Gastrointestinal Acute Radiation Syndrome: Mechanisms, Models, Markers, and Medical Countermeasures.

There have been a number of reported human exposures to high dose radiation, resulting from accidents at nuclear power plants (e.g., Chernobyl), atomic bombings (Hiroshima and Nagasaki), and mishaps in industrial and medical settings. If absorbed radiation doses are high enough, evolution of acute radiation syndromes (ARS) will likely impact both the bone marrow as well as the gastrointestinal (GI) tract. Damage incurred in the latter can lead to nutrient malabsorption, dehydration, electrolyte imbalance, altered microbiome and metabolites, and impaired barrier function, which can lead to septicemia and death. To prepare for a medical response should such an incident arise, the National Institute of Allergy and Infectious Diseases (NIAID) funds basic and translational research to address radiation-induced GI-ARS, which remains a critical and prioritized unmet need. Areas of interest include identification of targets for damage and mitigation, animal model development, and testing of medical countermeasures (MCMs) to address GI complications resulting from radiation exposure. To appropriately model expected human responses, it is helpful to study analogous disease states in the clinic that resemble GI-ARS, to inform on best practices for diagnosis and treatment, and translate them back to inform nonclinical drug efficacy models. For these reasons, the NIAID partnered with two other U.S. government agencies (the Biomedical Advanced Research and Development Authority, and the Food and Drug Administration), to explore models, biomarkers, and diagnostics to improve understanding of the complexities of GI-ARS and investigate promising treatment approaches. A two-day workshop was convened in August 2022 that comprised presentations from academia, industry, healthcare, and government, and highlighted talks from 26 subject matter experts across five scientific sessions. This report provides an overview of information that was presented during the conference, and important discussions surrounding a broad range of topics that are critical for the research, development, licensure, and use of MCMs for GI-ARS.

Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics.

The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.

The role of immune checkpoint receptors in the malignant phenotype of cutaneous T cell lymphoma.

Immune checkpoint receptors (ICR) modulate the immune response and are critical hubs for immunotherapy. However, data on their role in T lymphoid malignancies, such as cutaneous T cell lymphoma (CTCL), is sparse. We aimed to explore the role of ICR in the malignant features of transformed T lymphocytes and evaluate the effect of ICR-targeting monoclonal antibodies, often used as immunotherapy for solid tumors. We used the CTCL cell line HH and the Sézary cell line Hut78 to examine ICR expression and the effects of ICR inhibition on cell viability and proliferation. Despite their shared T cell progeny, the different CTCL cell lines exhibit markedly different ICR expression profiles. Programmed cell death-ligand 1 (PD-L1) was expressed by both cell lines, while programmed death-1 (PD-1) was expressed only by the HH cell line. Common to all malignant T cells was an autonomous hyper-proliferative state that did not require T cell receptor stimulation. A monoclonal antibody blocking PD-1 had a small but statistically significant augmenting effect on T cell proliferation. Of note, when the cells were exposed to ionizing radiation, healthy lymphocytes and those derived from the HH cell line were salvaged by anti-PD-L1. We show a regulatory role of ICR, mainly PD-1 and its ligand PD-L1, on cutaneous T cell malignancy.

Liquid biopsy reveals collateral tissue damage in cancer.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

A phase 1a/1b trial of CSF-1R inhibitor LY3022855 in combination with durvalumab or tremelimumab in patients with advanced solid tumors.

Background LY3022855 is a recombinant, immunoglobulin, human monoclonal antibody targeting the colony-stimulating factor-1 receptor. This phase 1 trial determined the safety, pharmacokinetics, and antitumor activity of LY3022855 in combination with durvalumab or tremelimumab in patients with advanced solid cancers who had received standard anti-cancer treatments. Methods In Part A (dose-escalation), patients received intravenous (IV) LY3022855 25/50/75/100 mg once weekly (QW) combined with durvalumab 750 mg once every two weeks (Q2W) IV or LY3022855 50 or 100 mg QW IV with tremelimumab 75/225/750 mg once every four weeks. In Part B (dose-expansion), patients with non-small cell lung cancer (NSCLC) or ovarian cancer (OC) received recommended phase 2 dose (RP2D) of LY3022855 from Part A and durvalumab 750 mg Q2W. Results Seventy-two patients were enrolled (median age 61 years): Part A = 33, Part B = 39. In Part A, maximum tolerated dose was not reached, and LY3022855 100 mg QW and durvalumab 750 mg Q2W was the RP2D. Four dose-limiting equivalent toxicities occurred in two patients from OC cohort. In Part A, maximum concentration, area under the concentration-time curve, and serum concentration showed dose-dependent increase over two cycles of therapy. Overall rates of complete response, partial response, and disease control were 1.4%, 2.8%, and 33.3%. Treatment-emergent anti-drug antibodies were observed in 21.2% of patients. Conclusions LY3022855 combined with durvalumab or tremelimumab in patients with advanced NSCLC or OC had limited clinical activity, was well tolerated. The RP2D was LY3022855 100 mg QW with durvalumab 750 mg Q2W. ClinicalTrials.gov ID: NCT02718911 (Registration Date: May 3, 2011).

ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

Checkpoint inhibitors: Better outcomes among advanced cutaneous head and neck melanoma patients.

OBJECTIVE: The aim of this study was to investigate if the treatment outcomes of checkpoint inhibitors (CPI) in patients with advanced-stage skin head and neck melanoma (HNM) differs from outcomes in patients with non-HNM. DESIGN: A retrospective cohort study of patients with unresectable AJCC stage III and stage IV, who received CPI between 2010 and 2017. PARTICIPANTS: Overall, 122 unresectable AJCC stage III and metastatic stage IV melanoma adult patients were treated with CPI during the study period (consecutive patients). The HNM group of patients was comparable with limbs and trunk melanoma group except different distant metastatic (M1a/b/c/d) pattern (p = 0.025). MAIN OUTCOMES: Comparison of overall survival and clinical response to CPI in patients with advanced-stage skin melanoma of the head and neck with non-HNM. RESULTS: We analyzed 38 patients with melanoma arising in the head and neck skin regions, 33 with melanoma of limbs and 51 with trunk melanoma. Most of the head and neck patients were men (89.5%), the average age of melanoma diagnosis was 61.4±16.7 years (range 16.4-85.6). More than a third of HNM group of patients (36.8%) were 70 years and older. Overall response rate (ORR) to CPI was 50% (CR 31.6% and PR 18.4%) in the head and neck study group of patients, compared to an ORR of 36.3% and 23.5% in melanoma of the limbs and of the trunk, respectively (p = 0.03). The median overall survival of HNM group of patients was 60.2±6.3 months, CI 95% [47.7-72.7], 63% were alive at 30 months, reaching a plateau. Whereas, the median survival time of limbs and trunk melanoma were 51.2 and 53.4 months, which did not reach significance. CONCLUSIONS AND RELEVANCE: Response rate to CPI is significantly improved in patients with melanoma of the head and neck and they have a trend towards improved, long standing, overall survival.

SLAMF6​ deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint.

SLAMF6 is a homotypic receptor of the Ig-superfamily whose exact role in immune modulation has remained elusive. Its constitutive expression on resting and activated T cells precludes it from being a bona fide exhaustion marker. By breeding Pmel-1 mice with SLAMF6 -/- mice, we generated donors for T cells lacking SLAMF6 and expressing a transgenic TCR for gp100-melanoma antigen. Activated Pmel-1xSLAMF6 -/- CD8+ T cells displayed improved polyfunctionality and strong tumor cytolysis. T-bet was the dominant transcription factor in Pmel-1 x SLAMF6 -/- cells, and upon activation, they acquired an effector-memory phenotype. Adoptive transfer of Pmel-1 x SLAMF6 -/- T cells to melanoma-bearing mice resulted in lasting tumor regression in contrast to temporary responses achieved with Pmel-1 T cells. LAG-3 expression was elevated in the SLAMF6 -/- cells, and the addition of the LAG-3-blocking antibody to the adoptive transfer protocol improved the SLAMF6 -/- T cells and expedited the antitumor response even further. The results from this study support the notion that SLAMF6 is an inhibitory immune receptor whose absence enables powerful CD8+ T cells to eradicate tumors.

The immune system helps to protect our bodies from illnesses and infections. Immunotherapies are medicines designed to treat diseases, such as cancer, by boosting the immune system against the condition. This is a powerful approach but so far immunotherapies have only had partial success and there is a need for further improvements. One protein called SLAMF6 is found on cells from the immune system that attack and kill cancer cells. Immunotherapies that suppress SLAMF6 on immune cells called killer T cells could increase immune system activity helping to treat cancers, particularly melanoma skin cancers. So far the potential for SLAMF6 as a target for immunotherapy has not been fully explored. Hajaj et al. created mice with killer T cells that recognized skin cancer cells and lacked SLAMF6. These modified cells were better at fighting cancer, producing more anti-cancer chemicals called cytokines and killing more cancer cells. The modified cells had a lasting effect on tumors and helped the mice to live longer. The effects could be further boosted by treating the mice in combination with other immunotherapies. SLAMF6 is a possible new target for skin cancer immunotherapy that could help more people to live longer following cancer diagnosis. The next step is to create a drug to target SLAMF6 in humans and to test it in clinical trials.

Intratumoral immune-biomarkers and mismatch repair status in leiyomyosarcoma -potential predictive markers for adjuvant treatment: a pilot study.

Leiomyosarcoma is the second most frequent soft-tissue sarcoma. Tumor lymphocytic infiltration (TIL) and programed cell death ligand-1 (PD-L1) have been associated with prognosis in different malignancies while DNA mismatch-repair deficiency (MMR-D) has been associated with response to check-point inhibitors. In this pilot study, we sought to examine TIL, PD-L1 and mismatch-repair (MMR) protein expression in 11 leiomyosarcoma and its association with outcome as potential biomarkers for adjuvant treatment. Eleven primary leiomyosarcoma archived-tissues were analyzed for expression of MMR proteins (MSH2, MLH1, MSH6 and PSM2), PD-L1 expression and PD-1, CD3 or CD8. MMR-D was detected in tumor tissue from 2/11 leiomyosarcoma patients. CD3 T-cells were present in all samples, whereas CD8 staining was positive in all but one. PDL-1 was positive in 4/11 and PD-L1 in 6/11. Interestingly, the three patients with the poorest outcome had strongly positive staining for PD-L1 and CD8 while in the two patients who are alive and recurrence-free, both PD-L1 and CD8 infiltration were lacking. We found an association between tumor infiltrating CD8 cytotoxic lymphocytes, strong PD-L1 staining and survival; suggesting a role as biomarkers for treatment decisions regarding peri-operative chemotherapy. We also identified MMR-D in two patients with leiomyosarcoma comprising 18% of our sample.

Adoptive cell therapy: past, present and future.

The immune system is a potent inhibitor of tumor growth with curative potential, constituting in many eyes the future of antineoplastic therapy. Adoptive cell therapy (ACT) is a form of immunotherapy in which autologous cancer-cognate lymphocytes are expanded and modified ex vivo and re-infused to combat the tumor. This review follows the evolvement of ACT and treatment protocols, focusing on unresolved dilemmas regarding this treatment while providing evidence for its effectiveness in refractory patients. Future directions of ACT are discussed, in particular with regard to genetic engineering of autologous cells, and the role of ACT in the era of checkpoint inhibitors is addressed.

Retrospective Study of the Predictive Value of Target Now in Systemic Therapy for Metastatic Colorectal and Gastric Carcinomas.

BACKGROUND: Predictive biomarkers for personalized treatment of neoplasms are suggested to be a major advancement in oncology and are increasingly used in clinical practice, albeit based on level II evidence. Target Now (TN) employs immunostaining and RNA expression on tumor samples to identify potentially beneficial or ineffective drugs. OBJECTIVES: To explore retrospectively the predictive value of TN for patients with colorectal and gastric carcinomas. METHODS: The study group comprised colorectal and gastric carcinoma patients with TN test reports. We identified chemotherapy regimens given for stage IV disease for which TN reports indicated prediction. Protocols were classified as having clinical benefit (CB; i.e., stable disease or any objective response) or progressive disease, and this was compared with the TN prediction. RESULTS: Nineteen patients--12 colorectal and 7 gastric carcinomas--met the inclusion criteria. There were 26 evaluable treatment protocols; of 18 with a CB15 were predicted to have a CB while 3 were predicted to have a lack of CB. Of eight protocols that had no CB, seven were predicted to have a CB and one a lack of CB. A chi-square test was non-significant (P = 0.78). An exploratory analysis yielded a positive predictive value of 68% and a sensitivity of 83% for the TN test. CONCLUSIONS: This study emphasizes the need for larger multi-center studies to validate the TN test before it is adopted into clinical practice.

Defective skeletal muscle growth in lamin A/C-deficient mice is rescued by loss of Lap2α.

Mutations in lamin A/C result in a range of tissue-specific disorders collectively called laminopathies. Of these, Emery-Dreifuss and Limb-Girdle muscular dystrophy 1B mainly affect striated muscle. A useful model for understanding both laminopathies and lamin A/C function is the Lmna(-/-) mouse. We found that skeletal muscle growth and muscle satellite (stem) cell proliferation were both reduced in Lmna(-/-) mice. Lamins A and C associate with lamina-associated polypeptide 2 alpha (Lap2α) and the retinoblastoma gene product, pRb, to regulate cell cycle exit. We found Lap2α to be upregulated in Lmna(-/-) myoblasts (MBs). To specifically test the contribution of elevated Lap2α to the phenotype of Lmna(-/-) mice, we generated Lmna(-/-)Lap2α(-/-) mice. Lifespan and body mass were increased in Lmna(-/-)Lap2α(-/-) mice compared with Lmna(-/-). Importantly, the satellite cell proliferation defect was rescued, resulting in improved myogenesis. Lmna(-/-) MBs also exhibited increased levels of Smad2/3, which were abnormally distributed in the cell and failed to respond to TGFß1 stimulation as in control cells. However, using SIS3 to inhibit signaling via Smad3 reduced cell death and augmented MB fusion. Together, our results show that perturbed Lap2α/pRb and Smad2/3 signaling are important regulatory pathways mediating defective muscle growth in Lmna(-/-) mice, and that inhibition of either pathway alone or in combination can ameliorate this deleterious phenotype.

Myogenesis in dysferlin-deficient myoblasts is inhibited by an intrinsic inflammatory response.

Limb-girdle muscular dystrophy type 2B results from mutations in dysferlin, a membrane-associated protein involved in cellular membrane repair. Primary myoblast cultures derived from dysferlinopathy patients show reduced myogenic potential, suggesting that dysferlin may regulate myotube fusion and be required for muscle regeneration. These observations contrast with the findings that muscle develops normally in pre-symptomatic dysferlinopathy patients. To better understand the role of dysferlin in myogenesis, we investigated this process in vitro using cells derived from two mouse models of dysferlinopathy: SJL/J and A/J mice. We observed that myotubes derived from dysferlin-deficient muscle were of significantly smaller diameters, contained fewer myonuclei, and displayed reduced myogenic gene expression compared to dysferlin-sufficient cells. Together, these findings suggest that the absence of dysferlin from myoblasts is detrimental to myogenesis. Pro-inflammatory NFκB signaling was upregulated in dysferlin-deficient myotubes; the anti-inflammatory agent celastrol reduced the NFκB activation and improved myogenesis in dysferlin-deficient cultures. The results suggest that decreased myotube fusion in dysferlin deficiency is attributable to intrinsic inflammatory activation and can be improved using anti-inflammatory mediators.

Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin-sensitive adenylyl cyclase.

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca(2+)/CaM. AC-AplC is most similar to the Ca(2+)-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca(2+), independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca(2+)-inhibited AC in SNs, together with a Ca(2+)/CaM-stimulated AC, would enhance the associative requirement for coincident Ca(2+) influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.

Activity-dependent neuron-glial signaling by ATP and leukemia-inhibitory factor promotes hippocampal glial cell development.

Activity-dependent signaling between neurons and astrocytes contributes to experience-dependent plasticity and development of the nervous system. However, mechanisms responsible for neuron-glial interactions and the releasable factors that underlie these processes are not well understood. The pro-inflammatory cytokine, leukemia-inhibitory factor (LIF), is transiently expressed postnatally by glial cells in the hippocampus and rapidly up-regulated by enhanced neural activity following seizures. To test the hypothesis that spontaneous neural activity regulates glial development in hippocampus via LIF signaling, we blocked spontaneous activity with the sodium channel blocker tetrodotoxin (TTX) in mixed hippocampal cell cultures in combination with blockers of LIF and purinergic signaling. TTX decreased the number of GFAP-expressing astrocytes in hippocampal cell culture. Furthermore, blocking purinergic signaling by P2Y receptors contributed to reduced numbers of astrocytes. Blocking activity or purinergic signaling in the presence of function-blocking antibodies to LIF did not further decrease the number of astrocytes. Moreover, hippocampal cell cultures prepared from LIF -/- mice had reduced numbers of astrocytes and activity-dependent neuron-glial signaling promoting differentiation of astrocytes was absent. The results show that endogenous LIF is required for normal development of hippocampal astrocytes, and this process is regulated by spontaneous neural impulse activity through the release of ATP.

Immune system evasion by peripheral nerve sheath tumor.

Mechanisms by which tumor cells evade detection by the host's immune system are thought to play a role in progression to malignancy, but this has not been investigated in the context of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant disorder, in which aggressive peripheral nerve tumors, known as malignant peripheral nerve sheath tumors (MPNSTs), develop in 5-10% of patients. Large scale gene expression profiling of a MPNST-derived cell line, T265, and normal human Schwann cells (hSCs) identified a large group of immune function genes down-regulated in T265 cells. Here we report that the aberrant expression of immune system related genes extends beyond MHC class I and II genes in T265 cells to include a transcription factor (MHC2TA) and other critical components of the antigen processing and presentation apparatus. TAP1, the transporter-activator protein that loads peptide antigens onto MHC class I molecules, is down-regulated, and CD74, a chaperone protein whose function is in processing and transport of MHC class II molecules, is down-regulated and alternatively spliced to produce an RNA transcript not evident in normal human Schwann cells. These findings reveal multiple molecular pathways and at least two cellular mechanisms acting to reduce the normal immune system molecules involved in antigen processing and presentation in cells derived from a peripheral nerve sheath tumor. Acquiring a "silent" immune signature may be a critical step in the progress towards malignancy in MPNSTs.

Serotonin receptor antagonists discriminate between PKA- and PKC-mediated plasticity in aplysia sensory neurons.

Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.