Iron homeostasis governs erythroid phenotype in polycythemia vera.

Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

Posttranslational Modification as a Critical Determinant of Cytoplasmic Innate Immune Recognition.

Cell surface innate immune receptors can directly detect a variety of extracellular pathogens to which cytoplasmic innate immune sensors are rarely exposed. Instead, within the cytoplasm, the environment is rife with cellular machinery and signaling pathways that are indirectly perturbed by pathogenic microbes to activate intracellular sensors, such as pyrin, NLRP1, NLRP3, or NLRC4. Therefore, subtle changes in key intracellular processes such as phosphorylation, ubiquitination, and other pathways leading to posttranslational protein modification are key determinants of innate immune recognition in the cytoplasm. This concept is critical to establish the "guard hypothesis" whereby otherwise homeostatic pathways that keep innate immune sensors at bay are released in response to alterations in their posttranslational modification status. Originally identified in plants, evidence that a similar guardlike mechanism exists in humans has recently been identified, whereby a mutation that prevents phosphorylation of the innate immune sensor pyrin triggers a dominantly inherited autoinflammatory disease. It is also noteworthy that even when a cytoplasmic innate immune sensor has a direct ligand, such as bacterial peptidoglycan (NOD1 or NOD2), RNA (RIG-I or MDA5), or DNA (cGAS or IFI16), it can still be influenced by posttranslational modification to dramatically alter its response. Therefore, due to their existence in the cytoplasmic milieu, posttranslational modification is a key determinant of intracellular innate immune receptor functionality.

Regulation of the Cell-Intrinsic DNA Damage Response by the Innate Immune Machinery.

Maintenance of genomic integrity is crucial for cell survival. As such, elegant DNA damage response (DDR) systems have evolved to ensure proper repair of DNA double-strand breaks (DSBs) and other lesions that threaten genomic integrity. Towards this end, most therapeutic studies have focused on understanding of the canonical DNA DSB repair pathways to enhance the efficacy of DNA-damaging therapies. While these approaches have been fruitful, there has been relatively limited success to date and potential for significant normal tissue toxicity. With the advent of novel immunotherapies, there has been interest in understanding the interactions of radiation therapy with the innate and adaptive immune responses, with the ultimate goal of enhancing treatment efficacy. While a substantial body of work has demonstrated control of the immune-mediated (extrinsic) responses to DNA-damaging therapies by several innate immune pathways (e.g., cGAS-STING and RIG-I), emerging work demonstrates an underappreciated role of the innate immune machinery in directly regulating tumor cell-intrinsic/cell-autonomous responses to DNA damage.

RIPLET, and not TRIM25, is required for endogenous RIG-I-dependent antiviral responses.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

Identification of a second binding site on the TRIM25 B30.2 domain.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Dose-Volume Predictors of Radiation Pneumonitis After Thoracic Hypofractionated Radiation Therapy.

PURPOSE: Hypofractionated radiation therapy (HFRT) is a common treatment for thoracic tumors, typically delivered as 60 Gy in 15 fractions. We aimed to identify dosimetric risk factors associated with radiation pneumonitis in patients receiving HFRT at 4 Gy per fraction, focusing on lung V20, mean lung dose (MLD), and lung V5 as potential predictors of grade ≥2 pneumonitis. METHODS AND MATERIALS: All patients were treated with thoracic HFRT to 60 Gy in 15 fractions or 72 Gy in 18 fractions at a single health care system from 2013 to 2020. Tumors near critical structures (trachea, proximal tracheobronchial tree, esophagus, spinal cord, or heart) were considered central (within 2 cm), and those closer were classified as ultracentral (within 1 cm). The primary endpoint was grade ≥2 pneumonitis. Logistic regression analyses, adjusting for target size and dosimetric variables, were used to establish a dose threshold associated with <20% risk of grade ≥2 pneumonitis. RESULTS: During a median 24.3-month follow-up, 18 patients (16.8%) developed grade ≥2 radiation pneumonitis, with no significant difference between the 2 dose regimens (17.3% vs 16.3%, P = .88). Four patients (3.7%) experienced grade ≥3 pneumonitis, including 2 grade 5 cases. Patients with grade ≥2 pneumonitis had significantly higher lung V20 (mean 23.4% vs 14.5%, P < .001), MLD (mean 13.0 Gy vs 9.5 Gy, P < .001), and lung V5 (mean 49.6% vs 40.6%, P = .01). Dose thresholds for a 20% risk of grade ≥2 pneumonitis were lung V20 <17.7%, MLD <10.6 Gy, and V5 <41.3%. Multivariable analysis revealed a significant association between lung V20 and grade ≥2 pneumonitis (adjusted odds ratio, 1.48, P = .03). CONCLUSIONS: To minimize the risk of grade ≥2 radiation pneumonitis when delivering 4 Gy per fraction at either 60 Gy or 72 Gy, it is advisable to maintain lung V20<17.7%. MLD <10.6 Gy and V5<41.3% can also be considered as lower-priority constraints. However, additional validation is necessary before incorporating these constraints into clinical practice or trial planning guidelines.

Radioresistant Pulmonary Oligometastatic and Oligoprogressive Lesions From Nonlung Primaries: Impact of Histology and Dose-Fractionation on Local Control After Radiation Therapy.

Purpose: We investigated whether pulmonary metastases from historically considered radioresistant primaries would have inferior local control after radiation therapy than those from nonradioresistant nonlung primaries, and whether higher biologically effective dose assuming alpha/beta=10 (BED10) would be associated with superior local control. Methods and Materials: We identified patients treated with radiation therapy for oligometastatic or oligoprogressive pulmonary disease to 1 to 5 lung metastases from nonlung primaries in 2013 to 2020 at a single health care system. Radioresistant primary cancers included colorectal carcinoma, endometrial carcinoma, renal cell carcinoma, melanoma, and sarcoma. Nonradioresistant primary cancers included breast, bladder, esophageal, pancreas, and head and neck carcinomas. The Kaplan-Meier estimator, log-rank test, and multivariable Cox proportional hazards regression were used to compare local recurrence-free survival (LRFS), new metastasis-free survival, progression-free survival, and overall survival. Results: Among 114 patients, 73 had radioresistant primary cancers. The median total dose was 50 Gy (IQR, 50-54 Gy) and the median number of fractions was 5 (IQR, 3-5). Median follow-up time was 59.6 months. One of 41 (2.4%) patients with a nonradioresistant metastasis experienced local failure compared with 18 of 73 (24.7%) patients with radioresistant metastasis (log-rank P = .004). Among radioresistant metastases, 12 of 41 (29.2%) patients with colorectal carcinoma experienced local failure compared with 6 of 32 (18.8%) with other primaries (log-rank P = .018). BED10 ≥100 Gy was associated with decreased risk of local recurrence. On univariable analysis, BED10 ≥100 Gy (hazard ratio [HR], 0.263; 95% CI, 0.105-0.656; P = .004) was associated with higher LRFS, and colorectal primary (HR, 3.060; 95% CI, 1.204-7.777; P = .019) was associated with lower LRFS, though these were not statistically significant on multivariable analysis. Among colorectal primary patients, BED10 ≥100 Gy was associated with higher LRFS (HR, 0.266; 95% CI, 0.072-0.985; P = .047) on multivariable analysis. Conclusions: Local control after radiation therapy was encouraging for pulmonary metastases from most nonlung primaries, even for many of those classically considered to be radioresistant. Those from colorectal primaries may benefit from testing additional strategies, such as resection or systemic treatment concurrent with radiation.

STING Agonists in Head and Neck Squamous Cell Carcinoma.

ABSTRACT: Despite the development of new treatment paradigms and improved biologic understanding of head and neck squamous cell carcinoma (HNSCC), therapeutic resistance remains a substantial problem, and novel treatment approaches are needed. Stimulator of interferon genes (STING) is a critical regulator of the antitumor response through regulation of both immune-dependent and tumor-intrinsic mechanisms. As such, the STING pathway has emerged as a rational pharmacologic target leading to the development of multiple STING agonists. These compounds have impressive preclinical efficacy as single agents and with PD-1 (programmed death-1) axis agents. However, clinical evaluation in this context has yet to show substantial efficacy. In contrast to monotherapy approaches, activation of STING in combination with DNA-damaging agents has been shown to enhance the effect of these agents in preclinical models and represents a promising approach to improve outcomes in patients with HNSCC. In this review, we will discuss the preclinical and clinical data supporting the use of STING agonists and highlight potential avenues of exploration to unlock the potential of these agents in HNSCC.

Combinations of immunotherapy and radiation therapy in head and neck squamous cell carcinoma: a narrative review.

Radiation therapy and systemic therapy are the primary non-surgical treatment modalities for head and neck squamous cell carcinoma (HNSCC). Despite advances in our biologic understanding of this disease and the development of novel therapeutics, treatment resistance remains a significant problem. It has become increasingly evident that the innate and adaptive immune systems play a significant role in the modulation of anti-tumor responses to traditional cancer-directed therapies. By inducing DNA damage and cell death, radiation therapy appears to activate both innate and adaptive immune responses. Immunotherapies targeting programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) also have yielded promising results, particularly in the recurrent/metastatic setting. In this review, we will discuss the rationale for combining radiotherapy with immunotherapy to harness the immunomodulatory effects of radiation therapy on HNSCC, as well as biomarkers for immune response. We will also review recent preclinical and clinical data exploring these combinations in various contexts, including recurrent/metastatic and locally advanced disease. Among those with locally advanced HNSCC, we will discuss clinical trials employing immunotherapy either concurrently with radiation therapy or as maintenance following chemoradiation in both the definitive and postoperative settings, with or without the use of cisplatin-based or non-cisplatin-based chemotherapy.

The translocon-associated protein (TRAP) complex regulates quality control of N-linked glycosylation during ER stress.

Asparagine (N)-linked glycosylation is required for endoplasmic reticulum (ER) homeostasis, but how this co- and posttranslational modification is maintained during ER stress is unknown. Here, we introduce a fluorescence-based strategy to detect aberrant N-glycosylation in individual cells and identify a regulatory role for the heterotetrameric translocon-associated protein (TRAP) complex. Unexpectedly, cells with knockout of SSR3 or SSR4 subunits restore N-glycosylation over time concurrent with a diminished ER stress transcriptional signature. Activation of ER stress or silencing of the ER chaperone BiP exacerbates or rescues the glycosylation defects, respectively, indicating that SSR3 and SSR4 enable N-glycosylation during ER stress. Protein levels of the SSR3 subunit are ER stress and UBE2J1 dependent, revealing a mechanism that coordinates upstream N-glycosylation proficiency with downstream ER-associated degradation and proteostasis. The fidelity of N-glycosylation is not static in both nontransformed and tumor cells, and the TRAP complex regulates ER glycoprotein quality control under conditions of stress.