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CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1β and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line–derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.

TSC2 immunohistochemistry assay validation and analysis of HCC xenograft and primary tumors. (A) TSC2 immunohistochemistry (IHC) assay with HCC cell lines. (B) TSC2 IHC assay with HCC xenograft tumors. (C) TSC2 expression by IHC assay in HCC xenografts and their corresponding primary tumors.

Race distribution of HCC patients in EVOLVE-1 trial. Percentage of Caucasian, Asian and other race in different groups of EVOLVE-1 patients (A) and TSC2 IHC H-score ranges (B).

Mechanisms of TSC2 loss in HCC cell lines and xenografts. (A) Taqman RT-PCR analysis of TSC1 and TSC2 mRNA levels in HCC cell lines. (B) Taqman RT-PCR analysis of MZB1, TSC1 and TSC2 mRNA levels in TSC2-null Li-7 cells treated with epigenetic inhibitors. (C) Taqman RT-PCR analysis and (D) immunoblot analysis of TSC2 G305V mutant in HEK293T cells.

Genetic characterization of 35 established HCC PTX models by targeted DNA sequencing (Excel file).

Supplementary Table 1. Quantification of phospho-protein levels in Figure 1B. Supplementary Table 2. Everolimus IC50 and growth inhibition at 1uM in HCC cell lines. Supplementary Table 3. Quantification of phospho-protein levels in Figure 2A.

TSC2 loss in HCC cell lines leads to mTOR activation. (A) Reverse Phase Protein Array (RPPA) analysis of nine HCC cell lines for TSC2 protein levels and phosphorylation levels of S6K1, 4E-BP1, S6 and AKT. (B) Immunoblot of TSC2 wild-type HuH-7 cells treated with inhibitors of PI3K, AKT or MEK. (C) p-S6K1(T389) immunoblot of TSC2-null HCC cell lines treated with varying concentrations of everolimus.

Model of mTOR activation and everolimus sensitivity caused by TSC2 loss.

Supplementary Figure 1 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 190K, JAK2-inhibitor trial (proliferation assay, colony assay)

Supplementary Figure 3 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 626K, Leukemia phenotype (FACS, immunohistochemistry)

Supplementary Table 5 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 339K, LSC sorting strategy

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Supplementary Methods, Figure and Table Legends 1-7 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Supplementary Table 7 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

PDF file - 58K, Colony assay with and without cytokines

Supplementary Table 6 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Supplementary Figure 4 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Supplementary Figure 2 from Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

OBJECTIVE A major obstacle to improving bedside neurosurgical procedure safety and accuracy with image guidance technologies is the lack of a rapidly deployable, real-time registration and tracking system for a moving patient. This deficiency explains the persistence of freehand placement of external ventricular drains, which has an inherent risk of inaccurate positioning, multiple passes, tract hemorrhage, and injury to adjacent brain parenchyma. Here, the authors introduce and validate a novel image registration and real-time tracking system for frameless stereotactic neuronavigation and catheter placement in the nonimmobilized patient. METHODS Computer vision technology was used to develop an algorithm that performed near-continuous, automatic, and marker-less image registration. The program fuses a subject’s preprocedure CT scans to live 3D camera images (Snap-Surface), and patient movement is incorporated by artificial intelligence–driven recalibration (Real-Track). The surface registration error (SRE) and target registration error (TRE) were calculated for 5 cadaveric heads that underwent serial movements (fast and slow velocity roll, pitch, and yaw motions) and several test conditions, such as surgical draping with limited anatomical exposure and differential subject lighting. Six catheters were placed in each cadaveric head (30 total placements) with a simulated sterile technique. Postprocedure CT scans allowed comparison of planned and actual catheter positions for user error calculation. RESULTS Registration was successful for all 5 cadaveric specimens, with an overall mean (± standard deviation) SRE of 0.429 ± 0.108 mm for the catheter placements. Accuracy of TRE was maintained under 1.2 mm throughout specimen movements of low and high velocities of roll, pitch, and yaw, with the slowest recalibration time of 0.23 seconds. There were no statistically significant differences in SRE when the specimens were draped or fully undraped (p = 0.336). Performing registration in a bright versus a dimly lit environment had no statistically significant effect on SRE (p = 0.742 and 0.859, respectively). For the catheter placements, mean TRE was 0.862 ± 0.322 mm and mean user error (difference between target and actual catheter tip) was 1.674 ± 1.195 mm. CONCLUSIONS This computer vision–based registration system provided real-time tracking of cadaveric heads with a recalibration time of less than one-quarter of a second with submillimetric accuracy and enabled catheter placements with millimetric accuracy. Using this approach to guide bedside ventriculostomy could reduce complications, improve safety, and be extrapolated to other frameless stereotactic applications in awake, nonimmobilized patients.

BACKGROUNDBehavioral comorbidities, such as anxiety and depression, are a prominent feature of IBD. The signals from the inflamed gut that cause changes in the brain leading to these behavioral comorbidities remain to be fully elucidated. We tested the hypothesis that enhanced leukocyte - cerebral endothelial cell interactions in experimental colitis initiate neuroimmune activation leading to anxiety-like behaviour.METHODSMale and female mice treated with dextran sodium sulfate were studied at the peak of acute colitis. Circulating leukocyte populations were determined using flow cytometry. Leukocyte - cerebral endothelial cell interactions were examined using intravital microscopy in mice treated with anti-integrin antibodies. Brain cytokine and chemokines were assessed using a multiplex assay in animals treated with anti-α4β7 integrin. Anxiety-like behavior was assessed using an elevated plus maze in animals after treatment with an intracerebroventricular injection of interleukin 1 receptor antagonist. RESULTSThe proportion of classical monocytes expressing α4β7 integrin was increased in peripheral blood of mice with colitis. An increase in the number of rolling and adherent leukocytes on cerebral endothelial cells was observed, the majority of which were neutrophils. Treatment with anti-α4β7 integrin significantly reduced the number of rolling leukocytes. After anti-Ly6C treatment to deplete monocytes, the number of rolling and adhering neutrophils were significantly reduced in mice with colitis. Interleukin-1β levels were elevated in the brain and treatment with anti-α4β7 significantly reduced them. Enhanced anxiety-like behaviour in mice with colitis was reversed by treatment with interleukin 1 receptor antagonist. CONCLUSIONSα4β7 integrin expressing monocytes direct the recruitment of neutrophils to the brain vasculature, leading to elevated cytokine levels that mediate anxiety-like behaviour in experimental colitis.

Robotic neurosurgery may improve the accuracy, speed, and availability of stereotactic procedures. We recently developed a computer vision and artificial intelligence-driven frameless stereotaxy for nonimmobilized patients, creating an opportunity to develop accurate and rapidly deployable robots for bedside cranial intervention.To validate a portable stereotactic surgical robot capable of frameless registration, real-time tracking, and accurate bedside catheter placement.Four human cadavers were used to evaluate the robot's ability to maintain low surface registration and targeting error for 72 intracranial targets during head motion, ie, without rigid cranial fixation. Twenty-four intracranial catheters were placed robotically at predetermined targets. Placement accuracy was verified by computed tomography imaging.Robotic tracking of the moving cadaver heads occurred with a program runtime of 0.111 ± 0.013 seconds, and the movement command latency was only 0.002 ± 0.003 seconds. For surface error tracking, the robot sustained a 0.588 ± 0.105 mm registration accuracy during dynamic head motions (velocity of 6.647 ± 2.360 cm/s). For the 24 robotic-assisted intracranial catheter placements, the target registration error was 0.848 ± 0.590 mm, providing a user error of 0.339 ± 0.179 mm.Robotic-assisted stereotactic procedures on mobile subjects were feasible with this robot and computer vision image guidance technology. Frameless robotic neurosurgery potentiates surgery on nonimmobilized and awake patients both in the operating room and at the bedside. It can affect the field through improving the safety and ability to perform procedures such as ventriculostomy, stereo electroencephalography, biopsy, and potentially other novel procedures. If we envision catheter misplacement as a "never event," robotics can facilitate that reality.

Microglia play an important role in the pathogenesis of multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). To more fully understand the role of microglia in EAE we characterized microglial transcriptomes before the onset of motor symptoms (pre-onset) and during symptomatic EAE. We compared the transcriptome in brain, where behavioral changes are initiated, and spinal cord, where damage is revealed as motor and sensory deficits. We used a RiboTag strategy to characterize ribosome-bound mRNA only in microglia without incurring possible transcriptional changes after cell isolation. Brain and spinal cord samples clustered separately at both stages of EAE, indicating regional heterogeneity. Differences in gene expression were observed in the brain and spinal cord of pre-onset and symptomatic animals with most profound effects in the spinal cord of symptomatic animals. Canonical pathway analysis revealed changes in neuroinflammatory pathways, immune functions and enhanced cell division in both pre-onset and symptomatic brain and spinal cord. We also observed a continuum of many pathways at pre-onset stage that continue into the symptomatic stage of EAE. Our results provide additional evidence of regional and temporal heterogeneity in microglial gene expression patterns that may help in understanding mechanisms underlying various symptomology in MS.

SRF388 is a first-in-class, anti-IL-27 antibody developed to enhance immune responses within the tumor microenvironment. Preclinical studies have demonstrated that IL-27 pathway blockade can inhibit tumor growth in mouse models of liver cancer and lung cancer metastases. There is also evidence that IL-27 pathway inhibition is accompanied by activation of the innate and adaptive arms of the immune system. An ongoing Phase I trial has shown good tolerability at all dose levels tested and preliminary monotherapy antitumor activity with SRF388 (NCT04374877). Here, we describe the preclinical and clinical data used to select a RP2D and characterize cytokine and chemokine changes observed in patients after treatment with SRF388. To guide selection of the RP2D, the relationship between maximal effective dose (MaxED), pharmacokinetics (PK), and whole blood inhibition of IL-27-mediated phosphorylation of STAT1 by SRF388 was evaluated in a mouse model of liver cancer. The concentration of SRF388 associated with optimal antitumor activity was ~20-fold above the concentration needed for complete inhibition (> 90%) of whole blood phosphorylated STAT1 (pSTAT1). These PK and activity relationships were also defined in patients during the dose-escalation phase of the trial and integrated with safety and efficacy data to select a monotherapy RP2D. In patients, PK were linear, no dose-limiting toxicities were reported, complete pSTAT1 inhibition was achieved throughout the first cycle at 0.3 mg/kg, and 1 patient experienced a confirmed partial response per RECIST version 1.1 at a dose of 10 mg/kg. In the MaxED mouse model, the area under the concentration versus time curve (AUC) associated with significant antitumor activity was 1720 day*μg/mL. In patients, the corresponding target AUC was achieved clinically at 10 mg/kg after a single dose of SRF388. Changes in the concentration of several serum cytokines and chemokines were observed after SRF388 treatment including an increase in IL-27, a phenomenon described for other anti-cytokine antibodies due to altered clearance of the cytokine-antibody complex. Changes in a subset of other serum cytokines/chemokines correlated with a reduction in target lesion size. Taken together, these data support the selection of a monotherapy RP2D of 10 mg/kg intravenously every 4 weeks for SRF388. These data highlight how complementary strategies utilizing preclinical and clinical biomarker evaluations can be employed to establish a monotherapy RP2D and assess biological activity of a first-in-class anti-cytokine antibody, SRF388, for patients with cancer. Citation Format: Jonathan A. Hill, Kerry F. White, Matthew Rausch, Jou-Ku Chung, Amita Patnaik, Aung Naing, Daniel Morgensztern, Charlene M. Mantia, Nizar M. Tannir, Lon S. Smith, Beth Bowers, Alex Alika, Lauren C. Harshman, Benjamin H. Lee. Determination of a recommended Phase 2 dose (RP2D) for SRF388, a first-in-class IL-27-blocking antibody, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1137.

Background IL-27 is a heterodimeric cytokine consisting of IL 27p28 and Epstein-Barr virus-induced gene 3 (EBI3) that binds the IL-27 receptor subunit alpha and glycoprotein 130. IL-27 is produced by activated macrophages and dendritic cells and limits the intensity and duration of immune responses in the tumor microenvironment by inducing the expression of immunoregulatory receptors (PD-L1, TIM3, LAG-3, TIGIT) and inhibiting production of proinflammatory cytokines (IFNγ, IL-17, TNFα). The IL-27 subunit EBI3 is elevated in plasma from patients with certain cancers including renal cell carcinoma, where it correlates with poor outcome. Based on high expression of IL-27 transcript in tumors from patients with hepatocellular carcinoma (HCC), the role of IL-27 was further explored in patient samples and a mouse model of HCC. Methods Gene expression profiles from the Cancer Genome Atlas (TCGA) were analyzed to identify tumors with elevated IL-27 transcripts. Serum from patients with HCC was analyzed for levels of the IL-27 subunit EBI3. The ability of SRF388, a first-in-class IL-27-blocking antibody that binds to IL-27p28, to reverse IL-27-induced inhibition of cytokine production in human immune cell cultures from patients with HCC was assessed in vitro. Finally, the anti-tumor activity of SRF388 was assessed in an orthotopic murine model of HCC. Results TCGA expression data revealed that IL-27p28 transcripts were elevated in tumors from patients with HCC relative to other indications. Serum levels of EBI3 were: 1) elevated in a subset of HCC patients; 2) inversely correlated with survival; 3) independent of serum alpha-fetoprotein levels; and 4) elevated in both hepatitis B/C virus positive and negative patients. Treatment with SRF388 stimulated increased cytokine production in activated peripheral blood mononuclear cells from patients with HCC that was further enhanced when combined with PD-1 blockade. Furthermore, SRF388 inhibited the growth of orthotopic Hepa1-6 liver tumors. mRNA transcriptional profiling of treated tumors revealed that SRF388 profoundly altered the transcriptional landscape in this model. In particular, treatment with SRF388 inhibited expression of immunoregulatory receptors PD-L1 and TIGIT, repressed transcripts associated with TGF-β signaling, and altered myeloid and natural killer cell transcripts. Conclusions These data indicate that elevated IL-27 subunit EBI3 is a hallmark of HCC and is associated with poor outcomes in these patients. Blockade of IL-27 with SRF388, currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04374877), may represent a promising therapy for patients with HCC where it can potentiate anti-tumor immune responses.

BACKGROUND: Novice anesthesiology residents must acquire new technical, cognitive, and behavioral skills as they transition into the high-stakes perioperative environment. Simulation-based education improves procedural skill and behavior, and it permits deliberate practice with feedback; exposure to uncommon, high-consequence events; assessment; reproducibility; and zero risk to patients. We introduced a 5-day, high-fidelity Simulation Boot Camp (SBC) in 2006 for first-year clinical anesthesia residents (CA-1s) and report over a decade of experience assessing its impact on self-efficacy, value, feasibility, and sustainability. METHODS: All CA-1s in our residency program participated in the SBC as part of orientation. Participants completed 2 individual high-fidelity simulations per day, each with a private debriefing session from an attending anesthesiologist in our simulation center. We measured their self-reported confidence, which we report as self-efficacy (SE), the belief in one’s own ability to successfully execute a skill or behavior necessary for a desired outcome, for 25 basic anesthesia skills before and after course completion. Participants also completed a postcourse evaluation. RESULTS: Of the 281 CA-1s who participated in the course from 2006 to 2016, we collected data on 267 (95%). SE improved over the course of SBC for all 25 individual skills (P < .001) and remained stable over the decade-long period of study. Univariate analysis revealed a strong association between increased SE and male sex (P < .001), video gaming experience (P < .001), and completion of a prior residency (P = .018). Males were also more likely to report video gaming experience (P < .001). Multivariable analysis revealed that although women had lower SE than did men, they had a greater increase in SE attributed to participation in SBC (P = .041). Participants strongly agreed SBC was a realistic and nonjudgmental learning tool, built confidence, and should be mandatory. Most comments were positive, reflecting overall satisfaction with SBC CONCLUSIONS: SBC increases SE, is feasible, valuable to participants, and sustainable with remarkably consistency over the study period.

Patients with rheumatoid arthritis experience chronic pain, depression and fatigue, even when inflammation of the joints is well controlled. To study the relationship between arthritis, depression, and sustained pain when articular inflammation is no longer observed, we tested the hypothesis that brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis. The murine model of antigen-induced arthritis (AIA) was used to evaluate the effects of knee inflammation on sustained pain and depression-like behavior. We measured joint pain using an automated dynamic plantar algesiometer and depression-like behavior with the tail suspension test. Cytokines were measured by Luminex assay and ELISA. TNF in the brain was blocked by intracerebroventricular injection of anti-TNF antibodies. Histological damage and elevated levels of cytokines were observed in the knee 24 h after antigen treatment, but not at 13 days. Reduced pain thresholds were seen 24 h and 13 days after treatment. Depression-like behavior was observed on day 13. Treatment with the antidepressant imipramine reduced both depression-like behavior and persistent pain. However, blocking joint pain with the analgesic dipyrone did not alter depression-like behavior. Elevated levels of TNF, CCL2, and CXCL-1 were observed in the hippocampus 24 h after treatment, with TNF remaining elevated at day 13. Intracerebroventricular infusion of an anti-TNF antibody blocked depression-like behavior and reduced persistent pain. We have demonstrated that depression-like behavior and pain is sustained in AIA mice after the resolution of inflammation. These changes are associated with elevated levels of TNF in the hippocampus and are dependent upon brain TNF. The findings reveal an important mechanistic link between the expression of chronic pain and depression in experimental arthritis. Furthermore, they suggest treating depression in rheumatoid arthritis may positively impact other debilitating features of this condition.

Emotional dysfunction is common in multiple sclerosis (MS) patients and in mouse models of MS, including experimental autoimmune encephalomyelitis (EAE); however, the etiology of these behaviors is poorly understood. To identify CNS changes associated with these behaviors, we focused on the basolateral amygdala (BLA) because of its central role in the regulation of emotional behavior. Whole-cell recordings were performed in the principal neurons of the BLA in early EAE, before demyelination, T-cell invasion, and motor dysfunction. EAE female mice displayed increased frequency of mEPSCs, with no alteration in amplitude or evoked EPSC paired-pulse ratio compared with controls. We found an increase in the AMPA-NMDA ratio and dendritic spine density, indicating increased numbers of glutamatergic synapses. We saw similar electrophysiological changes in BLA principal neurons after microglia were either inactivated (minocycline) or depleted (Mac1-Saporin) in the BLA. Microglia regulate synapses through pruning, directed by complement protein 3 (C3) expression. C3 was downregulated in the BLA in EAE. Ultrastructural analysis of microglia revealed more complex ramifications and reduced extracellular digestion of cellular elements. We also observed reduced IBA-1 and CD68 staining and lack of proinflammatory cytokine expression in the amygdala. Thus, early EAE is a state of microglial “deactivation” associated with reduced synaptic pruning. This contrasts with the prototypic microglial activation commonly associated with inflammatory CNS disease. Additionally, these data support a role for the acquired immune system to influence both neuronal and microglial function in early CNS autoimmunity.SIGNIFICANCE STATEMENTMicroglia help regulate synaptic homeostasis, but there has been little evidence for how this might be important in neuroinflammatory diseases. The data from this study reveal increased synaptic activity and spine density in early stages of experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis) in the basolateral amygdala, a nucleus important in the types of behavioral changes we have previously described. These electrophysiological and morphological effects occurred without significant elevation of local inflammatory cytokines or local demyelination. Unexpectedly, in the context of inflammatory state, we found that microglia were “deactivated.” This study provides strong evidence for a link between microglial activity and synaptic function; the conclusions contrast with the generally accepted view that microglia are activated in inflammatory disease.

1. Adam C. Adler, MS, MD*,‡ 2. Stephanie Leung, BS, CCLS† 3. Benjamin H. Lee, MD*,‡ 4. Scott R. Dubow, MD§ 1. *Department of Anesthesiology, Perioperative and Pain Medicine and 2. †Department of Child Life, Texas Children’s Hospital, Houston, TX 3. ‡Baylor College of Medicine, Houston, TX 4. §Department of Anesthesiology and Critical Care Medicine, The Children’s Hospital of Philadelphia; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA * Abbreviations: BiPAP: : bilevel positive airway pressure CLS: : child life specialist CPAP: : continuous positive airway pressure DEA: : Drug Enforcement Administration IV: : intravenous MRI: : magnetic resonance imaging OSA: : obstructive sleep apnea Clinicians should: identify the resources available to patients and families to allow for a smooth perioperative experience; understand the long-term effects and potential maladaptive changes associated with an anxiety-provoking perioperative period; understand the most common parental and patient concerns and misconceptions regarding surgery; understand the most common and up-to-date perioperative anesthesia practices; understand the indication for referral to the preoperative anesthesia clinic for patient evaluation to avoid unnecessary cancellation. Pediatricians are often tasked with preparing children and their families for the preoperative period and dealing with questions related to postoperative issues and concerns. Instruction based on current practice and new research and techniques may help patients and families cope with the stressors of surgery. Determining which patients may benefit from a medical or child life preoperative visit is of crucial importance. After completing this article, the reader should be able to: 1. Describe common preoperative anesthesia practice. 2. Recognize the role of a child life specialist in preparing children for surgery. 3. Differentiate children who would benefit from a preoperative anesthesia and/or child life evaluation. 4. Instruct patients and families on issues related to the perioperative period. 5. Address a wide array of parental concerns regarding surgery, anesthesia, and postoperative pain management. The period of time surrounding a planned surgical procedure can be tumultuous and anxiety provoking for both pediatric patients and their parents. The “mysterious place” that is the operating room often remains a mystery to many health care providers, including those tasked with caring for patients both before and after a procedure. As the health care provider most familiar to patients and their families, the pediatrician is in a …

BackgroundThe purine nucleotide adenosine plays an important role in dampening both the innate and adaptive arms of the immune system. In contrast, extracellular adenosine triphosphate (ATP), which can be generated at high levels due to cell stress, initiates proinflammatory responses. Extracellular ATP can be produced in cancerous tumors, and its conversion to adenosine monophosphate by CD39 (ENTPD1) and subsequent degradation to free adenosine by CD73 (NT5E) in the tumor microenvironment (TME) is a major contributor to tumor immune evasion. Accordingly, CD39 serves as an important immune regulator. CD39 is highly expressed in the TME of various tumor types, including lung, prostate, colon, ovarian, and pancreatic cancers, making CD39 an attractive target for immune-based anticancer therapy.SRF617 is a fully human anti-CD39 antibody designed to target human CD39 and inhibit its enzymatic activity. SRF617 provides a dual mechanism of immune activation by increasing inflammatory responses through accumulation of free ATP and alleviating immune suppression by reducing adenosine production. In this way, SRF617 shifts the TME to a proinflammatory state leading to reactivation of the immune system and antitumor function.MethodsA pharmacokinetic/pharmacodynamic profile was determined in CT26 tumor-bearing human CD39 knock-in (hCD39 KI) mice by measuring plasma SRF617 concentration, peripheral blood target occupancy (TO), and ATP hydrolysis activity in frozen tissue sections. Wild-type mice bearing subcutaneous (s.c.) KPC1245 tumors were treated with anti-murine CD39 antibody, alone or in combination with gemcitabine.ResultsCirculating SRF617 was observed in the plasma after intravenous injection of CT26 tumor-bearing hCD39 KI mice, which correlated with CD39-bound SRF617 on B cells. The presence of SRF617 correlated with a decrease in ATPase activity and CD39 expression in tissues. In the KPC tumor model, a combination effect was observed for anti-CD39 treatment with gemcitabine. Treatment with anti-CD39 antibody resulted in an increase in CD8+ T cells in the TME as measured by flow cytometry.ConclusionsSRF617 treatment of hCD39 KI mice led to reduction of ATPase activity in the TME that correlated with peripheral TO and plasma concentration. Anti-CD39 treatment alone or in combination with gemcitabine led to tumor growth inhibition and increased tumor-infiltrating lymphocytes as measured by flow cytometry in s.c. KPC tumors. These studies demonstrate the therapeutic potential of targeting CD39 for the treatment of cancer. SRF617 is currently being evaluated in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04336098) and in combination with chemotherapy in patients with pancreatic cancer.

BackgroundInterleukin (IL)-27 is a heterodimeric immunoregulatory cytokine that signals through the JAK/STAT pathway to increase the expression of coinhibitory receptors on immune cells (e.g. PD-L1, TIM-3, LAG-3) and dampen inflammatory cytokine production. Blockade of IL-27 leads to antitumor activity in preclinical mouse models of lung metastases. A Phase 1 trial of SRF388 (NCT04374877), a first-in-class anti–IL-27 antibody, has demonstrated monotherapy antitumor activity in a patient with non-small cell lung cancer (NSCLC).1 The current study aimed to characterize the immunoregulatory impact of IL-27 signaling by gene expression profiling.MethodsGene expression changes induced by IL-27 were examined in activated human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs), and the IL-27RA–expressing lung cancer cell line NCI-H2228 by microarray or single cell RNA-sequencing. The resulting IL-27 signature genes were interrogated by Gene Set Enrichment Analysis (GSEA) using publicly available datasets, including single cell RNA-seq analysis of the tumor microenvironment, from patients with NSCLC.2ResultsIL-27 induced a robust gene expression program in human immune cells that included several inhibitory receptors and canonical interferon regulated genes such as guanylate-binding proteins and interferon regulatory factors. GSEA and interferon signature analysis showed a striking overlap with those genes regulated by interferon-beta, a cytokine known to drive immune suppression associated with chronic viral infection and that is used therapeutically for controlling inflammation associated with the autoimmune disease multiple sclerosis. Moreover, interferon regulated pathways have recently emerged as a mechanism of resistance to immune checkpoint blockade in cancer. Exploration of the IL-27 gene signature in published datasets showed enrichment in macrophage populations associated with progressive disease in patients with NSCLC. While many of the properties of IL-27–mediated immune regulation have focused on hematopoietic cells, IL-27RA is also expressed on tumor cells from NSCLC patients with progressive disease as well as lung cancer cell lines in which IL-27 can upregulate PD-L1, IDO1, and other canonical interferon regulated genes.ConclusionsThese studies elucidate the transcriptional networks that are engaged after IL-27 signaling in immune and cancer cells and highlight the parallels with interferon-associated immune regulation. Blockade of IL-27 provides a novel therapeutic strategy to alleviate a gene transcriptional program implicated in immune suppression and checkpoint resistance.ReferencesPatnaik A, Morgensztern D, Mantia C, et al. Results of a phase 1 study of SRF388, a first-in-human, first-in-class, high-affinity anti-IL-27 antibody in advanced solid tumors. J Clin Oncol 2021;39:2551–2551.Maynard A, McCoach CE, Rotow JK, et al. Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing. Cell 2020;182:1232–1251.

Summary Lifestyle factors, including diet, play an important role in the survival of cancer patients. However, the molecular mechanisms underlying pathogenic links between diet and particular oncogenic mutations in human cancers remain unclear. We recently reported that the ketone body acetoacetate selectively enhances BRAF V600E mutant-dependent MEK1 activation in human cancers. Here we show that a high-fat ketogenic diet increased serum levels of acetoacetate, leading to enhanced tumor growth potential of BRAF V600E-expressing human melanoma cells in xenograft mice. Treatment with hypolipidemic agents to lower circulating acetoacetate levels or an inhibitory homolog of acetoacetate, dehydroacetic acid, to antagonize acetoacetate-BRAF V600E binding attenuated BRAF V600E tumor growth. These findings reveal a signaling basis underlying a pathogenic role of dietary fat in BRAF V600E-expressing melanoma, providing insights into the design of conceptualized "precision diets" that may prevent or delay tumor progression based on an individual's specific oncogenic mutation profile.

2551 Background: IL-27 is an immunosuppressive cytokine, consisting of two subunits p28 and EBI3, that upregulates immune checkpoint receptors (eg, PD-L1, TIGIT) and downregulates proinflammatory cytokines such as IFNγ, TNFα, and IL-17. SRF388 is a first-in-class, fully human IgG1 antibody to IL-27 that blocks the interaction between IL-27 and its receptor, thereby promoting immune activation in the tumor microenvironment. The IL-27 pathway is activated in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC), and high circulating levels of EBI3 are associated with inferior outcomes in both. Circulating EBI3 levels may serve as a predictive biomarker of SRF388 activity. Methods: Patients with advanced solid tumors refractory to standard therapy were enrolled in a phase 1 dose-escalation study (accelerated single patient followed by standard 3+3) to establish the preliminary safety of SRF388 as a monotherapy and to identify a dose suitable for expansion (NCT04374877). SRF388 was administered intravenously every 4 weeks. Tumor response was assessed by RECIST v1.1. SRF388 pharmacokinetic (PK) and pharmacodynamic (PD) [phospho-STAT (pSTAT) inhibition] analyses were performed. Results: As of January 26, 2021, 12 patients have received SRF388 at doses ranging from 0.003 to 10 mg/kg with 2 patients undergoing intra-patient dose escalation. Median age was 68 years, 67% were female, and ECOG PS was 0/1 (42%/58%). Median number of prior therapies was 2 (range 1–9), and 75% were anti-PD-(L)1 experienced (n = 9). The only treatment-related adverse events observed across dose levels were low-grade fatigue (n = 1, 8%), nausea (n = 1, 8%) and excess salivation (n = 1, 8%). No dose-limiting toxicities (DLTs) or ≥ Grade 3 related toxicity have occurred. Mean time on study is 12.5 weeks (range 4–40). One patient with RCC who received prior anti-PD-1 has prolonged stable disease for > 9 months. SRF388 PK are linear with estimated T1/2 ranging from 6–19 days. There is evidence of accumulation and no anti-drug antibody development to date. Maximal inhibition of the IL-27 signaling pathway as measured by > 90% pSTAT inhibition in whole blood was achieved starting at 0.3 mg/kg. Given combined evidence of near-complete pathway inhibition and preclinical human equivalent dose modeling projecting biologically active doses, additional slots were opened for RCC and HCC starting at 1 mg/kg. Conclusions: Preliminary results of IL-27 pathway blockade with a first-in-class therapeutic demonstrates that SRF388 is well tolerated at doses that achieve maximal inhibition of downstream pSTAT signaling through the dosing period. Expansions are planned in HCC and RCC. Updated data including the recommended phase 2 dose, clinical outcomes, PK/PD and correlative analyses will be presented. Clinical trial information: NCT04374877.

Background Anesthesia in West Africa is associated with high mortality rates. Critical shortages of adequately trained personnel, unreliable electrical supply, and lack of basic monitoring equipment are a few of the unique challenges to surgical care in this region. This study aims to describe the anesthesia practice at 2 tertiary care hospitals in Sierra Leone. Methods We conducted an observational study of anesthesia care at Connaught Hospital and Princess Christian Maternity Hospital in Freetown, Sierra Leone. Twenty-five percent of the anesthesia workforce in Sierra Leone, resident at both hospitals, was observed from June 2012 to February 2013. Perioperative assessments, anesthetic techniques, and intraoperative clinical and environmental irregularities were noted and analyzed. The postoperative status of observed cases was ascertained for morbidity and mortality. Results Between the 2 hospitals, 754 anesthesia cases and 373 general anesthetics were observed. Ketamine was the predominant IV anesthetic used. Both hospitals experienced infrastructural and environmental constraints to the delivery of anesthesia care during the observation period. Vital sign monitoring was irregular and dependent on age and availability of monitors. Perioperative mortality during the course of the study was 11.9 deaths/1000 anesthetics. Conclusions We identified gaps in the application of internationally recommended anesthesia practices at both hospitals, likely caused by lack of available resources. Mortality rates were similar to those in other resource-limited countries.

The oxidative pentose phosphate pathway (PPP) is crucial for cancer cell metabolism and tumor growth. We recently reported that targeting a key oxidative PPP enzyme, 6-phosphogluconate dehydrogenase (6PGD), using our novel small molecule 6PGD inhibitors Physcion and its derivative S3, shows anti-cancer effects. Notably, humans with genetic deficiency of either 6PGD or another oxidative PPP enzyme, glucose-6-phosphate dehydrogenase (G6PD), exhibit non-immune hemolytic anemia upon exposure to aspirin and various anti-malarial drugs. Inspired by these clinical observations, we examined the anti-cancer potential of combined treatment with 6PGD inhibitors and anti-malarial drugs. We found that stable knockdown of 6PGD sensitizes leukemia cells to anti-malarial agent dihydroartemisinin (DHA). Combined treatment with DHA and Physcion activates AMP-activated protein kinase, leading to synergistic inhibition of human leukemia cell viability. Moreover, our combined therapy synergistically attenuates tumor growth in xenograft nude mice injected with human K562 leukemia cells and cell viability of primary leukemia cells from human patients, but shows minimal toxicity to normal hematopoietic cells in mice as well as red blood cells and mononucleocytes from healthy human donors. Our findings reveal the potential for combined therapy using optimized doses of Physcion and DHA as a novel anti-leukemia treatment without inducing hemolysis.

Letter to the Editor.-- Dovey, Oliver M. et al., Internal tandem duplication (ITD) mutations in the juxtamembrane domain of the fms-like tyrosine kinase 3 (FLT3) gene occur in approximately one quarter of cases of acute myeloid leukemia (AML), are associated with constitutive activation of the kinase and confer a poor prognosis., BC is funded by the >China Scholarship Council> for his visiting studies in UK. AM is funded by the Kay Kendall Leukaemia Fund project grant. CG was funded by a Bloodwise Clinical Research Training Fellowship. IV is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal. We thank Servicio Santander Supercomputación for their support. OMD, JLC and GSV are funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA) and this work was also funded by the Wellcome Trust Sanger Institute

CD39 (ENTPD1) is a key enzyme responsible for the degradation of extracellular adenosine triphosphate (ATP) and is upregulated in the tumor microenvironment (TME). High levels of extracellular ATP, often generated in the TME as a result of tissue damage and immunogenic cell death, can initiate proinflammatory responses that are blocked by the enzymatic activity of CD39. In addition, extracellular adenosine accumulates in cancerous tissues through the degradation of ATP by CD39 and other ectonucleotidases (eg, CD73), and constitutes an important mechanism of tumor immune escape, induction of angiogenesis, and metastasis. Thus, inhibition of CD39 can convert a suppressive TME to a proinflammatory environment. SRF617 is an investigational fully human anti-CD39 antibody that binds to human CD39 with nanomolar affinity and potently inhibits its enzymatic function. Results of the current studies show that CD39 is predominantly expressed in tumor stroma and on tumor-infiltrating immune cells in samples from patients with cancer. Similar expression patterns are observed in various murine tumor models. In vivo, SRF617 has significant single-agent anti-tumor activity in a variety of cell line-derived xenograft models that express CD39. Cancer therapies, such as immunogenic cell death agents, can increase inflammation and ATP levels in the TME; combination studies of SRF617 with these agents showed improved preclinical efficacy and led to significant improvement in survival. In addition, a mouse-specific anti-CD39 surrogate antibody demonstrated potent binding and enzymatic inhibition of murine CD39 in vitro and significantly decreased tumor growth in a syngeneic murine tumor model. Pharmacodynamic studies demonstrated mechanistic changes in the tumor-infiltrating leukocytes and plasma chemokine levels. Combination treatment of a murine anti-CD39 surrogate and an anti-mouse PD-1 displayed improved activity and an increase in overall survival in the CT-26 mouse model. In addition, administration of SRF617 in combination with other immunotherapies also demonstrated a similar improvement in activity and survival. In summary, these studies demonstrate that SRF617 is a potent inhibitor of CD39 enzymatic activity both in vitro and in vivo. Inhibition of CD39 potentiates the activity of chemotherapy and immunotherapy agents to improve tumor growth inhibition and survival in mice. These findings support future clinical studies of SRF617 as monotherapy and in combination with other therapeutic agents in treating patients with cancer. Citation Format: Sonia G. Das, Austin Dulak, Gege Tan, Marc Johnson, Tauqeer H. Zaidi, Michael C. Warren, Secil Koseoglu, Erik Devereaux, Marisa O. Peluso, Alison M. Paterson, Benjamin H. Lee, Vito J. Palombella, Pamela M. Holland, Andrew C. Lake. SRF617, a potent enzymatic inhibitor of CD39, demonstrates single-agent activity and cooperates with various cancer therapies in both solid tumor and hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6639.

Background: CD47 is a type I integral membrane protein expressed on multiple human tumors, including ovarian cancer, and modulates cell processes such as cell migration, adhesion, T-cell function, and cell death via interaction with multiple ligands. Interaction of CD47 with SIRPα expressed on myeloid cells results in an inhibitory “don't eat me” signal that prevents phagocytosis of CD47-expressing cancer cells. Enhancement of the anti-tumor activity of chemotherapy has also been reported with CD47 antagonists. We investigated the effects of combining SRF231, an investigational fully human IgG4 antibody, with chemotherapy in models of human ovarian cancer. Methods: Expression of CD47 in 8 established platinum-resistant PDX models of ovarian cancer was measured by immunohistochemistry with the anti-CD47 antibody SP279. SRF231-mediated phagocytosis of ovarian cancer cell lines was assessed using a macrophage coculture system. Cell death from combining SRF231 with either doxorubicin or platinum in vitro was assessed by Annexin V assay. The activity of SRF231 combined with doxorubicin in vivo was compared to isotype control, SRF231, or doxorubicin monotherapy in an ovarian cancer subcutaneous xenograft model, OVCAR3. Additionally, the activity of SRF231 combined with platinum was compared to isotype control, SRF231, or platinum monotherapy in two luciferase-expressing intraperitoneal PDX ovarian cancer models, as measured by bioluminescent imaging. Results: CD47 expression was high across all 8 PDX models. SRF231 induced phagocytosis of ovarian cancer cell lines by human monocyte-derived macrophages. SRF231 potentiated doxorubicin- or oxaliplatin-mediated cell death in Jurkat cells in Annexin V assays. In the OVCAR3 xenograft model, the combination of SRF231 and doxorubicin resulted in significant tumor growth inhibition and improved survival compared to isotype control, SRF231 monotherapy, or doxorubicin monotherapy. In the PDX model DF216, which demonstrates no evidence of monotherapy response to SRF231, combination of SRF231 and carboplatin resulted in tumor regression with significantly enhanced anti-tumor activity compared to carboplatin or SRF231 monotherapy, as well as to isotype control. Similarly, in the PDX model DF86, where SRF231 demonstrates modest monotherapy activity, combination SRF231 and carboplatin resulted in tumor regression and significant increase in anti-tumor activity compared to carboplatin or SRF231 monotherapies. Conclusion: Anti-CD47 directed therapy with SRF231, a fully human antibody, demonstrated the ability to significantly increase the anti-tumor activity of standard chemotherapies in xenograft and platinum-resistant PDX models of ovarian cancer. Further exploration of combining anti-CD47 and platinum regimens in ovarian cancer is warranted. Citation Format: Joyce F. Liu, Kshama A. Doshi, Benjamin H. Lee, Marisa O. Peluso, Li Zhang, Shan Zhou, Qing Zeng, Stephen Wang, Paul T. Kirschmeier, Cam A. Tran, Cloud Paweletz, Matthew Rausch, Alison M. Paterson, Prafulla C. Gokhale, Ursula A. Matulonis. The anti-CD47 antibody SRF231 increases anti-tumor activity of standard of care chemotherapy in platinum-resistant PDX models of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4515.