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Abstract Exome or genome sequencing was performed to identify the genetic etiology for the clinical presentation of global developmental delay, intellectual disability, and sensorimotor neuropathy with associated distal weakness in two unrelated families. A homozygous frameshift variant c.186delA (p.A63Qfs*3) in the NUDT2 gene was identified in cases 1 and 2 from one family and a third case from another family. Variants in NUDT2 were previously shown to cause intellectual disability, but here we expand the phenotype by demonstrating its association with distal upper and lower extremity weakness due to a sensorimotor polyneuropathy with demyelinating and/or axonal features.

Abstract Whole‐exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group‐binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate‐4S (CS‐4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss‐of‐function by disrupting FIBCD1‐CS‐4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor‐related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal‐dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

Objective: Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging., Methods: In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE., Results: We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case., Interpretation: This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing., (© 2024 The Author(s). Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Regulatory T cells (Treg) are highly enriched within many tumors and suppress immune responses to cancer. There is intense interest in reprogramming Tregs to contribute to antitumor immunity. OX40 and CD137 are expressed highly on Tregs, activated and memory T cells, and NK cells. In this study, using a novel bispecific antibody targeting mouse OX40 and CD137 (FS120m), we show that OX40/CD137 bispecific agonism induces potent antitumor immunity partially dependent upon IFNγ production by functionally reprogrammed Tregs. Treatment of tumor-bearing animals with OX40/CD137 bispecific agonists reprograms Tregs into both fragile Foxp3+ IFNγ+ Tregs with decreased suppressive function and lineage-instable Foxp3- IFNγ+ ex-Tregs. Treg fragility is partially driven by IFNγ signaling, whereas Treg instability is associated with reduced IL2 responsiveness upon treatment with OX40/CD137 bispecific agonists. Importantly, conditional deletion of Ifng in Foxp3+ Tregs and their progeny partially reverses the antitumor efficacy of OX40/CD137 bispecific agonist therapy, revealing that reprogramming of Tregs into IFNγ-producing cells contributes to the anti-tumor efficacy of OX40/CD137 bispecific agonists. These findings provide insights into mechanisms by which bispecific agonist therapies targeting costimulatory receptors highly expressed by Tregs potentiate antitumor immunity in mouse models., Significance: The bispecific antibody FS120, an immunotherapy currently being tested in the clinic, partially functions by inducing anti-tumor activity of Tregs, which results in tumor rejection., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Fem1 homolog B (FEM1B) acts as a substrate recognition subunit for ubiquitin ligase complexes belonging to the CULLIN 2-based E3 family. Several biological functions have been proposed for FEM1B, including a structurally resolved function as a sensor for redox cell status by controlling mitochondrial activity, but its implication in human disease remains elusive., Methods: To understand the involvement of FEM1B in human disease, we made use of Matchmaker exchange platforms to identify individuals with de novo variants in FEM1B and performed their clinical evaluation. We performed functional validation using primary neuronal cultures and in utero electroporation assays, as well as experiments on patient's cells., Results: Five individuals with a recurrent de novo missense variant in FEM1B were identified: NM_015322.5:c.377G>A NP_056137.1:p.(Arg126Gln) (FEM1B R126Q ). Affected individuals shared a severe neurodevelopmental disorder with behavioral phenotypes and a variable set of malformations, including brain anomalies, clubfeet, skeletal abnormalities, and facial dysmorphism. Overexpression of the FEM1B R126Q variant but not FEM1B wild-type protein, during mouse brain development, resulted in delayed neuronal migration of the target cells. In addition, the individuals' cells exhibited signs of oxidative stress and induction of type I interferon signaling., Conclusion: Overall, our data indicate that p.(Arg126Gln) induces aberrant FEM1B activation, resulting in a gain-of-function mechanism associated with a severe syndromic developmental disorder in humans., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Immuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response., Methods: Using aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs., Results: We found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice., Discussion: These differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models., Competing Interests: This study was funded by AstraZeneca. The funder had the following involvement with the study: study design, data collection and analysis, decision to publish, and preparation of the manuscript. All authors were employees of and shareholders in AstraZeneca at the time of the study., (Copyright © 2023 Sitnikova, Walker, Prickett, Morrow, Valge-Archer, Robinson, Wilkinson and Dovedi.)

Craniosynostosis (CS) is the most common congenital cranial anomaly. Several Mendelian forms of syndromic CS are well described, but a genetic etiology remains elusive in a substantial fraction of probands. Analysis of exome sequence data from 526 proband-parent trios with syndromic CS identified a marked excess (observed 98, expected 33, p = 4.83 × 10 -20 ) of damaging de novo variants (DNVs) in genes highly intolerant to loss-of-function variation (probability of LoF intolerance > 0.9). 30 probands harbored damaging DNVs in 21 genes that were not previously implicated in CS but are involved in chromatin modification and remodeling (4.7-fold enrichment, p = 1.1 × 10 -11 ). 17 genes had multiple damaging DNVs, and 13 genes (CDK13, NFIX, ADNP, KMT5B, SON, ARID1B, CASK, CHD7, MED13L, PSMD12, POLR2A, CHD3, and SETBP1) surpassed thresholds for genome-wide significance. A recurrent gain-of-function DNV in the retinoic acid receptor alpha (RARA; c.865G>A [p.Gly289Arg]) was identified in two probands with similar CS phenotypes. CS risk genes overlap with those identified for autism and other neurodevelopmental disorders, are highly expressed in cranial neural crest cells, and converge in networks that regulate chromatin modification, gene transcription, and osteoblast differentiation. Our results identify several CS loci and have major implications for genetic testing and counseling., Competing Interests: Declaration of interests S.M., M.M., M.S., and P.K. are employees of GeneDx., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Many mothers, especially those with co-morbidities, do not achieve exclusive breastfeeding (EBF) for the first 6 months, with the loss of multiple health benefits including enhanced infant nutrition. We wished to evaluate whether proactive lactation consultant telephone advice in the first month postpartum improved breastfeeding rates for up to 6 months. A prospective cohort observational study was performed. Mother groupings included the following: Control (CG, n = 379)-standard postnatal care; Exposure (EG, n = 386)-standard postnatal care delivered by lactation consultant telephone contact for the first 3 weeks postpartum and then follow-up calls at 1, 3 and 6 months postpartum to ascertain breastfeeding status. Sore nipples (24%) and fussy/unsettled behaviour (14-19%) were common EG concerns. EG EBF rates were higher at 1 month (65% vs. 53%; p < 0.001), 3 months (57% vs. 49%; p = 0.041) and 6 months (45 vs. 33%; p < 0.001). EG EBF rates across the 6 months were higher for infants admitted to the NNU (52.9% vs. 37.5%, p = 0.003), obese mothers (58.3% vs. 37.2%, p < 0.001), mothers with depression (60.8% vs. 43.4%, p = 0.036) and all birth modes. Proactive early lactation advice significantly prolongs EBF and consequently enhances infant nutrition overall, including for mothers at risk of early breastfeeding cessation.

Purpose: This phase 1 study (NCT03440437) evaluated the safety, tolerability, pharmacokinetics (PK), and activity of FS118, a bispecific antibody-targeting LAG-3 and PD-L1, in patients with advanced cancer resistant to anti-PD-(L)1 therapy., Patients and Methods: Patients with solid tumors, refractory to anti-PD-(L)1-based therapy, received intravenous FS118 weekly with an accelerated dose titration design (800 μg to 0.3 mg/kg) followed by 3+3 ascending dose expansion (1 to 20 mg/kg). Primary objectives were safety, tolerability, and PK. Additional endpoints included antitumor activity, immunogenicity, and pharmacodynamics., Results: Forty-three patients with a median of three prior regimens in the locally advanced/metastatic setting, including at least one anti-PD-(L)1 regimen, received FS118 monotherapy. FS118 was well tolerated, with no serious adverse events relating to FS118 reported. No dose-limiting toxicities (DLT) were observed, and an MTD was not reached. The recommended phase 2 dose of FS118 was established as 10 mg/kg weekly. The terminal half-life was 3.9 days. Immunogenicity was transient. Pharmacodynamic activity was prolonged throughout dosing as demonstrated by sustained elevation of soluble LAG-3 and increased peripheral effector cells. The overall disease control rate (DCR) was 46.5%; this disease control was observed as stable disease, except for one late partial response. Disease control of 54.8% was observed in patients receiving 1 mg/kg or greater who had acquired resistance to PD-(L)1-targeted therapy., Conclusions: FS118 was well tolerated with no DLTs observed up to and including 20 mg/kg QW. Further studies are warranted to determine clinical benefit in patients who have become refractory to anti-PD-(L)1 therapy. See related commentary by Karapetyan and Luke, p. 835., (©2022 American Association for Cancer Research.)

Anaplastic thyroid cancer represents a rare, highly aggressive form of thyroid cancer with a poor prognosis and an overall survival ranging from 5 to 12 months. Unfortunately, treatment options remain limited, even for patients with a targetable driver mutation. Here, we present a case of a patient with a BRAF V600E-mutated, PD-L1 positive (tumor proportion score of 95%) anaplastic thyroid cancer refractory to standard therapies, including debulking surgery, followed by chemoradiation, who had further progressed on PD-1 monotherapy, and was unable to tolerate BRAF/MEK inhibition. Ongoing treatment with FS118, a bispecific LAG-3/PD-L1 antagonist, has afforded 3 years of disease control, including a late confirmed partial response, with excellent tolerability. Given this response, further investigation is required to delineate the mechanism by which dual PD-L1/LAG-3 blockade by FS118 overcomes initial PD-1 pathway resistance, and therefore, identify which patients are most likely to benefit. Simultaneously, expanded use should be considered for those with refractory disease, especially if PD-L1 positive. Insights Dual PD-L1/LAG-3 blockade may be an effective treatment strategy for refractory metastatic tumors, including anaplastic thyroid cancer., Competing Interests: Competing interests: MJK, J-BH and OS have no relevant competing interests. LK, MM, and CJS are employees of F-star Therapeutics and hold stock options in F-STAR Therapeutics. DJW has received research funding paid directly to the institution from FSTAR Therapeutics., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1 + activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1 + T cells versus PD-1 - T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo . Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1 + T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1 + T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy. See related commentary by Burton and Tawbi, p. 1008 . This article is highlighted in the In This Issue feature, p. 995 ., (©2021 American Association for Cancer Research.)

Preclinical studies of PD-L1 and CTLA-4 blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti-PD-L1 and anti-CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.

Purpose: Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb 2 ) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody., Experimental Design: This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb 2 )., Results: FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb 2 in blocking LAG-3- and PD-L1-mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb 2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb 2 , whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb 2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood., Conclusions: This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer., (©2020 American Association for Cancer Research.)

Following the success of immune checkpoint blockade therapy against cancer, agonistic antibodies targeting T-cell costimulatory pathways are in clinical trials. The TNF superfamily of receptors (TNFRSF) members CD137 and OX40 are costimulatory receptors that stimulate T-cell proliferation and activation upon interaction with their cognate ligands. Activating CD137 and OX40 with agonistic mAbs stimulates the immune system due to their broad expression on CD4 + and CD8 + T cells and natural killer cells and has antitumor effects in preclinical models. Most TNFRSF agonist antibodies require crosslinking via Fcγ receptors (FcγR), which can limit their clinical activity. FS120 mAb 2 , a dual agonist bispecific antibody targeting CD137 and OX40, activated both CD4 + and CD8 + T cells in an FcγR-independent mechanism, dependent on concurrent binding. A mouse surrogate version of the bispecific antibody displayed antitumor activity in syngeneic tumor models, independent of T regulatory cell depletion and of FcγR interaction, but associated with peripheral T-cell activation and proliferation. When compared with a crosslink-independent CD137 agonist mAb, the FS120 surrogate induced lower liver T-cell infiltration. These data support initiation of clinical development of FS120, a first-in-class dual agonist bispecific antibody for the treatment of human cancer., (©2020 American Association for Cancer Research.)

Purpose: Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown. Experimental Design: The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field. Results: We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells. Conclusions: These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy. Clin Cancer Res; 23(18); 5514-26. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy. Experimental Design: The EC 50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors. Results: The mGITRL-FP had an almost 50-fold higher EC 50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8 + and CD4 + T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR., (©2016 American Association for Cancer Research.)

Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR., (©2015 American Association for Cancer Research.)

Radiotherapy is a major part in the treatment of most common cancers, but many patients experience local recurrence with metastatic disease. In evaluating response biomarkers, we found that low doses of fractionated radiotherapy led to PD-L1 upregulation on tumor cells in a variety of syngeneic mouse models of cancer. Notably, fractionated radiotherapy delivered in combination with αPD-1 or αPD-L1 mAbs generated efficacious CD8(+) T-cell responses that improved local tumor control, long-term survival, and protection against tumor rechallenge. These favorable outcomes were associated with induction of a tumor antigen-specific memory immune response. Mechanistic investigations showed that IFNγ produced by CD8(+) T cells was responsible for mediating PD-L1 upregulation on tumor cells after delivery of fractionated radiotherapy. Scheduling of anti-PD-L1 mAb was important for therapeutic outcome, with concomitant but not sequential administration with fractionated radiotherapy required to improve survival. Taken together, our results reveal the mechanistic basis for an adaptive response by tumor cells that mediates resistance to fractionated radiotherapy and its treatment failure. With attention to scheduling, combination immunoradiotherapy with radiotherapy and PD-1/PD-L1 signaling blockade may offer an immediate strategy for clinical evaluation to improve treatment outcomes., (©2014 American Association for Cancer Research.)

The development of targeted therapies and immunotherapies has markedly advanced the treatment of metastasized melanoma. While treatment with selective BRAF(V600E) inhibitors (like vemurafenib or dabrafenib) leads to high response rates but short response duration, CTLA-4 blocking therapies induce sustained responses, but only in a limited number of patients. The combination of these diametric treatment approaches may further improve survival, but pre-clinical data concerning this approach is limited. We investigated, using Tyr::CreER(T2)PTEN(F-/-)BRAF(F-V600E/+) inducible melanoma mice, whether BRAF(V600E) inhibition can synergize with anti-CTLA-4 mAb treatment, focusing on the interaction between the BRAF(V600E) inhibitor PLX4720 and the immune system. While PLX4720 treatment strongly decreased tumor growth, it did not induce cell death in BRAF(V600E)/PTEN(-/-) melanomas. More strikingly, PLX4720 treatment led to a decreased frequency of tumor-resident T cells, NK-cells, MDSCs and macrophages, which could not be restored by the addition of anti-CTLA-4 mAb. As this effect was not observed upon treatment of BRAF wild-type B16F10 tumors, we conclude that the decreased frequency of immune cells correlates to BRAF(V600E) inhibition in tumor cells and is not due to an off-target effect of PLX4720 on immune cells. Furthermore, anti-CTLA-4 mAb treatment of inducible melanoma mice treated with PLX4720 did not result in enhanced tumor control, while anti-CTLA-4 mAb treatment did improve the effect of tumor-vaccination in B16F10-inoculated mice. Our data suggest that vemurafenib may negatively affect the immune activity within the tumor. Therefore, the potential effect of targeted therapy on the tumor-microenvironment should be taken into consideration in the design of clinical trials combining targeted and immunotherapy.

Background: This is the first controlled study to explore whether adjunctive immersive virtual reality (VR) can reduce excessive pain of soldiers with combat-related burn injuries during wound debridement., Methods: Patients were US soldiers burned in combat attacks involving explosive devices in Iraq or Afghanistan. During the same wound care session using a within-subject experimental design, 12 patients received half of their severe burn wound cleaning procedure (~6 minutes) with standard of care pharmacologies and half while in VR (treatment order randomized). Three 0 to 10 Graphic Rating Scale pain scores for each of the treatment conditions served as the primary variables., Results: Patients reported significantly less pain when distracted with VR. "Worst pain" (pain intensity) dropped from 6.25 of 10 to 4.50 of 10. "Pain unpleasantness" ratings dropped from "moderate" (6.25 of 10) to "mild" (2.83 of 10). "Time spent thinking about pain" dropped from 76% during no VR to 22% during VR. Patients rated "no VR" as "no fun at all" (<1 of 10) and rated VR as "pretty fun" (7.5 of 10). Follow-up analyses showed VR was especially effective for the six patients who scored 7 of 10 or higher (severe to excruciating) on the "worst pain" (pain intensity) ratings., Conclusions: These preliminary results provide the first evidence from a controlled study that adjunctive immersive VR reduced pain of patients with combat-related burn injuries during severe burn wound debridement. Pain reduction during VR was greatest in patients with the highest pain during no VR. These patients were the first to use a unique custom robot-like arm mounted VR goggle system.

TEL-AML1 is an oncogenic fusion protein associated with childhood pre-B acute lymphoblastic leukaemia. From published microarray datasets we identified the Rho Guanine Nucleotide Exchange Factor (RhoGEF) Asef to be associated with TEL-AML1 leukaemia. However, the Asef gene is not a direct target of TEL-AML1 transcriptional control. Forced expression of Asef in vitro induced and maintained proliferation of haemaopoietic progenitor cells and co-operated with TEL-AML1 greatly to enhance proliferation and haemopoietic colony size. In haemopoietic transplantation reconstitution assays Asef and TEL-AML1 together failed to induce a leukaemic phenotype., (2009 Elsevier Ltd. All rights reserved.)

Ets-related gene (ERG) is a member of the ETS transcription factor gene family located on Hsa21. ERG is known to have a crucial role in establishing definitive hematopoiesis and is required for normal megakaryopoiesis. Truncated forms of ERG are associated with multiple cancers such as Ewing's sarcoma, prostate cancer, and leukemia as part of oncogenic fusion translocations. Increased expression of ERG is highly indicative of poor prognosis in acute myeloid leukemia and ERG is expressed in acute megakaryoblastic leukemia (AMKL); however, it is unclear if expression of ERG per se has a leukemogenic activity. We show that ectopic expression of ERG in fetal hematopoietic progenitors promotes megakaryopoiesis and that ERG alone acts as a potent oncogene in vivo leading to rapid onset of leukemia in mice. We observe that the endogenous ERG is required for the proliferation and maintenance of AMKL cell lines. ERG also strongly cooperates with the GATA1s mutated protein, found in Down syndrome AMKL, to immortalize megakaryocyte progenitors, suggesting that the additional copy of ERG in trisomy 21 may have a role in Down syndrome AMKL. These data suggest that ERG is a hematopoietic oncogene that may play a direct role in myeloid leukemia pathogenesis.

AML1-ETO and TEL-AML1 are chimeric proteins resulting from the t(8;21)(q22;q22) in acute myeloid leukemia, and the t(12;21)(p13;q22) in pre-B-cell leukemia, respectively. The Runt domain of AML1 in both proteins mediates DNA binding and heterodimerization with the core binding factor beta (CBFbeta) subunit. To determine whether CBFbeta is required for AML1-ETO and TEL-AML1 activity, we introduced amino acid substitutions into the Runt domain that disrupt heterodimerization with CBFbeta but not DNA binding. We show that CBFbeta contributes to AML1-ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and is indispensable for its cooperativity with the activated receptor tyrosine kinase TEL-PDGFbetaR in generating acute myeloid leukemia in mice. Similarly, CBFbeta is essential for TEL-AML1's ability to promote self-renewal of B cell precursors in vitro. These studies validate the Runt domain/CBFbeta interaction as a therapeutic target in core binding factor leukemias.

The t[(11;19)(p22;q23)] translocation, which gives rise to the MLL-ENL fusion protein, is commonly found in infant acute leukemias of both the myeloid and lymphoid lineage. To investigate the molecular mechanism of immortalization by MLL-ENL we established a Tet-regulatable system of MLL-ENL expression in primary hematopoietic progenitor cells. Immortalized myeloid cell lines were generated, which are dependent on continued MLL-ENL expression for their survival and proliferation. These cells either terminally differentiate or die when MLL-ENL expression is turned off with doxycycline. The expression profile of all 39 murine Hox genes was analyzed in these cells by real-time quantitative PCR. This analysis showed that loss of MLL-ENL was accompanied by a reduction in the expression of multiple Hoxa genes. By comparing these changes with Hox gene expression in cells induced to differentiate with granulocyte colony-stimulating factor, we show for the first time that reduced Hox gene expression is specific to loss of MLL-ENL and is not a consequence of differentiation. Our data also suggest that the Hox cofactor Meis-2 can substitute for Meis-1 function. Thus, MLL-ENL is required to initiate and maintain immortalization of myeloid progenitors and may contribute to leukemogenesis by aberrantly sustaining the expression of a "Hox code" consisting of Hoxa4 to Hoxa11.

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis, fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro, preferentially promoting B-lymphocyte development, enhancing self-renewal of B-cell precursors, and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However, it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis, TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion.