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Ataxia with anti-regulator of G-protein signaling 8 autoantibodies (RGS8-Abs) is an autoimmune disease recently described in four patients. The present study aimed to identify other patients with RGS8-Abs, describe their clinical features, including the link between RGS8-related autoimmune cerebellar ataxia (ACA) and cancer. Patients with RGS8-Abs were identified retrospectively in the biological collections of the French Reference Center for Paraneoplastic Neurological Syndrome and the University of California San Francisco Center for Encephalitis and Meningitis. Clinical data were collected, and cerebrospinal fluid, serum, and tumor pathological samples were retrieved to characterize the autoantibodies and the associated malignancies. Only three patients with RGS8-Abs were identified. All of them presented with a pure cerebellar ataxia of mild to severe course, unresponsive to current immunotherapy regimens for ACA. Two patients presented with a Hodgkin lymphoma of the rare specific subtype called nodular lymphocyte-predominant Hodgkin lymphoma, with very mild extension. Autoantibodies detected in all patients enriched the same epitope on the RGS8 protein, which is an intracellular protein physiologically expressed in Purkinje cells but also ectopically expressed specifically in lymphoma cells of patients with RGS8-related ACA. The present results and those of the four cases previously described suggest that RGS8-Abs define a new paraneoplastic neurological syndrome of extreme rarity found mostly in middle-aged males that associates pure cerebellar ataxia and a particular lymphoma specifically expressing the RGS8 antigen. As in other paraneoplastic ACA with intracellular antigen, the disease course is severe, and patients tend to exhibit a poor response to immune therapy.

The Phase 3 randomized controlled trial, TBTC Study 31/ACTG A5349 (NCT02410772) demonstrated that a 4-month rifapentine-moxifloxacin regimen for drug-susceptible pulmonary tuberculosis was safe and effective. The primary efficacy outcome was 12-month tuberculosis disease free survival, while the primary safety outcome was the proportion of grade 3 or higher adverse events during the treatment period. We conducted an analysis of demographic, clinical, microbiologic, radiographic, and pharmacokinetic data and identified risk factors for unfavorable outcomes and adverse events. Among participants receiving the rifapentine-moxifloxacin regimen, low rifapentine exposure is the strongest driver of tuberculosis-related unfavorable outcomes (HR 0.65 for every 100 µg∙h/mL increase, 95%CI 0.54-0.77). The only other risk factors identified are markers of higher baseline disease severity, namely Xpert MTB/RIF cycle threshold and extent of disease on baseline chest radiography (Xpert: HR 1.43 for every 3-cycle-threshold decrease, 95%CI 1.07-1.91; extensive disease: HR 2.02, 95%CI 1.07-3.82). From these risk factors, we developed a simple risk stratification to classify disease phenotypes as easier-, moderately-harder, or harder-to-treat TB. Notably, high rifapentine exposures are not associated with any predefined adverse safety outcomes. Our results suggest that the easier-to-treat subgroup may be eligible for further treatment shortening while the harder-to-treat subgroup may need higher doses or longer treatment.

BackgroundPoor treatment outcomes among people with drug-resistant TB (DR-TB) are a major concern. Heteroresistance (presence of susceptible and resistant Mycobacterium tuberculosis in the same sample) has been identified in some people with TB, but its impact on treatment outcomes is unknown.MethodsWe used targeted deep sequencing to identify mutations associated with DR-TB and heteroresistance in culture samples of 624 people with DR-TB. We evaluated the association between heteroresistance and time to unfavorable treatment outcome using Cox proportional hazards regression.ResultsThe proportion of drug-resistant isolates with a known mutation conferring resistance was lower for streptomycin (45.2%) and second-line injectables (79.1%) than for fluoroquinolones (86.7%), isoniazid (93.2%) and rifampin (96.5%). Fifty-two (8.3%) had heteroresistance, and it was more common for fluoroquinolones (4.6%) than rifampin (2.2%), second-line injectables (1.4%), streptomycin (1.7%), or isoniazid (1.3%). There was no association between heteroresistance and time to unfavorable outcome among people with multidrug-resistant TB (adjusted hazard ratio [aHR] 1.74, 95% CI 0.39-7.72) or pre-extensively DR-TB (aHR 0.65, 95% CI 0.24-1.72).ConclusionsHeteroresistance was relatively common (8.3%) among people with DR-TB in the Philippines. However, we found insufficient evidence to demonstrate an impact on unfavorable treatment outcomes.

BackgroundStandard treatment for patients with newly diagnosed glioblastoma includes surgery, radiotherapy (RT), and temozolomide (TMZ) chemotherapy (TMZ/RT→TMZ). The proteasome has long been considered a promising therapeutic target because of its role as a central biological hub in tumor cells. Marizomib is a novel pan-proteasome inhibitor that crosses the blood-brain barrier.MethodsEuropean Organisation for Research and Treatment of Cancer 1709/Canadian Cancer Trials Group CE.8 was a multicenter, randomized, controlled, open-label phase 3 superiority trial. Key eligibility criteria included newly diagnosed glioblastoma, age > 18 years and Karnofsky performance status > 70. Patients were randomized in a 1:1 ratio. The primary objective was to compare overall survival (OS) in patients receiving marizomib in addition to TMZ/RT→TMZ with patients receiving the only standard treatment in the whole population and in the subgroup of patients with MGMT promoter-unmethylated tumors.ResultsThe trial was opened at 82 institutions in Europe, Canada, and the U.S. A total of 749 patients (99.9% of the planned 750) were randomized. OS was not different between the standard and the marizomib arm (median 17 vs. 16.5 months; HR = 1.04; P = .64). PFS was not statistically different either (median 6.0 vs. 6.3 months; HR = 0.97; P = .67). In patients with MGMT promoter-unmethylated tumors, OS was also not different between standard therapy and marizomib (median 14.5 vs. 15.1 months, HR = 1.13; P = .27). More CTCAE grade 3/4 treatment-emergent adverse events were observed in the marizomib arm than in the standard arm.ConclusionsAdding marizomib to standard temozolomide-based radiochemotherapy resulted in more toxicity, but did not improve OS or PFS in patients with newly diagnosed glioblastoma.

BackgroundGastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.MethodsTwo patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.ResultsM5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.ConclusionsHumanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the

PurposeThe purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC).Experimental designRB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss.ResultsRB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1.ConclusionsCo-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

PurposeUnderstanding the complex bidirectional interactions between neurons and glioma cells could help to identify new therapeutic targets. Herein, the techniques and application of novel neuroscience tools implemented to study the complex interactions between brain and malignant gliomas, their results, and the potential therapeutic opportunities were reviewed.MethodsLiterature search was performed on PubMed between 2001 and 2023 using the keywords "glioma", "glioblastoma", "circuit remodeling", "plasticity", "neuron networks" and "cortical networks". Studies including grade 2 to 4 gliomas, diffuse midline gliomas, and diffuse intrinsic pontine gliomas were considered.ResultsGlioma cells are connected through tumour microtubes and form a highly connected network within which pacemaker cells drive tumorigenesis. Unconnected cells have increased invasion capabilities. Glioma cells are also synaptically integrated within neural circuitry. Neurons promote tumour growth via paracrine and direct electrochemical mechanisms, including glutamatergic AMPA-receptors. Increased glutamate release in the tumor microenvironment and loss of peritumoral GABAergic inhibitory interneurons result in network hyperexcitability and secondary epilepsy. Functional imaging, local field potentials and subcortical mapping, performed in awake patients, have defined patterns of malignant circuit remodeling. Glioma-induced remodeling is frequent in language and even motor cortical networks, depending on tumour biological parameters, and influences functional outcomes.ConclusionThese data offer new insights into glioma tumorigenesis. Future work will be needed to understand how tumor intrinsic molecular drivers influence neuron-glioma interactions but also to integrate these results to design new therapeutic options for patients.

Glioblastoma (GBM) is the deadliest form of primary brain tumor with limited treatment options. Recent studies have profiled GBM tumor heterogeneity, revealing numerous axes of variation that explain the molecular and spatial features of the tumor. Here, we seek to bridge descriptive characterization of GBM cell type heterogeneity with the functional role of individual populations within the tumor. Our lens leverages a gene program-centric meta-atlas of published transcriptomic studies to identify commonalities between diverse tumors and cell types in order to decipher the mechanisms that drive them. This approach led to the discovery of a tumor-derived stem cell population with mixed vascular and neural stem cell features, termed a neurovascular progenitor (NVP). Following in situ validation and molecular characterization of NVP cells in GBM patient samples, we characterized their function in vivo. Genetic depletion of NVP cells resulted in altered tumor cell composition, fewer cycling cells, and extended survival, underscoring their critical functional role. Clonal analysis of primary patient tumors in a human organoid tumor transplantation system demonstrated that the NVP has dual potency, generating both neuronal and vascular tumor cells. Although NVP cells comprise a small fraction of the tumor, these clonal analyses demonstrated that they strongly contribute to the total number of cycling cells in the tumor and generate a defined subset of the whole tumor. This study represents a paradigm by which cell type-specific interrogation of tumor populations can be used to study functional heterogeneity and therapeutically targetable vulnerabilities of GBM.

Nineteen genetic therapies have been approved by the U.S. Food and Drug Administration (FDA) to date, a number that now includes the first CRISPR genome editing therapy for sickle cell disease, CASGEVY (exagamglogene autotemcel). This extraordinary milestone is widely celebrated because of the promise for future genome editing treatments of previously intractable genetic disorders and cancers. At the same time, such genetic therapies are the most expensive drugs on the market, with list prices exceeding $4 million per patient. Although all approved cell and gene therapies trace their origins to academic or government research institutions, reliance on for-profit pharmaceutical companies for subsequent development and commercialization results in prices that prioritize recouping investments, paying for candidate product failures, and meeting investor and shareholder expectations. To increase affordability and access, sustainable discovery-to-market alternatives are needed that address system-wide deficiencies. Here, we present recommendations of a multi-disciplinary task force assembled to chart such a path. We describe a pricing structure that, once implemented, could reduce per-patient cost tenfold and propose a business model that distributes responsibilities while leveraging diverse funding sources. We also outline how academic licensing provisions, manufacturing innovation and supportive regulations can reduce cost and enable broader patient treatment.

ObjectivePatients with relapsed or metastatic head and neck squamous cell carcinoma (HNSCC) after primary local therapy have low response rates with cetuximab, systemic chemotherapy or check point inhibitor therapy. Novel combination therapies with the potential to improve outcomes for patients with HNSCC is an area of high unmet need.MethodsThis is a phase II single-arm clinical trial of locally advanced or metastatic HNSCC patients treated with a combination of soluble EphB4-human serum albumin (sEphB4-HSA) fusion protein and pembrolizumab after platinum-based chemotherapy with up to 2 prior lines of treatment. The primary endpoints were safety and tolerability and the primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included progression free survival (PFS) and overall survival (OS). HPV status and EphrinB2 expression were evaluated for outcome.ResultsTwenty-five patients were enrolled. Median follow up was 40.4 months (range 9.8 - 40.4). There were 6 responders (ORR 24%). There were 5 responders in the 11 HPV-negative and EphrinB2 positive patients, (ORR 45%) with 2 of these patients achieving a complete response (CR). The median PFS in HPV-negative/EphrinB2 positive patients was 3.2 months (95% CI 1.1, 7.3). Median OS in HPV-negative/EphrinB2 positive patients was 10.9 months (95% CI 2.0, 13.7). Hypertension, transaminitis and fatigue were the most common toxicities.DiscussionThe combination of sEphB4-HSA and pembrolizumab has a favorable toxicity profile and favorable activity particularly among HPV-negative EphrinB2 positive patients with HNSCC.

The early 2-factor (E2F) family of transcription factors, including E2F1 through 8, plays a critical role in apoptosis, metabolism, proliferation, and angiogenesis within glioblastoma (GBM). However, the specific functions of E2F transcription factors (E2Fs) and their impact on the malignancy of Bevacizumab (BVZ)-responsive GBM subtypes remain unclear. This study used data from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI), and Gene Expression Omnibus (GEO) to explore the impact of eight E2F family members on the clinical characteristics of BVZ-responsive GBM subtypes and possible mechanisms of recurrence after BVZ treatment. Using machine learning algorithms, including TreeBagger and deep neural networks, we systematically predicted and validated GBM patient survival terms based on the expression profiles of E2Fs across BVZ-responsive GBM subtypes. Our bioinformatics analyses suggested that a significant increase in E2F8 post-BVZ treatment may enhance the function of angiogenesis and stem cell proliferation, implicating this factor as a candidate mechanism of GBM recurrence after treatment. In addition, BVZ treatment in unresponsive GBM patients may potentially worsen disease progression. These insights underscore that E2F family members play important roles in GBM malignancy and BVZ treatment response, highlighting their potential as prognostic biomarkers, therapeutic targets, and recommending precision BVZ treatment to individual GBM patients.

Glioblastoma (GBM) is the most common malignant primary brain tumor and remains incurable. Previous work has shown that systemic administration of Decitabine (DAC) induces sufficient expression of cancer-testis antigens (CTA) in GBM for targeting by adoptive T-cell therapy in vivo. However, the mechanisms by which DAC enhances immunogenicity in GBM remain to be elucidated. Using New York esophageal squamous cell carcinoma 1 (NY-ESO-1) as a representative inducible CTA, we demonstrate in patient tissue, immortalized glioma cells, and primary patient-derived gliomaspheres that basal CTA expression is restricted by promoter hypermethylation in gliomas. DAC treatment of glioma cells specifically inhibits DNA methylation silencing to render NY-ESO-1 and other CTA into inducible tumor antigens at single-cell resolution. Functionally, NY-ESO-1 T-cell receptor-engineered effector cell targeting of DAC-induced antigen in primary glioma cells promotes specific and polyfunctional T-cell cytokine profiles. In addition to induction of CTA, DAC concomitantly reactivates tumor-intrinsic human endogenous retroviruses, interferon response signatures, and MHC-I. Overall, we demonstrate that DAC induces targetable tumor antigen and enhances T-cell functionality against GBM, ultimately contributing to the improvement of targeted immune therapies in glioma.SignificanceThis study dissects the tumor-intrinsic epigenetic and transcriptional mechanisms underlying enhanced T-cell functionality targeting decitabine-induced cancer-testis antigens in glioma. Our findings demonstrate concomitant induction of tumor antigens, reactivation of human endogenous retroviruses, and stimulation of interferon signaling as a mechanistic rationale to epigenetically prime human gliomas to immunotherapeutic targeting.

Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases (NMT). Myristoylation is emerging as a promising cancer therapeutic target; however, the molecular determinants of sensitivity to NMT inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that NMTs are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS-mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation, and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter translocase of inner mitochondrial membrane 17 homolog A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis NMT-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.SignificanceKRAS-mutant lung carcinomas with LKB1 and/or KEAP1 co-mutations have intrinsic therapeutic resistance. We show that these tumors are sensitive to NMT inhibitors, which slow tumor growth in vivo and sensitize cells to platinum-based chemotherapy in vitro. Inhibition of myristoylation causes death by parthanatos and thus has the potential to kill apoptosis and ferroptosis-resistant cancer cells. Our findings warrant investigation of NMT as a therapeutic target in highly aggressive lung carcinomas.

Glioblastoma (GBM) is the deadliest adult brain cancer. Under the current standard of care, almost all patients succumb to the disease and novel treatments are urgently needed. Recognizing that GBMs are addicted to cholesterol, past clinical trials have repurposed statins against GBM but failed. The purpose of this study was to test whether treatments that upregulate the cholesterol biosynthesis pathway in GBM would generate a metabolic vulnerability that can be exploited using statins and to determine the underlying mechanisms.Effects of radiotherapy and temozolomide or dopamine receptor antagonists on the mevalonate pathway in GBM were assessed in vitro and in vivo. The impact of statins on self-renewal of glioma stem cells and median survival was studied. Branches of the mevalonate pathway were probed to identify relevant effector proteins.Cells surviving combination treatments that converge in activating the immediate early response, universally upregulated the mevalonate pathway and increased stemness of GBM cells through activation of the Rho-GTPase Rac-1. Activation of the mevalonate pathway and Rac-1 was inhibited by statins, which led to improved survival in mouse models of glioblastoma when combined with radiation and drugs that target the glioma stem cell pool and plasticity of glioma cells.We conclude that a combination of dopamine receptor antagonists and statins could potentially improve radiotherapy outcome and warrants further investigation.SignificanceCombination therapies that activate the mevalonate pathway in GBM cells after sublethal treatment enhance self-renewal and migratory capacity through Rac-1 activation, which creates a metabolic vulnerability that can be further potentially exploited using statins.

PurposeIn 2013, afatinib was approved for non-small-cell lung cancer with subsequent indication expansion. We investigated published afatinib clinical trials to assess risk and benefit profiles for the drug in its approved indication of non-small-cell lung cancer as well as in off-label uses. Previous literature demonstrates excessive patient burden and limited benefit as afatinib has spread into more indications. A trial analysis is needed to establish efficacy and risk.MethodsIn this investigation, we screened literature databases and clinical trial registries for trials of afatinib as monotherapy or in combination interventions for cancer treatment. We extracted participant demographics, adverse event characteristics, as well as clinical and surrogate endpoints for each trial. Studies were deemed positive, negative, or indeterminate based on their achieving of primary endpoints as well as their safety.ResultsOur search yielded 2444 articles; we excluded 2352 articles for a final inclusion of 92 trials of 8859 patients. Our sample had 49 (53%) positive trials, 27 (29%) negative trials, and 16 (17%) indeterminate trials. The most common off-label indications for afatinib were breast cancer and squamous cell carcinoma of head and neck. The median OS for all trials was 8.4 months, median PFS 3.4 months, and the total ORR was 29.6%. Our study found that trials performed in disease states beyond the initial indications were largely negative with little patient benefit. The adverse events within our trial sample appear to be in line with expectations for toxicity.ImplicationsThese results are consistent with other studies that present similar findings, such as in Carlisle et al which indicate limited efficacy in nonapproved indications. Future trials should keep this potential evidence and patient burden in mind before initiation of those trials. This study contributes to the understanding of afatinib's risk-benefit profile across many clinical applications.

BackgroundAutoimmune diseases have been linked to an increased risk of pregnancy-related complications. A family history of autoimmune diseases may be related to the risk of childhood cancer based on similar histocompatibility antigens. We utilized data from national registries in Denmark to examine associations between maternal autoimmune disease and cancer in their offspring.MethodsWe linked data from several national registries in Denmark to identify childhood cancer cases in children

Glioblastoma is a clinically and molecularly heterogeneous disease, and new predictive biomarkers are needed to identify those patients most likely to respond to specific treatments. Through prospective genomic profiling of 459 consecutive primary treatment-naïve IDH-wildtype glioblastomas in adults, we identified a unique subgroup (2%, 9/459) defined by somatic hypermutation and DNA replication repair deficiency due to biallelic inactivation of a canonical mismatch repair gene. The deleterious mutations in mismatch repair genes were often present in the germline in the heterozygous state with somatic inactivation of the remaining allele, consistent with glioblastomas arising due to underlying Lynch syndrome. A subset of tumors had accompanying proofreading domain mutations in the DNA polymerase POLE and resultant "ultrahypermutation". The median age at diagnosis was 50 years (range 27-78), compared with 63 years for the other 450 patients with conventional glioblastoma (p

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines.ImportanceMelioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .

IDH1mut gliomas produce high levels of D-2-hydroxyglutarate (D-2-HG), an oncometabolite capable of inhibiting α-ketoglutarate-dependent dioxygenases critical to a range of cellular functions involved in gliomagenesis. IDH1mut gliomas also exhibit slower growth rates and improved treatment sensitivity compared with their IDH1wt counterparts. This study explores the mechanism driving apparent reduced growth in IDH1mut gliomas. Specifically, we investigated the relationship between IDH1mut and the RNA N6-methyladenosine (m6A) demethylases FTO and ALKBH5, and their potential for therapeutic targeting. We investigated the role of D-2-HG and m6A in tumor proliferation/viability using glioma patient tumor samples, patient-derived gliomaspheres, and U87 cells, as well as with mouse intracranial IDH1wt gliomasphere xenografts. Methylation RNA immunoprecipitation sequencing (MeRIP-seq) RNA sequencing was used to identify m6A-enriched transcripts in IDH1mut glioma. We show that IDH1mut production of D-2-HG is capable of reducing glioma cell growth via inhibition of the m6A epitranscriptomic regulator, FTO, with resultant m6A hypermethylation of a set of mRNA transcripts. On the basis of unbiased MeRIP-seq epitranscriptomic profiling, we identify ATF5 as a hypermethylated, downregulated transcript that potentially contributes to increased apoptosis. We further demonstrate how targeting this pathway genetically and pharmacologically reduces the proliferative potential of malignant IDH1wt gliomas, both in vitro and in vivo. Our work provides evidence that selective inhibition of the m6A epitranscriptomic regulator FTO attenuates growth in IDH1wt glioma, recapitulating the clinically favorable growth phenotype seen in the IDH1mut subtype.SignificanceWe show that IDH1mut-generated D-2-HG can reduce glioma growth via inhibition of the m6A demethylase, FTO. FTO inhibition represents a potential therapeutic target for IDH1wt gliomas and possibly in conjunction with IDH1mut inhibitors for the treatment of IDH1mut glioma. Future studies are necessary to demonstrate the role of ATF5 downregulation in the indolent phenotype of IDH1mut gliomas, as well as to identify other involved gene transcripts deregulated by m6A hypermethylation.

Zika virus (ZIKV) infection during pregnancy causes severe neurological and ocular abnormalities in infants, yet no vaccine or antivirals are available. Our transcriptomic analysis of ZIKV-infected retinal pigment epithelial (RPE) cells revealed alterations in the cholesterol pathway. Thus, we investigated the functional roles of ATP binding cassette transporter G1 (ABCG1) and sterol response element binding protein 2 (SREPB-2), two key players in cholesterol metabolism, during ocular ZIKV infection. Our in vitro data showed that increased ABCG1 activity via liver X receptors (LXRs), reduced ZIKV replication, while ABCG1 knockdown increased replication with elevated intracellular cholesterol. Conversely, inhibiting SREBP-2 or its knockdown reduced ZIKV replication by lowering cholesterol levels. In vivo, LXR agonist or SREBP-2 inhibitor treatment mitigated ZIKV-induced chorioretinal lesions in mice, concomitant with decreased expression of inflammatory mediators and increased activation of antiviral response genes. In summary, our study identifies ABCG1's antiviral role and SREBP-2's proviral effects in ocular ZIKV infection, offering cholesterol metabolism as a potential target to develop antiviral therapies.

Survival from ovarian cancer depends on the resection status after primary surgery. We performed genome-wide association analyses for resection status of 7705 ovarian cancer patients, including 4954 with high-grade serous carcinoma (HGSOC), to identify variants associated with residual disease. The most significant association with resection status was observed for rs72845444, upstream of MGMT, in HGSOC (p = 3.9 × 10-8). In gene-based analyses, PPP2R5C was the most strongly associated gene in HGSOC after stage adjustment. In an independent set of 378 ovarian tumours from the AGO-OVAR 11 study, variants near MGMT and PPP2R5C correlated with methylation and transcript levels, and PPP2R5C mRNA levels predicted progression-free survival in patients with residual disease. MGMT encodes a DNA repair enzyme, and PPP2R5C encodes the B56γ subunit of the PP2A tumour suppressor. Our results link heritable variation at these two loci with resection status in HGSOC.

PurposeMultiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models.Experimental designIn vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity.Results[89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups.ConclusionsOur study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric Plasmodium export element (PEXEL)/host-targeting motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here, we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood stage. While the N termini of exported proteins containing the PEXEL and immediately downstream ~10 residues are sufficient to mediate translocation into the RBC, the equivalent UIS2 N terminus does not promote the export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position, which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RIL↓DE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N terminus mediates export, not PEXEL processing per se. Prompted by this observation, we further show that PEXEL sequences in the N termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.IMPORTANCEHost erythrocyte remodeling by malaria parasite-exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/host-targeting motif by the aspartic protease Plasmepsin V, which exposes a mature N terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate that PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing, reinforcing that features of the mature N terminus, and not PEXEL cleavage, identify cargo for export. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

Hodgkin lymphoma variant of Richter’s transformation (HvRT) is a rare complication for patients with chronic lymphocytic leukemia (CLL), with an overall poor prognosis. We present the first known case series of patients with HvRT treated with the combination of brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine (A + AVD). In our series of 4 patients, two patients treated with A + AVD for HvRT had durable remissions of 40 and 42 months, while two patients had disease progression and ultimately died. Continued investigation into the optimal management for patients with HvRT is still needed.

PurposeModels to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, multiple models are needed to fully elucidate key aspects of disease biology and to recapitulate clinically relevant phenotypes.Experimental designMatched patient samples, patient-derived xenografts (PDX), and PDX-derived cell lines were comprehensively evaluated using whole-genome sequencing and RNA sequencing. The in vivo metastatic phenotype of the PDX-derived cell lines was characterized in both an intravenous and an orthotopic murine model. As a proof-of-concept study, we tested the preclinical effectiveness of a cyclin-dependent kinase inhibitor on the growth of metastatic tumors in an orthotopic amputation model.ResultsPDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication in a subset of cell lines. The cell lines were heterogeneous in their metastatic capacity, and heterogeneous tissue tropism was observed in both intravenous and orthotopic models. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden.ConclusionsThe variation in metastasis predilection sites between osteosarcoma PDX-derived cell lines demonstrates their ability to recapitulate the spectrum of the disease observed in patients. We describe here a panel of new osteosarcoma PDX-derived cell lines that we believe will be of wide use to the osteosarcoma research community.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.To assess long-term risk of local tumor regrowth, we report updated organ preservation rate and oncologic outcomes of the OPRA trial (ClinicalTrials.gov identifier: NCT02008656). Patients with stage II/III rectal cancer were randomly assigned to receive induction chemotherapy followed by chemoradiation (INCT-CRT) or chemoradiation followed by consolidation chemotherapy (CRT-CNCT). Patients who achieved a complete or near-complete response after finishing treatment were offered watch-and-wait (WW). Total mesorectal excision (TME) was recommended for those who achieved an incomplete response. The primary end point was disease-free survival (DFS). The secondary end point was TME-free survival. In total, 324 patients were randomly assigned (INCT-CRT, n = 158; CRT-CNCT, n = 166). Median follow-up was 5.1 years. The 5-year DFS rates were 71% (95% CI, 64 to 79) and 69% (95% CI, 62 to 77) for INCT-CRT and CRT-CNCT, respectively (P = .68). TME-free survival was 39% (95% CI, 32 to 48) in the INCT-CRT group and 54% (95% CI, 46 to 62) in the CRT-CNCT group (P = .012). Of 81 patients with regrowth, 94% occurred within 2 years and 99% occurred within 3 years. DFS was similar for patients who underwent TME after restaging (64% [95% CI, 53 to 78]) and patients in WW who underwent TME after regrowth (64% [95% CI, 53 to 78]; P = .94). Updated analysis continues to show long-term organ preservation in half of the patients with rectal cancer treated with total neoadjuvant therapy. In patients who enter WW, most cases of tumor regrowth occur in the first 2 years.

BackgroundCentral nervous system (CNS) WHO grade 2 low-grade glioma (LGG) patients are at high risk for recurrence and with unfavorable long-term prognosis due to the treatment resistance and malignant transformation to high-grade glioma. Considering the relatively intact systemic immunity and slow-growing nature, immunotherapy may offer an effective treatment option for LGG patients.MethodsWe conducted a prospective, randomized pilot study to evaluate the safety and immunological response of the multipeptide IMA950 vaccine with agonistic anti-CD27 antibody, varlilumab, in CNS WHO grade 2 LGG patients. Patients were randomized to receive combination therapy with IMA950 + poly-ICLC and varlilumab (Arm 1) or IMA950 + poly-ICLC (Arm 2) before surgery, followed by adjuvant vaccines.ResultsA total of 14 eligible patients were enrolled in the study. Four patients received pre-surgery vaccines but were excluded from postsurgery vaccines due to the high-grade diagnosis of the resected tumor. No regimen-limiting toxicity was observed. All patients demonstrated a significant increase of anti-IMA950 CD8+ T-cell response postvaccine in the peripheral blood, but no IMA950-reactive CD8+ T cells were detected in the resected tumor. Mass cytometry analyses revealed that adding varlilumab promoted T helper type 1 effector memory CD4+ and effector memory CD8+ T-cell differentiation in the PBMC but not in the tumor microenvironment.ConclusionThe combinational immunotherapy, including varlilumab, was well-tolerated and induced vaccine-reactive T-cell expansion in the peripheral blood but without a detectable response in the tumor. Further developments of strategies to overcome the blood-tumor barrier are warranted to improve the efficacy of immunotherapy for LGG patients.

Pharmacokinetic (PK) studies in children are usually small and have ethical constraints due to the medical complexities of drawing blood in this special population. Often, population PK models for the drug(s) of interest are available in adults, and these models can be extended to incorporate the expected deviations seen in children. As a consequence, there is increasing interest in the use of optimal design methodology to design PK sampling schemes in children that maximize information using a small sample size and limited number of sampling times per dosing period. As a case study, we use the novel tuberculosis drug delamanid, and show how applications of optimal design methodology can result in highly efficient and model-robust designs in children for estimating PK parameters using a limited number of sampling measurements. Using developed population PK models based on available data from adults living with and without HIV, and limited data on children without HIV, competing designs for children living with HIV were derived and assessed based on robustness to model uncertainty.

A major barrier to the impact of genomic diagnosis in patients with congenital malformations is the lack of understanding regarding how sequence variants contribute to disease pathogenesis and whether this information could be used to generate patient-specific therapies. Congenital diaphragmatic hernia (CDH) is among the most common and severe of all structural malformations; however, its underlying mechanisms are unclear. We identified loss-of-function sequence variants in the epigenomic regulator gene SIN3A in two patients with complex CDH. Tissue-specific deletion of Sin3a in mice resulted in defects in diaphragm development, lung hypoplasia, and pulmonary hypertension, the cardinal features of CDH and major causes of CDH-associated mortality. Loss of SIN3A in the lung mesenchyme resulted in reduced cellular differentiation, impaired cell proliferation, and increased DNA damage. Treatment of embryonic Sin3a mutant mice with anacardic acid, an inhibitor of histone acetyltransferase, reduced DNA damage, increased cell proliferation and differentiation, improved lung and pulmonary vascular development, and reduced pulmonary hypertension. These findings demonstrate that restoring the balance of histone acetylation can improve lung development in the Sin3a mouse model of CDH.

Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.

AbstractRNA-binding proteins (RBPs) are emerging as a novel class of therapeutic targets in cancer, including in leukemia, given their important role in posttranscriptional gene regulation, and have the unexplored potential to be combined with existing therapies. The RBP insulin-like growth factor 2 messenger RNA-binding protein 3 (IGF2BP3) has been found to be a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Here, we study the combined effects of targeting IGF2BP3 and menin-MLL interaction in MLL-AF4-driven leukemia in vitro and in vivo, using genetic inhibition with CRISPR-Cas9-mediated deletion of Igf2bp3 and pharmacologic inhibition of the menin-MLL interaction with multiple commercially available inhibitors. Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the effects of menin-MLL inhibition on cell growth and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and menin-MLL inhibition led to increased differentiation in vitro and in vivo, seen in functional readouts and by gene expression analyses. IGF2BP3 knockdown had a greater effect on increasing survival and attenuating disease than pharmacologic menin-MLL inhibition with small molecule MI-503 alone and showed enhanced antileukemic effects in combination. Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4-mediated leukemogenesis and a potent therapeutic target, providing a paradigm for targeting leukemia at both the transcriptional and posttranscriptional level.

The intestinal parasites Giardia lamblia and Entamoeba histolytica are major causes of morbidity and mortality associated with diarrheal diseases. Metronidazole is the most common drug used to treat giardiasis and amebiasis. Despite its efficacy, treatment failures in giardiasis occur in up to 5%-40% of cases. Potential resistance of E. histolytica to metronidazole is an increasing concern. Therefore, it is critical to search for more effective drugs to treat giardiasis and amebiasis. We identified antigiardial and antiamebic activities of the rediscovered nitroimidazole compound, fexinidazole, and its sulfone and sulfoxide metabolites. Fexinidazole is equally active against E. histolytica and G. lamblia trophozoites, and both metabolites were 3- to 18-fold more active than the parent drug. Fexinidazole and its metabolites were also active against a metronidazole-resistant strain of G. lamblia. G. lamblia and E. histolytica cell extracts exhibited decreased residual nitroreductase activity when metabolites were used as substrates, indicating nitroreductase may be central to the mechanism of action of fexinidazole. In a cell invasion model, fexinidazole and its metabolites significantly reduced the invasiveness of E. histolytica trophozoites through basement membrane matrix. A q.d. oral dose of fexinidazole and its metabolites at 10 mg/kg for 3 days reduced G. lamblia infection significantly in mice compared to control. The newly discovered antigiardial and antiamebic activities of fexinidazole, combined with its FDA-approval and inclusion in the WHO Model List of Essential Medicines for the treatment of human African trypanosomiasis, offer decreased risk and a shortened development timeline toward clinical use of fexinidazole for treatment of giardiasis or amebiasis.

Cough is a common and commonly ignored symptom of lung disease. Cough is often perceived as difficult to quantify, frequently self-limiting, and non-specific. However, cough has a central role in the clinical detection of many lung diseases including tuberculosis (TB), which remains the leading infectious disease killer worldwide. TB screening currently relies on self-reported cough which fails to meet the World Health Organization (WHO) accuracy targets for a TB triage test. Artificial intelligence (AI) models based on cough sound have been developed for several respiratory conditions, with limited work being done in TB. To support the development of an accurate, point-of-care cough-based triage tool for TB, we have compiled a large multi-country database of cough sounds from individuals being evaluated for TB. The dataset includes more than 700,000 cough sounds from 2,143 individuals with detailed demographic, clinical and microbiologic diagnostic information. We aim to empower researchers in the development of cough sound analysis models to improve TB diagnosis, where innovative approaches are critically needed to end this long-standing pandemic.

Medulloblastoma is the most common malignant brain tumor in children. It has WNT-driven, SHH-driven/TP53 mutant, SHH-driven/TP53 wildtype, and non-WNT/non-SHH subgroups. MAGMAS (Mitochondrial Associated Granulocyte Macrophage colony-stimulating factor Signaling molecules) encodes a mitochondrial import inner membrane translocase subunit and is responsible for the translocation of matrix proteins across the inner membrane. We previously reported that a small molecule MAGMAS inhibitor, BT9, decreases cell proliferation, migration, and oxidative phosphorylation in adult glioblastoma cell lines. The aim of our study was to investigate whether the chemotherapeutic effect of BT9 can be extended to pediatric medulloblastoma.MethodsDAOY (SHH driven/tp53 mutant) and D425 (non-SHH group 3) were treated with BT9. For in vitro analysis, cell proliferation, death, migration, invasion, and metabolic activity were assessed using MTT assay, TUNEL staining, scratch wound assay, Matrigel invasion chambers, and seahorse assay, respectively. A D425 orthotopic xenograft mouse model was used to evaluate BT9 efficacy in vivo.ResultsBT9 treatment resulted in a significant decrease in cell proliferation (DAOY, 24 hours IC50: 3.6 μM, 48 hours IC50: 2.3 μM, 72 hours IC50: 2.1 μM; D425 24 hours IC50: 3.4 μM, 48 hours IC50: 2.2 μM, 72 hours IC50: 2.1 μM) and a significant increase in cell death (DAOY, 24 hours p = 0.0004, 48 hours p

Phosphoinositide-3-kinase γ (PI3Kγ) plays a critical role in pancreatic ductal adenocarcinoma (PDA) by driving the recruitment of myeloid-derived suppressor cells (MDSC) into tumor tissues, leading to tumor growth and metastasis. MDSC also impair the efficacy of immunotherapy. In this study we verify the hypothesis that MDSC targeting, via PI3Kγ inhibition, synergizes with α-enolase (ENO1) DNA vaccination in counteracting tumor growth.Mice that received ENO1 vaccination followed by PI3Kγ inhibition had significantly smaller tumors compared to those treated with ENO1 alone or the control group, and correlated with i) increased circulating anti-ENO1 specific IgG and IFNγ secretion by T cells, ii) increased tumor infiltration of CD8+ T cells and M1-like macrophages, as well as up-modulation of T cell activation and M1-like related transcripts, iii) decreased infiltration of Treg FoxP3+ T cells, endothelial cells and pericytes, and down-modulation of the stromal compartment and T cell exhaustion gene transcription, iv) reduction of mature and neo-formed vessels, v) increased follicular helper T cell activation and vi) increased "antigen spreading", as many other tumor-associated antigens were recognized by IgG2c "cytotoxic" antibodies. PDA mouse models genetically devoid of PI3Kγ showed an increased survival and a pattern of transcripts in the tumor area similar to that of pharmacologically-inhibited PI3Kγ-proficient mice. Notably, tumor reduction was abrogated in ENO1 + PI3Kγ inhibition-treated mice in which B cells were depleted.These data highlight a novel role of PI3Kγ in B cell-dependent immunity, suggesting that PI3Kγ depletion strengthens the anti-tumor response elicited by the ENO1 DNA vaccine.

In the original publication [...].

Despite recent advancements in cancer immunotherapy, many patients with gliomas and glioblastomas have yet to experience substantial therapeutic benefits. Modulating the tumor microenvironment (TME) of gliomas, which is typically "cold", is crucial for improving treatment outcomes. Clinical tumor specimens obtained post-immunotherapy provide invaluable insights. However, access to such post-immunotherapy samples remains limited, even in clinical trials, as tumor tissues are often collected only at tumor relapse. Recent studies of neoadjuvant immunotherapy provided important insights by incorporating surgical resections of post-treatment tumors. Moreover, pre-surgical immunotherapies are increasingly integrated into clinical trial designs to evaluate treatment efficacy. These investigations reveal critical information, particularly regarding the delivery success of therapeutic agents, the expansion and persistence of immune products, and the cellular and molecular changes induced in the TME. In this review, we assess the findings on post-treatment tumor specimens obtained from recent immunotherapy clinical trials on gliomas, highlight the importance of these samples for understanding therapeutic impacts, and discuss proactive investigation approaches for future clinical trials.

Therapeutic antibodies designed to target three immune checkpoint proteins have been applied in the treatment of various malignancies, including small and non-small cell lung cancers, melanoma, renal cell carcinoma, and others. These treatments combat cancers by reactivating cytotoxic T cells. Nevertheless, this mode of action was found to be associated with a broad range of immune-related adverse events (irAEs), including pneumonitis, sarcoidosis, myocarditis, nephritis, colitis, and hepatitis. Depending on their severity, these irAEs often necessitate the suspension or discontinuation of treatment and, in rare instances, may lead to fatalities. We analyzed over nineteen million reports and identified over eighty thousand adverse event reports from patients treated with immune checkpoint inhibitors submitted to the Food and Drug Administration's Adverse Event Reporting System MedWatch. Reports concerning pembrolizumab, nivolumab, cemiplimab, avelumab, durvalumab, atezolizumab, and ipilimumab revealed a statistically significant association between the irAEs and concurrent infectious diseases for five out of seven treatments. Furthermore, the association trend was preserved across all three types of checkpoint inhibitors and each of the five individual therapeutic agent cohorts, while the remaining two showed the same trend, but an increased confidence interval, due to an insufficient number of records.

IntroductionCancer combination treatments involving immunotherapies with targeted radiation therapy are at the forefront of treating cancers. However, dosing and scheduling of these therapies pose a challenge. Mathematical models provide a unique way of optimizing these therapies.MethodsUsing a preclinical model of multiple myeloma as an example, we demonstrate the capability of a mathematical model to combine these therapies to achieve maximum response, defined as delay in tumor growth. Data from mice studies with targeted radionuclide therapy (TRT) and chimeric antigen receptor (CAR)-T cell monotherapies and combinations with different intervals between them was used to calibrate mathematical model parameters. The dependence of progression-free survival (PFS), overall survival (OS), and the time to minimum tumor burden on dosing and scheduling was evaluated. Different dosing and scheduling schemes were evaluated to maximize the PFS and optimize timings of TRT and CAR-T cell therapies.ResultsTherapy intervals that were too close or too far apart are shown to be detrimental to the therapeutic efficacy, as TRT too close to CAR-T cell therapy results in radiation related CAR-T cell killing while the therapies being too far apart result in tumor regrowth, negatively impacting tumor control and survival. We show that splitting a dose of TRT or CAR-T cells when administered in combination is advantageous only if the first therapy delivered can produce a significant benefit as a monotherapy.DiscussionMathematical models are crucial tools for optimizing the delivery of cancer combination therapy regimens with application along the lines of achieving cure, maximizing survival or minimizing toxicity.

Sorafenib, FDA-approved therapy for patients with advanced hepatocellular carcinoma (HCC), leads to limited improvement in overall survival. However, it may indirectly impact the expansion and activity of natural killer (NK) cells. While NK cell-based immunotherapies generally exhibit favorable safety profiles, their effectiveness in controlling solid tumor growth is constrained, primarily due to the absence of antigen specificity and suboptimal expansion and persistence within the tumor microenvironment. In this study, we postulated that enhancing NK cell functionality via cytokine activation could bolster their viability and cytotoxic capabilities, leading to an improved therapeutic response when combined with sorafenib. Memory-like (ML)-NK cells were generated through the supplementation of optimal concentrations of interleukin (IL)-12 and IL-18 cytokines. Following a single day of treatment, cytotoxicity against rat and human HCC cells was evaluated via flow cytometry analysis. A rat HCC model was developed in 30 animals via subcapsular implantation and assigned to control, NK, sorafenib, ML-NK, and combination groups. Sorafenib was administered orally, and NK cells were delivered via the intrahepatic artery. Tumor growth was measured one week after treatment evaluation. Therapeutic efficacy during in-vitro and in-vivo analysis was investigated through a one-way ANOVA test, followed by pairwise two-tailed Student t-tests, considering P < 0.05 statistically significant. The in-vitro experiment results demonstrated that sorafenib and conventional NK cell therapies induced more substantial cell death than the control group (P < 0.01). ML NK cells significantly improved cell death compared to conventional NK cell immunotherapy. Furthermore, sorafenib in combination with ML-NK cells significantly decreased the viability of HCC cells (P < 0.05) compared to sorafenib plus conventional NK cell combination therapy. In vivo experiments have shown that sorafenib and ML-NK cell immunotherapy reduced the growth rate of HCC tumors compared to conventional NK immunotherapy and control groups. Notably, a combination of sorafenib and ML-NK cell immunochemotherapy resulted in the most significant suppression of tumor growth when compared to other therapies. In conclusion, our experimental findings demonstrate that the concurrent administration of sorafenib and ML-NK immunotherapy enhances cytotoxicity against HCC by optimizing the therapeutic response through cytokine activation, resulting in a significant decrease in tumor growth.

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6βrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.

BackgroundPatients with multiple myeloma are immunosuppressed due to both the disease itself and immunosuppressive therapies. Thus, when presenting with respiratory failure and pulmonary opacities, pneumonia must be considered. However, while rare, immunomodulating medications used in the treatment of multiple myeloma can also cause potentially life-threatening respiratory failure, a distinction which has important treatment implications.Case presentationAn 80-year-old male with recently diagnosed multiple myeloma undergoing treatment with lenalidomide and daratumumab presented with acute, rapidly progressive hypoxic respiratory failure ultimately requiring intubation and mechanical ventilatory support. Imaging revealed bilateral pulmonary opacities, however infectious workup was negative, and he was ultimately diagnosed with lenalidomide-induced interstitial pneumonitis, a rare but serious adverse effect of this medication. He was treated with drug discontinuation and methylprednisolone, and quickly recovered.ConclusionLenalidomide is an immunomodulating medication used in the treatment of multiple myeloma, and is associated with rare but serious cases of drug-induced interstitial pneumonitis. Thus, if a patient receiving lenalidomide develops shortness of breath and/or hypoxia, drug-induced pneumonitis must be on the differential. Permanent drug discontinuation with or without corticosteroids is the mainstay of treatment, and patients are often able to fully recover, underscoring the need for early recognition of this condition.

BackgroundNeuroblastoma is the most common extra-cranial pediatric solid tumor. 131I-metaiodobenzylguanidine (MIBG) is a targeted radiopharmaceutical highly specific for neuroblastoma tumors, providing potent radiotherapy to widely metastatic disease. Aurora kinase A (AURKA) plays a role in mitosis and stabilization of the MYCN protein in neuroblastoma. We aimed to study the impact of AURKA inhibitors on DNA damage and tumor cell death in combination with 131I-MIBG therapy in a pre-clinical model of high-risk neuroblastoma.ResultsUsing an in vivo model of high-risk neuroblastoma, we demonstrated a marked combinatorial effect of 131I-MIBG and alisertib on tumor growth. In MYCN amplified cell lines, the combination of radiation and an AURKA A inhibitor increased DNA damage and apoptosis and decreased MYCN protein levels.ConclusionThe combination of AURKA inhibition with 131I-MIBG treatment is active in resistant neuroblastoma models.

IntroductionNon-Hodgkin's lymphoma (NHL) encompasses a diverse group of lymphoma subtypes with a wide range in disease course. Previous studies show that hypogammaglobulinemia in treatment-naïve patients is associated with poorer survival in high grade B-cell non-Hodgkin's lymphomas, though it is not known how this applies across all B-cell lymphoid malignancies.MethodsWe conducted a retrospective study of immunoglobulin levels and clinical outcomes including survival, hospitalization, and infection rates in patients diagnosed with B-cell non-Hodgkin lymphomas of all grades at our institution.ResultsTwo-hundred twenty-three adults (aged = 18 years) with available pre-treatment IgG levels were selected, with hypogammaglobulinemia defined as IgG< 500 mg/mL. For this analysis, we grouped DLBCL (n=90), Primary CNS (n=5), and Burkitt lymphoma (n=1) together as high-grade, while CLL (n=52), mantle cell (n=20), marginal zone (n=25), follicular (n=21), and Waldenstrom macroglobulinemia (n=5) were low-grade. The incidence of hypogammaglobulinemia in our cohort of both high and low-grade lymphoma patients was 13.5% (n=30). Across all NHL subtypes, individuals with baseline IgG< 500 mg/dL showed an increased rate of hospitalization (4.453, CI: 1.955-10.54, p= 0.0005) and higher mortality (3.325, CI: 1.258, 8.491, p= 0.013), yet no association in number of infections when compared with those with IgG=500 mg/dL. There was a higher hospitalization rate (3.237, CI: 1.77-6.051, p=0.0017) in those with high-grade lymphoma with hypogammaglobulinemia when compared with low-grade. There was no statistically significant difference in individuals who were alive after three years in those with baseline IgG

Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule sarcomere inhibitors are promising novel therapeutics for the management of obstructive HCM (oHCM) patients and have shown efficacy in left ventricular outflow tract obstruction (LVOTO) relief. The objective of this study was to explore the 6-, 24-, and 48-hour (h) pharmacodynamic effects of the cardiac myosin inhibitor, CK-586, in six purpose-bred cats with naturally occurring oHCM. A blinded, randomized, five-treatment group, crossover preclinical trial was conducted to assess the pharmacodynamic effects of CK-586 in this oHCM model. Dose assessments and select echocardiographic variables were assessed five times over a 48-h period. Treatment with oral CK-586 safely ameliorated LVOTO in oHCM cats. CK-586 treatment dose-dependently eliminated obstruction (reduced LVOTOmaxPG), increased measures of systolic chamber size (LVIDs Sx), and decreased select measures of heart function (LV FS% and LV EF%) in the absence of impact on heart rate. At all tested doses, a single oral CK-586 dose resulted in improved or resolved LVOTO with well-tolerated, dose-dependent, reductions in LV systolic function. The results from this study pave the way for the potential use of CK-586 in both the veterinary and human clinical setting.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKƴ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

Autophagy is a form of programmed cell degradation that enables the maintenance of homeostasis in response to extracellular stress stimuli. Autophagy is primarily activated by starvation and mediates the degradation, removal, or recycling of cell cytoplasm, organelles, and intracellular components in eukaryotic cells. Autophagy is also involved in the pathogenesis of human diseases, including several cancers. Human uveal melanoma (UM) is the primary intraocular malignancy in adults and has an extremely poor prognosis; at present there are no effective therapies. Several studies have suggested that autophagy is important in UM. By understanding the mechanisms of activation of autophagy in UM it may be possible to develop biomarkers to provide more definitive disease prognoses and to identify potential drug targets for the development of new therapeutic strategies. This article reviews the current information regarding autophagy in UM that could facilitate biomarker and drug development.