A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

The impact of institutional measures on optimal use of intravenous immunoglobulin.

BACKGROUND: Intravenous immunoglobulin (IVIG) shortage represents an emerging issue in transfusion medicine. Limited data are available to determine effective strategies for optimal use. The objective of this retrospective observational study was to determine the impact of institutional measures on IVIG use at a large academic center. METHODS: IVIG infusions from November 26, 2018 to September 25, 2022 were categorized according to their appropriateness (Recommended, Option of treatment, or Unrecommended), based on provincial guidelines, and separated into three phases: Reference, Transition, and Post-Implementation phases, the latter following the adoption of restrictive measures, including mandatory standardized order forms, a blood bank gatekeeping strategy, and the creation of a stewardship committee. RESULTS: A total of 5431 IVIG infusions were administered to 544 patients, accounting for 295,033 g. The most common indication categories were neurology (30.4%), immunology (29.0%), and hematology (17.4%). From Reference to Post-Implementation phase, IVIG infusions decreased from 2275 to 2000 with unrecommended indications dropping from 9.5% to 7.4% (p = 0.01), and a global reduction of 23.0% (from 131,163 g to 100,936 g of IVIG). Decrease in chronic immunomodulation accounted for 48.3% of total reduction (14,610 g of 30,227 g), whereas single-use immunomodulation, 40.5% (12,237 g of 30,227 g). Moreover, an absolute reduction of 16.9% was observed in orders exceeding the recommended doses (20.8% to 3.9%; p < 0.0001). Together, the unrecommended and excessive IVIG doses decreased from 19,975 g (15.2%) to 6670 g (6.6%). CONCLUSIONS: A global reduction in IVIG use and a preferential decrease in the unrecommended orders were observed, most likely attributable to the bundle of restrictive strategies implemented.

Following your gut: the emerging role of the gut microbiota in predicting and treating immune-related adverse events.

PURPOSE OF REVIEW: Although immune checkpoint inhibition has reshaped the therapeutic landscape leading to improved outcomes across an array of both solid and hematologic malignancies, a significant source of morbidity is caused by immune-related adverse events (irAEs) caused by these agents. RECENT FINDINGS: The gut microbiota has emerged as a biomarker of response to these agents, and more recently, also as a key determinant of development of irAEs. Emerging data have revealed that enrichment of certain bacterial genera is associated with an increased risk of irAEs, with the most robust evidence pointing to an intimate connection with the development of immune-related diarrhea and colitis. These bacteria include Bacteroides , Enterobacteriaceae, and Proteobacteria (such as Klebsiella and Proteus ) . Lachnospiraceae spp. and Streptococcus spp. have been implicated irAE-wide in the context of ipilimumab. SUMMARY: We review recent lines of evidence pointing to the role of baseline gut microbiota on the development of irAE, and the potentials for therapeutic manipulation of the gut microbiota in order to reduce irAE severity. The connections between gut microbiome signatures of response and toxicity will need to be untangled in further studies.

Physiologic colonic uptake of 18F-FDG on PET/CT is associated with clinical response and gut microbiome composition in patients with advanced non-small cell lung cancer treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICI) represent the backbone treatment for advanced non-small cell lung cancer (NSCLC). Emerging data suggest that increased gut microbiome diversity is associated with favorable response to ICI and that antibiotic-induced dysbiosis is associated with deleterious outcomes. 18F-FDG physiologic colonic uptake on PET/CT increases following treatment with antibiotics (ATB) and could act as a surrogate marker for microbiome composition and predict prognosis. The aim of this study was to determine if 18F-FDG physiologic colonic uptake prior to ICI initiation correlates with gut microbiome profiling and clinical outcomes in patients with advanced NSCLC. METHODS: Seventy-one patients with advanced NSCLC who underwent a PET/CT prior to ICI were identified. Blinded colonic contouring was performed for each colon segment and patients were stratified according to the median of the average colon SUVmax as well as for each segment in low vs. high SUVmax groups. Response rate, progression-free survival (PFS), and overall survival (OS) were compared in the low vs. high SUVmax groups. Gut microbiome composition was analyzed for 23 patients using metagenomics sequencing. RESULTS: The high colon SUVmax group had a higher proportion of non-responders (p = 0.033) and significantly shorter PFS (4.1 vs. 11.3 months, HR 1.94, 95% CI 1.11-3.41, p = 0.005). High caecum SUVmax correlated with numerically shorter OS (10.8 vs. 27.6 months, HR 1.85, 95% CI 0.97-3.53, p = 0.058). Metagenomics sequencing revealed distinctive microbiome populations in each group. Patients with low caecum SUVmax had higher microbiome diversity (p = 0.046) and were enriched with Bifidobacteriaceae, Lachnospiraceae, and Bacteroidaceae. CONCLUSIONS: Lower colon physiologic 18F-FDG uptake on PET/CT prior to ICI initiation was associated with better clinical outcomes and higher gut microbiome diversity in patients with advanced NSCLC. Here, we propose that 18F-FDG physiologic colonic uptake on PET/CT could serve as a potential novel marker of gut microbiome composition and may predict clinical outcomes in this population.

Transcriptome analysis reveals TMPRSS6 isoforms with distinct functionalities.

TMPRSS6 (matriptase-2) is a type II transmembrane serine protease involved in iron homoeostasis. At the cell surface of hepatocytes, TMPRSS6 cleaves haemojuvelin (HJV) and regulates the BMP/SMAD signalling pathway leading to production of hepcidin, a key regulator of iron absorption. Although four TMPRSS6 human isoforms and three mice Tmprss6 isoforms are annotated in databases (Ensembl and RefSeq), their relative expression or activity has not been studied. Analyses of RNA-seq data and RT-PCR from human tissues reveal that TMPRSS6 isoform 1 (TMPRSS6-1) and 3 are mostly expressed in human testis while TMPRSS6-2 and TMPRSS6-4 are the main transcripts expressed in human liver, testis and pituitary. Furthermore, we confirm the existence and analyse the relative expression of three annotated mice Tmprss6 isoforms. Using heterologous expression in HEK293 and Hep3B cells, we show that all human TMPRSS6 isoforms reach the cell surface but only TMPRSS6-1 undergoes internalization. Moreover, truncated TMPRSS6-3 or catalytically altered TMPRSS6-4 interact with HJV and prevent its cleavage by TMPRSS6-2, suggesting their potential role as dominant negative isoforms. Taken together, our results highlight the importance of understanding the precise function of each TMPRSS6 isoforms both in human and in mouse.

The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to ß-amyloid peptide.

Recent studies have reported that many proteases, besides the canonical α-, ß-, and γ-secretases, cleave the amyloid precursor protein (APP) and modulate ß-amyloid (Aß) peptide production. Moreover, specific APP isoforms contain Kunitz protease-inhibitory domains, which regulate the proteolytic activity of serine proteases. This prompted us to investigate the role of matriptase, a member of the type II transmembrane serine protease family, in APP processing. Using quantitative RT-PCR, we detected matriptase mRNA in several regions of the human brain with an enrichment in neurons. RNA sequencing data of human dorsolateral prefrontal cortex revealed relatively high levels of matriptase RNA in young individuals, whereas lower levels were detected in older individuals. We further demonstrate that matriptase and APP directly interact with each other and that matriptase cleaves APP at a specific arginine residue (Arg-102) both in vitro and in cells. Site-directed (Arg-to-Ala) mutagenesis of this cleavage site abolished matriptase-mediated APP processing. Moreover, we observed that a soluble, shed matriptase form cleaves endogenous APP in SH-SY5Y cells and that this cleavage significantly reduces APP processing to Aß40. In summary, this study identifies matriptase as an APP-cleaving enzyme, an activity that could have important consequences for the abundance of Aß and in Alzheimer's disease pathology.

The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction.

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

Prostasin is required for matriptase activation in intestinal epithelial cells to regulate closure of the paracellular pathway.

The type II transmembrane serine protease matriptase is a key regulator of epithelial barriers in skin and intestine. In skin, matriptase acts upstream of the glycosylphosphatidylinositol-anchored serine protease, prostasin, to activate the prostasin zymogen and initiate a proteolytic cascade that is required for stratum corneum barrier functionality. Here, we have investigated the relationship between prostasin and matriptase in intestinal epithelial barrier function. We find that similar to skin, matriptase and prostasin are components of a common intestinal epithelial barrier-forming pathway. Depletion of prostasin by siRNA silencing in Caco-2 intestinal epithelium inhibits barrier development similar to loss of matriptase, and the addition of recombinant prostasin to the basal side of polarized Caco-2 epithelium stimulates barrier forming changes similar to the addition of recombinant matriptase. However, in contrast to the proteolytic cascade in skin, prostasin functions upstream of matriptase to activate the endogenous matriptase zymogen. Prostasin is unable to proteolytically activate the matriptase zymogen directly but induces matriptase activation indirectly. Prostasin requires expression of endogenous matriptase to stimulate barrier formation since matriptase depletion by siRNA silencing abrogates prostasin barrier-forming activity. Active recombinant matriptase, however, does not require the expression of endogenous prostasin for barrier-forming activity. Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades.

Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Repotrectinib: Redefining the therapeutic landscape for patients with ROS1 fusion-driven non-small cell lung cancer.

The ROS1 proto-oncogene encodes a receptor tyrosine kinase with structural homology to other oncogenic drivers, including ALK and TRKA-B-C. The FDA-approved tyrosine kinase inhibitors (TKIs) crizotinib and entrectinib have demonstrated efficacy in treating ROS1 fusion-positive NSCLC. However, limitations such as poor blood-brain barrier penetration and acquired resistance, particularly the ROS1 G2032R solvent-front mutation, hinder treatment durability. Repotrectinib, a next-generation macrocyclic TKI, was rationally designed to overcome on-target resistance mutations and improve brain distribution through its low molecular weight. In the TRIDENT-1 clinical trial, repotrectinib demonstrated significant efficacy in both TKI-naïve and TKI-pretreated patients with ROS1-rearranged NSCLC, including those with CNS metastases and G2032R resistance mutations. In the TKI-naïve cohort (n = 71), 79% of patients achieved an objective response, with a median progression-free survival (PFS) of 35.7 months, surpassing all previously approved ROS1 TKIs. In patients who had received one prior ROS1 TKI but were chemotherapy-naïve (n = 56), objective responses were observed in 38%, and median PFS was 9.0 months. The safety profile of repotrectinib was consistent with earlier-generation ROS1 TKIs and common adverse events included anemia, neurotoxicity, increased creatine kinase levels, and weight gain. These findings underscore the potential of repotrectinib to address unmet needs in ROS1-rearranged NSCLC, offering durable responses and improved intracranial activity. Future research should prioritize developing next-generation, selective ROS1 inhibitors to reduce Trk-mediated toxicities and improve treatment tolerance.

Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from µM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

Phase 2 trial of regorafenib in recurrent or metastatic adenoid cystic carcinoma.

BACKGROUND: There is a significant need for effective therapies to treat recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC). This study evaluated the multi-targeted, vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI) regorafenib in patients with R/M ACC. METHODS: Patients with progressive R/M ACC were treated with regorafenib until disease progression, consent withdrawal, or excessive toxicity. The primary endpoints were best overall response (BOR) and 6-month progression-free survival (PFS). Genomic and transcriptomic biomarker analyses were performed in tumors from trial participants. RESULTS: Thirty-eight patients were enrolled, including 7 (18%) patients with prior VEGFR-TKIs. No objective responses were observed. Six-month PFS was 45%, and median PFS was 7.2 months (95%CI 5.2-11.9 months). Presence of either activating NOTCH1 (22%) or KDM6A alterations (24%) was associated with decreased PFS (HR 2.6, 95%CI 1.1-6.1, p=0.03). Bulk RNA sequencing of pre-treatment tumors revealed that regorafenib clinical benefit (CB; PFS≥6 months; n=11) was associated with native enrichment of immune-related signatures. Immune deconvolution revealed a greater degree of macrophage and T-cell infiltration in CB tumors. Tumors from patients with no clinical benefit (NCB; PFS<6 months; n=9) had greater expression of signatures related to cell cycle progression (E2F targets, G2/M checkpoint). CONCLUSION: The trial failed to meet the pre-specified 6-month PFS and BOR targets. We hypothesize that TKI efficacy may be reliant upon an interplay between kinase inhibition and the ACC immune microenvironment, while programs promoting cell cycle progression may contribute to TKI resistance. These observations suggest that trials evaluating CDK4/6 inhibition plus a VEGFR-TKI should be considered.

Development of ketobenzothiazole-based peptidomimetic TMPRSS13 inhibitors with low nanomolar potency.

TMPRSS13, a member of the Type II Transmembrane Serine Proteases (TTSP) family, is involved in cancer progression and in cell entry of respiratory viruses. To date, no inhibitors have been specifically developed toward this protease. In this study, a chemical library of 65 ketobenzothiazole-based peptidomimetic molecules was screened against a proteolytically active form of recombinant TMPRSS13 to identify novel inhibitors. Following an initial round of screening, subsequent synthesis of additional derivatives supported by molecular modelling, uncovered important molecular determinants involved in TMPRSS13 inhibition. One inhibitor, N-0430, achieved low nanomolar affinity towards TMPRSS13 activity in a cellular context. Using a SARS-CoV-2 pseudovirus cell entry model, we further show the ability of N-0430 to block TMPRSS13-dependent entry of the pseudovirus. The identified peptidomimetic inhibitors and the molecular insights of their potency gained from this study will aid in the development of specific TMPRSS13 inhibitors.

Nanomolar anti-SARS-CoV-2 Omicron activity of the host-directed TMPRSS2 inhibitor N-0385 and synergistic action with direct-acting antivirals.

SARS-CoV-2 Omicron subvariants with increased transmissibility and immune evasion are spreading globally with alarming persistence. Whether the mutations and evolution of spike (S) Omicron subvariants alter the viral hijacking of human TMPRSS2 for viral entry remains to be elucidated. This is particularly important to investigate because of the large number and diversity of mutations of S Omicron subvariants reported since the emergence of BA.1. Here we report that human TMPRSS2 is a molecular determinant of viral entry for all the Omicron clinical isolates tested in human lung cells, including ancestral Omicron subvariants (BA.1, BA.2, BA.5), contemporary Omicron subvariants (BQ.1.1, XBB.1.5, EG.5.1) and currently circulating Omicron BA.2.86. First, we used a co-transfection assay to demonstrate the endoproteolytic cleavage by TMPRSS2 of spike Omicron subvariants. Second, we found that N-0385, a highly potent TMPRSS2 inhibitor, is a robust entry inhibitor of virus-like particles harbouring the S protein of Omicron subvariants. Third, we show that N-0385 exhibits nanomolar broad-spectrum antiviral activity against live Omicron subvariants in human Calu-3 lung cells and primary patient-derived bronchial epithelial cells. Interestingly, we found that N-0385 is 10-20 times more potent than the repositioned TMPRSS2 inhibitor, camostat, against BA.5, EG.5.1, and BA.2.86. We further found that N-0385 shows broad synergistic activity with clinically approved direct-acting antivirals (DAAs), i.e., remdesivir and nirmatrelvir, against Omicron subvariants, demonstrating the potential therapeutic benefits of a multi-targeted treatment based on N-0385 and DAAs.

Antibiotics are associated with worse outcomes in lung cancer patients treated with chemotherapy and immunotherapy.

Anti-PD(L)-1 inhibition combined with platinum doublet chemotherapy (Chemo-IO) has become the most frequently used standard of care regimen in patients with non-small cell lung cancer (NSCLC). The negative impact of antibiotics on clinical outcomes prior to anti-PD(L)-1 inhibition monotherapy (IO) has been demonstrated in multiple studies, but the impact of antibiotic exposure prior to initiation of Chemo-IO is controversial. We assessed antibiotic exposures at two time windows: within 60 days prior to therapy (-60 d window) and within 60 days prior to therapy and 42 days after therapy (-60 + 42d window) in 2028 patients with advanced NSCLC treated with Chemo-IO and IO monotherapy focusing on objective response rate (ORR: rate of partial response and complete response), progression-free survival (PFS), and overall survival (OS). We also assessed impact of antibiotic exposure in an independent cohort of 53 patients. Univariable and multivariable analyses were conducted along with a meta-analysis from similar studies. For the -60 d window, in the Chemo-IO group (N = 769), 183 (24%) patients received antibiotics. Antibiotic exposure was associated with worse ORR (27% vs 40%, p = 0.001), shorter PFS (3.9 months vs. 5.9 months, hazard ratio [HR] 1.35, 95%CI 1.1,1.6, p = 0.0012), as well as shorter OS (10 months vs. 15 months, HR 1.50, 95%CI 1.2,1.8, p = 0.00014). After adjusting for known prognostic factors in NSCLC, antibiotic exposure was independently associated with worse PFS (HR 1.39, 95%CI 1.35,1.7, p = 0.002) and OS (HR 1.61, 95%CI 1.28,2.03, p < 0.001). Similar results were obtained in the -60 + 42d window, and also in an independent cohort. In a meta-analysis of patients with NSCLC treated with Chemo-IO (N = 4) or IO monotherapy (N = 13 studies) antibiotic exposure before treatment was associated with worse OS among all patients (n = 11,351) (HR 1.93, 95% CI 1.52, 2.45) and Chemo-IO-treated patients (n = 1201) (HR 1.54, 95% CI 1.28, 1.84). Thus, antibiotics exposure prior to Chemo-IO is common and associated with worse outcomes, even after adjusting for other factors. These results highlight the need to implement antibiotic stewardship in routine oncology practice.

A phase 1 study of concurrent cabozantinib and cetuximab in recurrent or metastatic head and neck squamous cell cancer.

OBJECTIVES: Epidermal growth factor receptor (EGFR) inhibition with cetuximab is a standard treatment for head and neck squamous cell carcinoma (HNSCC). Activation of the receptor tyrosine kinases AXL, MET and VEGFR can mediate resistance to cetuximab. Cabozantinib, a multikinase inhibitor (MKI) targeting AXL/MET/VEGFR, has demonstrated antitumor activity in preclinical models of HNSCC. This investigator- initiated phase I trial evaluated the safety and efficacy of cetuximab plus cabozantinib in patients with recurrent/metastatic (R/M) HNSCC. MATERIALS AND METHODS: Patients received cetuximab concurrently with cabozantinib daily on a 28-day cycle. Using a 3 + 3 dose-escalation design, the primary endpoint was to determine the maximally tolerated dose (MTD) of cabozantinib. Secondary endpoints included overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) RESULTS: Among the 20 patients enrolled, most had prior disease progression on immune checkpoint inhibitors (95 %), platinum-based chemotherapy (95 %), and cetuximab (80 %). No dose-limiting toxicities were recorded and the MTD for cabozantinib was established to be 60 mg. Grade ≥ 3 adverse events occurred in 65 % of patients (n = 13). ORR was 20 %, with 4 partial responses (PRs). Two PRs were observed in cetuximab-naïve patients (n = 4), with an ORR of 50 % in this subgroup. In the overall population, DCR was 75 %, median PFS was 3.4 months and median OS was 8.1 months. CONCLUSION: Cetuximab plus cabozantinib demonstrated a manageable toxicity profile and preliminary efficacy in patients with heavily treated R/M HNSCC. The combination of cetuximab with MKIs targeting the AXL/MET/VEGFR axis warrants further investigation, including in cetuximab-naïve patients.

Rational In Silico Design of Selective TMPRSS6 Peptidomimetic Inhibitors via Exploitation of the S2 Subpocket.

TMPRSS6 is a potential therapeutic target for the treatment of iron overload due to its role in regulating levels of hepcidin. Although potent TMPRSS6 inhibitors have been previously developed, their lack of specificity requires optimization to avoid potential side effects before pursuing preclinical development with in vivo models. Here, using computer-aided drug design based on a TMPRSS6 homology model, we reveal that the S2 position of TMPRSS6 offers a potential avenue to achieve selectivity against other members of the TTSP family. Accordingly, we synthesized novel peptidomimetic molecules containing lipophilic amino acids at the P2 position to exploit this unexplored pocket. This enabled us to identify TMPRSS6-selective small molecules with low nanomolar affinity. Finally, pharmacokinetic parameters were determined, and a compound was found to be potent in cellulo toward its primary target while retaining TTSP-subtype selectivity and showing no signs of alteration in in vitro TEER experiments.

Gut microbiome predicts gastrointestinal toxicity outcomes from chemoradiation therapy in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Chemoradiation (CRT) in patients with locally advanced head and neck squamous cell cancer (HNSCC) is associated with significant toxicities, including mucositis. The gut microbiome represents an emerging hallmark of cancer and a potentially important biomarker for CRT-related adverse events. This prospective study investigated the association between the gut microbiome composition and CRT-related toxicities in patients with HNSCC, including mucositis. MATERIALS AND METHODS: Stool samples from patients diagnosed with locally advanced HNSCC were prospectively collected prior to CRT initiation and analyzed using shotgun metagenomic sequencing to evaluate gut microbiome composition at baseline. Concurrently, clinicopathologic data, survival outcomes and the incidence and grading of CRT-emergent adverse events were documented in all patients. RESULTS: A total of 52 patients were included, of whom 47 had baseline stool samples available for metagenomic analysis. Median age was 62, 83 % patients were men and 54 % had stage III-IV disease. All patients developed CRT-induced mucositis, including 42 % with severe events (i.e. CTCAE v5.0 grade ≥ 3) and 25 % who required enteral feeding. With a median follow-up of 26.5 months, patients with severe mucositis had shorter overall survival (HR = 3.3, 95 %CI 1.0-10.6; p = 0.02) and numerically shorter progression-free survival (HR = 2.8, 95 %CI, 0.8-9.6; p = 0.09). The gut microbiome beta-diversity of patients with severe mucositis differed from patients with grades 1-2 mucositis (p = 0.04), with enrichment in Mediterraneibacter (Ruminococcus gnavus) and Clostridiaceae family members, including Hungatella hathewayi. Grade 1-2 mucositis was associated with enrichment in Eubacterium rectale, Alistipes putredinis and Ruminococcaceae family members. Similar bacterial profiles were observed in patients who required enteral feeding. CONCLUSION: Patients who developed severe mucositis had decreased survival and enrichment in specific bacteria associated with mucosal inflammation. Interestingly, these same bacteria have been linked to immune checkpoint inhibitor resistance.

Tumor-Agnostic Genomic and Clinical Analysis of BRAF Fusions Identifies Actionable Targets.

PURPOSE: Even though BRAF fusions are increasingly detected in standard multigene next-generation sequencing panels, few reports have explored their structure and impact on clinical course. EXPERIMENTAL DESIGN: We collected data from patients with BRAF fusion-positive cancers identified through a genotyping protocol of 97,024 samples. Fusions were characterized and reviewed for oncogenic potential (in-frame status, non-BRAF partner gene, and intact BRAF kinase domain). RESULTS: We found 241 BRAF fusion-positive tumors from 212 patients with 82 unique 5' fusion partners spanning 52 histologies. Thirty-nine fusion partners were not previously reported, and 61 were identified once. BRAF fusion incidence was enriched in pilocytic astrocytomas, gangliogliomas, low-grade neuroepithelial tumors, and acinar cell carcinoma of the pancreas. Twenty-four patients spanning multiple histologies were treated with MAPK-directed therapies, of which 20 were evaluable for RECIST. Best response was partial response (N = 2), stable disease (N = 11), and progressive disease (N = 7). The median time on therapy was 1 month with MEK plus BRAF inhibitors [(N = 11), range 0-18 months] and 8 months for MEK inhibitors [(N = 14), range 1-26 months]. Nine patients remained on treatment for longer than 6 months [pilocytic astrocytomas (N = 6), Erdheim-Chester disease (N = 1), extraventricular neurocytoma (N = 1), and melanoma (N = 1)]. Fifteen patients had acquired BRAF fusions. CONCLUSIONS: BRAF fusions are found across histologies and represent an emerging actionable target. BRAF fusions have a diverse set of fusion partners. Durable responses to MAPK therapies were seen, particularly in pilocytic astrocytomas. Acquired BRAF fusions were identified after targeted therapy, underscoring the importance of postprogression biopsies to optimize treatment at relapse in these patients.