module. Comparison of Mediator structures with and without pol II reveals conformational changes that propagate across the entire Mediator, from Head to Tail, coupling activator- and pol II-interacting regions.

Lock and key to transcription: σ-DNA interaction.

How does RNA polymerase recognize a promoter in duplex DNA? How are the DNA strands pried apart to enable RNA synthesis? A crystal structure by Feklistov and Darst unexpectedly reveals that these two processes are interconnected.

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion.

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.

High-specific-activity 131iodine-metaiodobenzylguanidine for therapy of unresectable pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPG) are rare cancers arising from the adrenal medulla (pheochromocytoma) or autonomic ganglia (paraganglioma). They have highly variable biological behavior. Most PPG express high-affinity norepinephrine transporters, allowing active uptake of the norepinephrine analog, 131iodine-metaiodobenzylguanidine (131I-MIBG). Low-specific-activity forms of 131I-MIBG have been used since 1983 for therapy of PPG. High-specific-activity 131I-MIBG therapy improves hypertension management, induces partial radiological response or stable disease, decreases biochemical markers of disease activity and is well tolerated by patients. This drug, approved in the USA in July 2018, is the first approved agent for patients with unresectable, locally advanced or metastatic PPG and imaging evidence of metaiodobenzylguanidine uptake, who require systemic anticancer therapy.

Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera.

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Šfrom a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Šlevel to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.

Phase 3 Trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors.

BACKGROUND: Patients with advanced midgut neuroendocrine tumors who have had disease progression during first-line somatostatin analogue therapy have limited therapeutic options. This randomized, controlled trial evaluated the efficacy and safety of lutetium-177 (177Lu)-Dotatate in patients with advanced, progressive, somatostatin-receptor-positive midgut neuroendocrine tumors. METHODS: We randomly assigned 229 patients who had well-differentiated, metastatic midgut neuroendocrine tumors to receive either 177Lu-Dotatate (116 patients) at a dose of 7.4 GBq every 8 weeks (four intravenous infusions, plus best supportive care including octreotide long-acting repeatable [LAR] administered intramuscularly at a dose of 30 mg) (177Lu-Dotatate group) or octreotide LAR alone (113 patients) administered intramuscularly at a dose of 60 mg every 4 weeks (control group). The primary end point was progression-free survival. Secondary end points included the objective response rate, overall survival, safety, and the side-effect profile. The final analysis of overall survival will be conducted in the future as specified in the protocol; a prespecified interim analysis of overall survival was conducted and is reported here. RESULTS: At the data-cutoff date for the primary analysis, the estimated rate of progression-free survival at month 20 was 65.2% (95% confidence interval [CI], 50.0 to 76.8) in the 177Lu-Dotatate group and 10.8% (95% CI, 3.5 to 23.0) in the control group. The response rate was 18% in the 177Lu-Dotatate group versus 3% in the control group (P<0.001). In the planned interim analysis of overall survival, 14 deaths occurred in the 177Lu-Dotatate group and 26 in the control group (P=0.004). Grade 3 or 4 neutropenia, thrombocytopenia, and lymphopenia occurred in 1%, 2%, and 9%, respectively, of patients in the 177Lu-Dotatate group as compared with no patients in the control group, with no evidence of renal toxic effects during the observed time frame. CONCLUSIONS: Treatment with 177Lu-Dotatate resulted in markedly longer progression-free survival and a significantly higher response rate than high-dose octreotide LAR among patients with advanced midgut neuroendocrine tumors. Preliminary evidence of an overall survival benefit was seen in an interim analysis; confirmation will be required in the planned final analysis. Clinically significant myelosuppression occurred in less than 10% of patients in the 177Lu-Dotatate group. (Funded by Advanced Accelerator Applications; NETTER-1 ClinicalTrials.gov number, NCT01578239 ; EudraCT number 2011-005049-11 .).

Impact of liver tumour burden, alkaline phosphatase elevation, and target lesion size on treatment outcomes with 177Lu-Dotatate: an analysis of the NETTER-1 study.

PURPOSE: To assess the impact of baseline liver tumour burden, alkaline phosphatase (ALP) elevation, and target lesion size on treatment outcomes with 177Lu-Dotatate. METHODS: In the phase 3 NETTER-1 trial, patients with advanced, progressive midgut neuroendocrine tumours (NET) were randomised to 177Lu-Dotatate (every 8 weeks, four cycles) plus octreotide long-acting release (LAR) or to octreotide LAR 60 mg. Primary endpoint was progression-free survival (PFS). Analyses of PFS by baseline factors, including liver tumour burden, ALP elevation, and target lesion size, were performed using Kaplan-Meier estimates; hazard ratios (HRs) with corresponding 95% CIs were estimated using Cox regression. RESULTS: Significantly prolonged median PFS occurred with 177Lu-Dotatate versus octreotide LAR 60 mg in patients with low (< 25%), moderate (25-50%), and high (> 50%) liver tumour burden (HR 0.187, 0.216, 0.145), and normal or elevated ALP (HR 0.153, 0.177), and in the presence or absence of a large target lesion (diameter > 30 mm; HR, 0.213, 0.063). Within the 177Lu-Dotatate arm, no significant difference in PFS was observed amongst patients with low/moderate/high liver tumour burden (P = 0.7225) or with normal/elevated baseline ALP (P = 0.3532), but absence of a large target lesion was associated with improved PFS (P = 0.0222). Grade 3 and 4 liver function abnormalities were rare and did not appear to be associated with high baseline liver tumour burden. CONCLUSIONS: 177Lu-Dotatate demonstrated significant prolongation in PFS versus high-dose octreotide LAR in patients with advanced, progressive midgut NET, regardless of baseline liver tumour burden, elevated ALP, or the presence of a large target lesion. Clinicaltrials.gov : NCT01578239, EudraCT: 2011-005049-11.

Structure of an RNA polymerase II preinitiation complex.

The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription.

Polymorphic HLA-C Receptors Balance the Functional Characteristics of KIR Haplotypes.

The human killer cell Ig-like receptor (KIR) locus comprises two groups of KIR haplotypes, termed A and B. These are present in all human populations but with different relative frequencies, suggesting they have different functional properties that underlie their balancing selection. We studied the genomic organization and functional properties of the alleles of the inhibitory and activating HLA-C receptors encoded by KIR haplotypes. Because every HLA-C allotype functions as a ligand for KIR, the interactions between KIR and HLA-C dominate the HLA class I-mediated regulation of human NK cells. The C2 epitope is recognized by inhibitory KIR2DL1 and activating KIR2DS1, whereas the C1 epitope is recognized by inhibitory KIR2DL2 and KIR2DL3. This study shows that the KIR2DL1, KIR2DS1, and KIR2DL2/3 alleles form distinctive phylogenetic clades that associate with specific KIR haplotypes. KIR A haplotypes are characterized by KIR2DL1 alleles that encode strong inhibitory C2 receptors and KIR2DL3 alleles encoding weak inhibitory C1 receptors. In striking contrast, KIR B haplotypes are characterized by KIR2DL1 alleles that encode weak inhibitory C2 receptors and KIR2DL2 alleles encoding strong inhibitory C1 receptors. The wide-ranging properties of KIR allotypes arise from substitutions throughout the KIR molecule. Such substitutions can influence cell surface expression, as well as the avidity and specificity for HLA-C ligands. Consistent with the crucial role of inhibitory HLA-C receptors in self-recognition, as well as NK cell education and response, most KIR haplotypes have both a functional C1 and C2 receptor, despite the considerable variation that occurs in ligand recognition and surface expression.

Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry.

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II.

The X-ray crystal structure of the Head module, one-third of the Mediator of transcriptional regulation, has been determined as a complex with the C-terminal domain (CTD) of RNA polymerase II. The structure reveals multiple points of interaction with an extended conformation of the CTD; it suggests a basis for regulation by phosphorylation of the CTD. Biochemical studies show a requirement for Mediator-CTD interaction for transcription.

Subunit architecture of general transcription factor TFIIH.

Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain.

RNA polymerase II transcription: structure and mechanism.

A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

Peptide Receptor Radionuclide Therapy Is Effective for Clinical Control of Symptomatic Metastatic Insulinoma: A Long-Term Retrospective Analysis.

Metastatic insulinoma is a rare malignant neuroendocrine tumor characterized by inappropriate insulin secretion, resulting in life-threatening hypoglycemia, which is often difficult to treat. There is currently very limited information about the efficacy of peptide receptor radionuclide therapy (PRRT) for clinical control of hypoglycemia. The aim of this long-term retrospective study was to evaluate the therapeutic efficacy of PRRT for improving hypoglycemia, to evaluate the change of medication after PRRT, and to calculate progression-free survival (PFS) and overall survival (OS). Methods: Inclusion criteria were histologically proven somatostatin receptor-positive metastatic malignant insulinoma and at least 2 cycles of [90Y]Y-DOTATOC or [177Lu]Lu-DOTATOC therapy from early 2000 to early 2022. A semiquantitative scoring system was used to quantify the severity and frequency of hypoglycemic episodes under background antihypoglycemic therapy (somatostatin analog, diazoxide, everolimus, corticosteroids): score 0, no hypoglycemic episodes; score 1, hypoglycemic events requiring additional conservative treatment with optimization of nutrition; score 2, severe hypoglycemia necessitating hospitalization and combined medication or history of hypoglycemic coma. Hypoglycemic score before and after PRRT was analyzed. Time of benefit was defined as a time range of fewer hypoglycemic episodes in the observation period than at baseline. Information on antihypoglycemic medication before and after therapy, PFS, and OS was recorded. Results: Twenty-six of 32 patients with a total of 106 [90Y]Y-DOTATOC/[177Lu]Lu-DOTATOC cycles were included. The average observation period was 21.5 mo (range, 2.3-107.4 mo). Before therapy, 81% (n = 21) of the patients had a hypoglycemia score of 2 and 19% (n = 5) had a score of 1. After PRRT, 81% of patients (n = 21) had a decreased score, and the remaining 5 patients showed a stable situation. There was temporary worsening of hypoglycemia just after injection of [90Y]Y-DOTATOC/[177Lu]Lu-DOTATOC in 19% of patients. The average time of benefit in the observation period was 17.2 mo (range, 0-70.2 mo). Antihypoglycemic medication reduction was achieved in 58% (n = 15) of patients. The median OS and PFS after the start of PRRT were 19.7 mo (95% CI, 6.5-32.9 mo) and 11.7 mo (95% CI, 4.9-18.5 mo), respectively. Conclusion: To our knowledge, our study included the largest cohort of patients with malignant insulinoma to be evaluated. Long-lasting symptom control and reduction of antihypoglycemic medications were shown in most patients after late-line PRRT.

Efficacy and Safety of Esmethadone (REL-1017) in Patients With Major Depressive Disorder and Inadequate Response to Standard Antidepressants: A Phase 3 Randomized Controlled Trial.

Objective: To test esmethadone (REL-1017) as adjunctive treatment in patients with major depressive disorder (MDD) and inadequate response to standard antidepressants.Methods: In this phase 3, double-blind, placebo-controlled trial, outpatients with MDD (DSM-5) were randomized to daily oral esmethadone (75 mg on day 1, followed by 25 mg daily on days 2 through 28) or placebo between December 2020 and December 2022. The primary efficacy measure was change from baseline (CFB) to day 28 in the Montgomery-Asberg Depression Rating Scale (MADRS) score. The intent-to-treat (ITT) population included all randomized participants. The per-protocol (PP) population included completers without major protocol deviations impacting assessment. Post hoc analyses included participants with severe depression (baseline MADRS score ≥35).Results: For the ITT analysis (n = 227), mean CFB was 15.1 (SD 11.3) for esmethadone (n = 113) and 12.9 (SD 10.4) for placebo (n = 114), with a mean difference (MD) of 2.3, which was not statistically significant (P = .154; Cohen effect size [ES] = 0.21). Remission rates were 22.1% and 13.2% (P = .076), and response rates were 39.8% and 27.2% (P = .044) with esmethadone and placebo, respectively. For the PP analysis (n = 198), mean CFB was 15.6 (SD 11.2) for esmethadone (n = 101) and 12.5 (SD 9.9) for placebo (n = 97), with an MD of 3.1 (P = .051; ES =0.29). In post hoc analyses of patients with baseline MADRS ≥35 in the ITT population (n = 112), MD was 6.9; P = .0059; ES = 0.57, and for the PP population (n = 98), MD was 7.9; P = .0015; ES = 0.69. Adverse events (AEs) were predominantly mild or moderate and transient, with no significant differences between groups.Conclusions: The primary end point was not met. Esmethadone showed stronger efficacy in PP than in ITT analyses, with the discrepancy not attributable to AEs impacting treatment adherence. Significant efficacy occurred in post hoc analyses of patients with severe depression. Esmethadone was well tolerated, consistent with prior studies.Trial Registration: ClinicalTrials.gov identifier: NCT04688164.

AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery.

AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference Fourier maps.

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription.

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the beta-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity.

Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells.

On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.

Consensus report of the 2021 National Cancer Institute neuroendocrine tumor clinical trials planning meeting.

Important progress has been made over the last decade in the classification, imaging, and treatment of neuroendocrine neoplasm (NENs), with several new agents approved for use. Although the treatment options available for patients with well-differentiated neuroendocrine tumors (NETs) have greatly expanded, the rapidly changing landscape has presented several unanswered questions about how best to optimize, sequence, and individualize therapy. Perhaps the most important development over the last decade has been the approval of 177Lu-DOTATATE for treatment of gastroenteropancreatic-NETs, raising questions around optimal sequencing of peptide receptor radionuclide therapy (PRRT) relative to other therapeutic options, the role of re-treatment with PRRT, and whether PRRT can be further optimized through use of dosimetry among other approaches. The NET Task Force of the National Cancer Institute GI Steering Committee convened a clinical trial planning meeting in 2021 with multidisciplinary experts from academia, the federal government, industry, and patient advocates to develop NET clinical trials in the era of PRRT. Key clinical trial recommendations for development included 1) PRRT re-treatment, 2) PRRT and immunotherapy combinations, 3) PRRT and DNA damage repair inhibitor combinations, 4) treatment for liver-dominant disease, 5) treatment for PRRT-resistant disease, and 6) dosimetry-modified PRRT.