A pre-B acute lymphoblastic leukemia cell line model reveals the mechanism of thalidomide therapy-related B-cell leukemogenesis.

Lenalidomide, a thalidomide derivative, is prescribed as maintenance therapy for multiple myeloma (MM). Patients with MM receiving lenalidomide were found to develop a distinct therapy-related B cell acute lymphoblastic leukemia (B-ALL). However, the molecular mechanism by which lenalidomide drives B-ALL is unknown. We show that thalidomide treatment of B cell lines increased CD34 expression and fibronectin adhesion. This resembled the effects of Ikzf1 loss of function mutations in B-ALL. IKZF1 is a transcription factor that can act as both a transcriptional activator and a repressor depending upon the target loci. In our experiments, thalidomide-induced degradation of IKZF1 increased the expression of its transcriptional repression targets Itga5 and CD34 explaining the increased adhesion and stemness. Strikingly, withdrawal of thalidomide lead to the mis-localization of IKZF1 to the cytoplasm. Moreover, chromatin immunoprecipitation data showed a long-term effect of thalidomide treatment on IKZF1 target loci. This included decreased chromatin occupancy at early B cell factor 1 (EBF1) and Spi1 (PU.1). Consequently, B-cell lineage specifying transcription factors including Pax5, Spi1 and EBF1 were downregulated even after 7 days of thalidomide withdrawal. Our study thus provides a molecular mechanism of thalidomide-induced B-ALL whereby thalidomide alters the chromatin occupancy of IKZF1 at key B-cell lineage transcription factors leading to a persistent block in B-cell differentiation.

Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

The emerging role of targeted protein degradation to treat and study cancer.

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in-depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues' action and CRBN's neo-targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non-Hodgkin's lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein-degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity-induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Targeting the microenvironment in the treatment of arteriovenous malformations.

Extracranial arteriovenous malformations (AVMs) are regarded as rare diseases and are prone to complications such as pain, bleeding, relentless growth, and high volume of shunted blood. Due to the high vascular pressure endothelial cells of AVMs are exposed to mechanical stress. To control symptoms and lesion growth pharmacological treatment strategies are urgently needed in addition to surgery and interventional radiology. AVM cells were isolated from three patients and exposed to cyclic mechanical stretching for 24 h. Thalidomide and bevacizumab, both VEGF inhibitors, were tested for their ability to prevent the formation of circular networks and proliferation of CD31+ endothelial AVM cells. Furthermore, the effect of thalidomide and bevacizumab on stretched endothelial AVM cells was evaluated. In response to mechanical stress, VEGF gene and protein expression increased in patient AVM endothelial cells. Thalidomide and bevacizumab reduced endothelial AVM cell proliferation. Bevacizumab inhibited circular network formation of endothelial AVM cells and lowered VEGF gene and protein expression, even though the cells were exposed to mechanical stress. With promising in vitro results, bevacizumab was used to treat three patients with unresectable AVMs or to prevent regrowth after incomplete resection. Bevacizumab controlled bleeding, pulsation, and pain over the follow up of eight months with no patient-reported side effects. Overall, mechanical stress increases VEGF expression in the microenvironment of AVM cells. The monoclonal VEGF antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. The clinical application of bevacizumab in AVM treatment demonstrates effective symptom control with no side effects.

Thalidomide attenuates radiation-induced apoptosis and pro-inflammatory cytokine secretion in oral epithelial cells by promoting LZTS3 expression.

Radiation-induced oral mucositis (RIOM) is a prevalent oral complication that occurs in individuals undergoing radiotherapy or radiation treatment for head and neck tumors. The presence of oral mucosal rupture and ulcerative lesions, which are the defining features of this condition, can significantly affect the quality of life of patients. Additionally, it can interfere with tumor therapy and contribute to an unfavorable prognosis. Current evidence suggests that cellular inflammation and programmed cell death are important factors in disease development. Moreover, thalidomide (THD) has been revealed to reduce the incidence and severity of RIOM in patients undergoing chemoradiotherapy for nasopharyngeal carcinoma. However, the mechanism through which THD improves RIOM remains unknown. This study aimed to investigate the role of LZTS3 in RIOM by analyzing various sequencing datasets and conducting knockdown and overexpression experiments. We used small interfering RNA transfection and LZTS3 overexpression, followed by validation through polymerase chain reaction, western blotting, flow cytometry, and enzyme-linked immunosorbent assay. In this study, we identified LZTS3 as a potential target for THD regulation in RIOM. Through a series of experiments, we confirmed that LZTS3 has the ability to inhibit the inflammatory response and apoptosis of cells. In addition, we also found that THD can regulate the expression of LZTS3 by upregulating, thereby affecting inflammatory response and apoptosis. We repeated these results in a live animal model. In summary, THD has the potential to reduce the occurrence of oral mucositis in patients by upregulating LZTS3 levels. These findings provide a promising avenue for future drug research and development to treat RIOM.

Tofacitinib versus thalidomide for mucocutaneous lesions of systemic lupus erythematosus: A real-world CSTAR cohort study XXVII.

OBJECTIVE: Thalidomide is an effective medication for refractory mucocutaneous lesions of systemic lupus erythematosus (SLE) and can treat arthritis in some autoimmune diseases, but it has some adverse reactions. Recently, the effectiveness of tofacitinib in treating mucocutaneous lesions of SLE has been reported. We aimed to compare the efficacy and safety of tofacitinib with thalidomide in treating mucocutaneous and musculoskeletal lesions in patients with SLE. METHODS: This study was a real-world cohort study based on the Chinese SLE Treatment and Research group (CSTAR) registry. SLE patients who manifested mucocutaneous and/or musculoskeletal symptoms and were prescribed tofacitinib or thalidomide were included. We retrospectively conducted comparisons between the tofacitinib and thalidomide groups regarding clinical improvements, SLE disease activity, serological indicators, glucocorticoid doses, and adverse events at the 1, 3, and 6-months time points. RESULTS: At 3 and 6 months, the tofacitinib group exhibited a higher proportion of patients with improvement in mucocutaneous and musculoskeletal issues. Additionally, a greater percentage of patients in the tofacitinib group achieved remission or a low disease activity state (LLDAS) at these time points. No significant serological improvements were observed in either the tofacitinib or thalidomide groups. Fewer adverse events were observed in the tofacitinib group than in the thalidomide group. CONCLUSIONS: Tofacitinib might be superior to thalidomide in the improvement of mucocutaneous and musculoskeletal lesions in SLE, and had a good safety profile.

Safety and efficacy of thalidomide in treatment of gastrointestinal bleeding secondary to angioectasias: a systematic review and meta-analysis.

BACKGROUND: Thalidomide has been used for angioectasia-associated refractory gastrointestinal bleeding (GIB), with studies showing variable efficacy and side effects profile. We conducted a meta-analysis to reconcile the data. METHODS: Online databases were searched for studies evaluating thalidomide in patients with refractory/recurrent GIB due to angioectasias. The outcomes of interest were cessation of bleeding, rebleeding, need for blood transfusion, hospitalization and adverse events. Pooled proportions for incidence, and odds ratios (OR) for comparison with control were calculated along with 95% confidence interval (CI). RESULTS: A total of seven studies with 346 patients (n = 269 thalidomide, n = 77 control) were included. Thalidomide dose was usually started at 50-100mg/day. The mean age was 65 years, 45% patients were men, and mean follow-up was 1.8 years. The pooled clinical outcomes with thalidomide were: cessation of bleeding 42.2% (95% CI 36.02 to 48.41), rebleeding 30%, need for blood transfusion 20.1%, hospitalization 40% and adverse events 55.9%. When compared with the control group in 2 studies, patients on thalidomide had significantly higher odds of cessation of bleeding (OR 21.40, 95% CI 5.78 to 79.29, p < 0.00001) and adverse events, with lower need for blood transfusion and hospitalization. DISCUSSION: In patients with angioectasias-related refractory/recurrent GIB, the use of thalidomide results in significantly decreased bleeding risk and may play a role in the management of such patients.

Thalidomide for the Management of Gastrointestinal Bleeding in a Palliative Care Setting.

BACKGROUND: Palliative care patients frequently present with clinically significant gastrointestinal bleeding. Due to the existence of confounding comorbidities and a remarkably reduced state of general health in many cases, the management of gastrointestinal bleeding in this population is often challenging. SUMMARY: This review summarizes and discusses the role of thalidomide in gastrointestinal bleeding with a special focus on palliative care patients. In addition, an illustrative case report is presented. Thalidomide may be beneficial in gastrointestinal bleeding by exerting antiangiogenic effects. The drug has an acceptable safety profile. Side effects like neurotoxicity may limit its use but can be monitored safely. Due to thalidomide's thrombin generation potential, patients managed with thalidomide-containing regimes should be closely monitored for deep venous thrombosis. Given its teratogenicity, thalidomide should not be administered to women of childbearing potential who are not using adequate contraception. KEY MESSAGE: Physicians caring for patients in a palliative care setting should be aware of thalidomide as an effective therapeutic option when endoscopy fails to find a bleeding source or for those patients who cannot or refuse to undergo endoscopy but present with recurrent or obscure gastrointestinal bleeding.

Old drug, new use: The thalidomide-based fluorescent probe for cysteine detection and imaging in living cells.

Thalidomide, as a high-profile cereblon (CRBN) ligand, has attracted much attention because of its ability to target protein degradation. In this study, we are committed to developing a new fluorescent probe THD-1 based on thalidomide, aiming at improving the performance of cysteine fluorescent probe in optical properties and biocompatibility. The experimental results showed that THD-1, as a cysteine fluorescent probe, owned the characteristics of obvious colorimetric change, fast response time, good selectivity and high sensitivity. The mechanism of THD-1 sensing cysteine was further verified to ensure its reliability and effectiveness. It was also worth mentioning that THD-1 was successfully applied to the biological imaging of cysteine in living A549 cells, which highlighted its value in practical application. Overall, thalidomide, as a clinically approved drug, not only enriches the fluorescent skeleton library, but also paves a new way for the further development of fluorescent probes.

Teratogenicity is more likely a function of primary and secondary pharmacology than caused by chemically reactive metabolites: a critical evaluation of 40 years of scientific research.

The number of therapeutic drugs known to be human teratogens is actually relatively small. This may reflect the rigorous animal testing and well defined labelling. Some of these drugs were identified to have reactive metabolites and this has been postulated, historically, to be their teratogenic mechanism. These drugs include thalidomide, various anticonvulsants and retinoic acid derivatives.Many of these experiments were conducted in a period where chemically reactive metabolites were being intensely investigated and associated with all forms of toxicity. The legacy of this is that these examples are routinely cited as well established mechanisms.Examination of mechanism leads to the conclusion that the teratogenicity in humans of these compounds is likely due to the primary and secondary pharmacology of the parent drug and stable circulating metabolites and that association of reactive metabolites to this toxicity is unwarranted.

Environmental Signals.

Environmental factors have long been known to play a role in the pathogenesis of congenital heart disease (CHD), but this has not been a major focus of research in the modern era. Studies of human exposures and animal models demonstrate that demographics (age, race, socioeconomic status), diseases (e.g., diabetes, hypertension, obesity, stress, infection, high altitude), recreational and therapeutic drug use, and chemical exposures are associated with an increased risk for CHD. Unfortunately, although studies suggest that exposures to these factors may cause CHD, in most cases, the data are not strong, are inconclusive, or are contradictory. Although most studies concentrate on the effects of maternal exposure, paternal exposure to some agents can also modify this risk. From a mechanistic standpoint, recent delineation of signaling and genetic controls of cardiac development has revealed molecular pathways that may explain the effects of environmental signals on cardiac morphogenesis and may provide further tools to study the effects of environmental stimuli on cardiac development. For example, environmental factors likely regulate cellular signaling pathways, transcriptional and epigenetic regulation, proliferation, and physiologic processes that can control the development of the heart and other organs. However, understanding of the epidemiology and risk of these exposures and the mechanistic basis for any effects on cardiac development remains incomplete. Further studies defining the relationship between environmental exposures and human CHD and the mechanisms involved should reveal strategies to prevent, diagnose, and treat CHD induced by environmental signals.

Dynamic salivary cytokine profile of recurrent aphthous stomatitis patients in thalidomide maintenance treatment.

OBJECTIVE: To dynamically compare the longitudinal (time axis) and transverse (between groups) differences of the salivary cytokines during thalidomide maintenance treatment of recurrent aphthous stomatitis. METHODS: A randomized, controlled, clinical trial was performed. After the initial prednisone treatment, thalidomide (50 mg/d vs. 25 mg/d) was used as a maintenance drug for 4 or 8 weeks. The salivary IL-4, 5, 6, 10, TNF-α, and IFN-γ were dynamically detected with a cytometric bead array. RESULTS: Overall, the level of six elevated salivary cytokines after prednisone treatment was significantly downregulated, remained low during thalidomide maintenance, and rebounded at recurrence. The effect of 50 mg/d thalidomide on the salivary cytokines was not superior to 25 mg/d medication. The relapse-free period following drug withdrawal was the longest in the subgroup using 25 mg/d thalidomide for 8 weeks. The order of magnitude of IL-6 was the most obvious, and at week 8, only the level of IL-6 in the group (25 mg/d thalidomide for 8 weeks) continued to decline compared with the other groups. CONCLUSION: Thalidomide maintenance treatment can effectively sustain low levels of salivary IL-4, 5, 6, 10, TNF-α, and IFN-γ of recurrent aphthous stomatitis patients. IL-6 displayed a good correlation with the disease and is expected to become an index for diagnosis and follow-up. CLINICAL RELEVANCE: Low-dose long-term thalidomide maintenance treatment was supported for recurrent aphthous stomatitis. TRIAL REGISTRATION: Trial registration number of ChiCTR-IPR-16009759 at http://www.chictr.org/index.aspx .

Thalidomide and Hydroxyurea in Transfusion-Dependent Thalassemia: Efficacy, Safety Profile and Impact on Quality of Life.

Transfusion-dependent thalassemia (TDT) is a major public health concern in India, requiring regular transfusions for survival. There is also significant morbidity caused by iron overload and transfusion related infections. Novel therapies targeting fetal hemoglobin induction are the need of the hour in resource-poor institutions for patients where transplant is not feasible for various reasons. This single arm, non-randomised prospective trial evaluated the efficacy and safety of a combination of low dose thalidomide and hydroxyurea in TDT along with the impact on quality of life (QoL). It included 41 TDT patients, who failed a reasonable trial of hydroxyurea. Complete response (CR) was defined as transfusion independence and partial response (PR) denoted at least a 50% reduction in transfusion requirement. The rest were defined as non-responders (NR). The mean age of the cohort was 20.78 years (range 12-45 years). There were 13 males and 28 females. Nineteen (46.3%), 7 (17.1%), and 15 (36.6%) patients achieved CR, PR, and no response respectively. The overall response rate (CR + PR) was 63.4%. There was a significant increase in hemoglobin levels with decrement in transfusion burden and ferritin levels. There were no significant adverse reactions. No significant predictors of response were found including amongst genetic modifiers. It improved the health related QoL amongst responders. The combination of thalidomide and hydroxyurea appear safe and effective in the reduction in transfusion requirement of TDT patients. The judicious use of these drugs can improve the quality of life and pave the way for patients not eligible for a stem cell transplant.

Efficacy and safety of thalidomide for recurrent aphthous stomatitis: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is considered as the most common oral mucosal lesion affecting up to 25% of people worldwide. Thalidomide has been reported for the treatment of RAS, but the evidence has not been systematically evaluated. We first systematically reviewed the efficacy and safety of thalidomide for the treatment of RAS. METHODS: We searched The Cochrane Library, PubMed, Scopus, Web of Science, Embase, China National Knowledge Infrastructure (CNKI), Chinese BioMedical Literature Database (CBM), Wanfang Data, and VIP information from inception to December 2023. Randomized controlled trials (RCTs) comparing thalidomide with control for RAS were included in the analysis. The primary outcome were complete response and overall response, and the secondary outcome were recurrence interval (RI), ulcer number and size, healing time, visual analogue scale (VAS), immunological data, and adverse events. Meta-analysis was conducted using the Review Manager 5.4 software. RESULTS: Twenty-one trials involving 1668 patients were included in this review. The results of our meta-analysis showed that thalidomide significantly improved the complete response rate and overall response rate, prolonged the recurrence interval, accelerated the healing process, reduced the number and size of ulcers, and lowered TNF-α levels in the treatment of RAS. However, thalidomide significantly increases adverse events. RESULTS: Thalidomide has a significant benefit in the treatment of RAS. However, considering the potential side effects of thalidomide, it may be an optimal treatment option for major RAS patients or cases that do not respond to topical agents. TRIAL REGISTRATION: PROSPERO registration number: CRD42024495038.

Genetic and therapeutic for oral lichen planus and diabetes mellitus: a comprehensive study.

BACKGROUND: This study employed a bidirectional Mendelian Randomization (MR) approach to explore the causal relationships between Oral Lichen Planus (OLP), diabetes mellitus (DM), and glycemic control. It also aims to identify potential pharmacological and herbal treatments that effectively address both OLP and the metabolic dysfunctions associated with DM. METHODS: This study employs a two-way MR approach to investigate the potential causal relationships between diabetes type and glycated hemoglobin (HbA1c) levels, and the risk of OLP. We analyzed differentially expressed genes from the OLP dataset in the Genomics Expression Omnibus (GEO) database, cross-referencing these with HbA1c-related genes for enrichment analysis. Additionally, the Drug-Gene Interaction Database (DGIdb) and Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) were utilized to assess the effectiveness of specific drugs, herbs, and ingredients in treating OLP while managing blood glucose levels. RESULTS: The MR analysis revealed a significant association between Type 1 Diabetes mellitus (T1DM) and an increased risk of OLP, unlike Type 2 Diabetes mellitus (T2DM). This finding indicates a unique immunological interaction in T1DM that may predispose individuals to OLP. The drug prediction analysis focused on core targets linked to OLP and HbA1c, evaluating the therapeutic potential of retinoic acid, prednisone, and thalidomide for treating OLP and regulating blood glucose levels. Additionally, herbal medicines such as Ecliptae herbaand Amygdalus communis vas, along with herbal ingredients like quercetin, luteolin, and 17-beta-estradiol, were identified for their anti-inflammatory properties and potential to mitigate metabolic dysfunction in diabetes. CONCLUSION: The study highlighted a complex interplay between diabetes and OLP, underscoring the efficacy of integrated therapeutic strategies that target both conditions. The findings suggest that both pharmaceutical and herbal treatments can effectively manage the clinical manifestations of OLP and associated metabolic challenges. This holistic approach to treatment could significantly enhance patient outcomes by addressing the interconnected aspects of these chronic conditions.

Thalidomide as an Effective Treatment in Sideroblastic Anemia, Immunodeficiency, Periodic Fevers, and Developmental Delay (SIFD).

PURPOSE: Sideroblastic anemia, immunodeficiency, periodic fevers, and developmental delay (SIFD) is an autosomal recessive syndrome caused by biallelic loss-of-function variant of tRNA nucleotidyl transferase 1 (TRNT1). Efficacious methods to treat SIFD are lacking. We identified two novel mutations in TRNT1 and an efficacious and novel therapy for SIFD. METHODS: We retrospectively summarized the clinical records of two patients with SIFD from different families and reviewed all published cases of SIFD. RESULTS: Both patients had periodic fever, developmental delay, rash, microcytic anemia, and B cell lymphopenia with infections. Whole-exome sequencing of patient 1 identified a previously unreported homozygous mutation of TRNT1 (c.706G > A/p.Glu236Lys). He received intravenous immunoglobulin (IVIG) replacement and antibiotics, but died at 1 year of age. Gene testing in patient 2 revealed compound heterozygous mutations (c.907C > G/p.Gln303Glu and c.88A > G/p.Met30Val) in TRNT1, the former of which is a novel mutation. Periodic fever was controlled in the first month after adalimumab therapy and IVIG replacement, but recurred in the second month. Adalimumab was discontinued and replaced with thalidomide, which controlled the periodic fever and normalized inflammatory markers effectively. A retrospective analysis of reported cases revealed 69 patients with SIFD carrying 46 mutations. The male: female ratio was 1: 1, and the mean age of onset was 3.0 months. The most common clinical manifestations in patients with SIFD were microcytic anemia (82.6%), hypogammaglobulinemia/B cell lymphopenia (75.4%), periodic fever (66.7%), and developmental delay (60.0%). In addition to the typical tetralogy, SIFD features several heterogeneous symptoms involving multiple systems. Corticosteroids, immunosuppressants, and anakinra have low efficacy, whereas etanercept suppressed fever and improved anemia in reports. Bone-marrow transplantation can be used to treat severe SIFD, but carries a high risk. In total, 28.2% (20/71) of reported patients died, mainly because of multi-organ failure. Biallelic mutations located in exon1-intron5 lead to more severe phenotypes and higher mortality. Furthermore, 15.5% (11/71) patients survived to adulthood. The symptoms could be resolved spontaneously in five patients. CONCLUSIONS: Thalidomide can control the inflammation of SIFD and represents a new treatment for SIFD.

Thalidomide promotes NLRP3/caspase-1-mediated pyroptosis of macrophages in Talaromyces marneffei infection.

Macrophage-derived inflammatory cytokines are critical for host defense against Talaromyces marneffei (T. marneffei) infection among HIV/AIDS patients, and excessive inflammatory cytokines are associated with poor outcomes of AIDS-associated talaromycosis. However, the underlying mechanisms of macrophage-caused pyroptosis and cytokine storm are poorly understood. Here, in the T. marneffei-infected mice and macrophages, we show that T. marneffei induced pyroptosis in macrophages through the NLRP3/caspase-1 pathway. The immunomodulatory drug thalidomide could promote the pyroptosis of macrophages infected T. marneffei. In T. marneffei-infected mice, the splenic macrophages underwent increasing pyroptosis as talaromycosis deteriorated. Thalidomide ameliorated inflammation of mice, while amphotericin B (AmB) in combination with thalidomide did not improve overall survival compared with AmB alone. Taken together, our findings suggest that thalidomide promotes NLRP3/caspase-1-mediated pyroptosis of macrophages in T. marneffei infection.

SALL4 promotes angiogenesis in gastric cancer by regulating VEGF expression and targeting SALL4/VEGF pathway inhibits cancer progression.

BACKGROUND: Spalt-like protein 4 (SALL4) is a stemness-related transcription factor whose abnormal re-expression contributes to cancer initiation and progression. However, the role of SALL4 in cancer angiogenesis remains unknown. METHODS: Analyses of clinical specimens via TCGA datasets were performed to determine the expression level and clinical significance of SALL4 in STAD (Stomach Adenocarcinoma). SALL4 knockdown, knockout, and overexpression were achieved by siRNA, CRISPR/Cas9, and plasmid transfection. The effects of conditioned medium (CM) from SALL4 knockdown or overexpression of gastric cancer cells on endothelial cell proliferation, migration, and tube formation were investigated by CCK-8 assay, transwell migration assay, and tube formation assay. The regulation of VEGF gene expression by SALL4 was studied by qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assay, and electrophoretic mobility shift assay (EMSA). Engineered exosomes from 293T cells loaded with si-SALL4-B and thalidomide were produced to test their therapeutic effect on gastric cancer progression. RESULTS: SALL4 expression was increased in STAD and positively correlated with tumor progression and poor prognosis. SALL4-B knockdown or knockout decreased while over-expression increased the promotion of human umbilical vein endothelial cells (HUVEC) cell proliferation, migration, and tube formation by gastric cancer cell-derived CM. Further investigation revealed a widespread association of SALL4 with angiogenic gene transcription through the TCGA datasets. Additionally, SALL4-B knockdown reduced, while over-expression enhanced the expression levels of VEGF-A, B, and C genes. The results of ChIP and EMSA assays indicated that SALL4 could directly bind to the promoters of VEGF-A, B, and C genes and activate their transcription, which may be associated with increased histone H3-K79 and H3-K4 modifications in their promoter regions. Furthermore, si-SALL4-B and thalidomide-loaded exosomes could be efficiently uptaken by gastric cancer cells and significantly reduced SALL4-B and Vascular Endothelial Growth Factor (VEGF) expression levels in gastric cancer cells, thus inhibiting the pro-angiogenic role of their derived CM. CONCLUSION: These findings suggest that SALL4 plays an important role in angiogenesis by transcriptionally regulating VEGF expression. Co-delivery of the functional siRNA and anticancer drug via exosomes represents a useful approach to inhibiting cancer angiogenesis by targeting SALL4/VEGF pathway.

Novel, thalidomide-like, non-cereblon binding drug tetrafluorobornylphthalimide mitigates inflammation and brain injury.

BACKGROUND: Quelling microglial-induced excessive neuroinflammation is a potential treatment strategy across neurological disorders, including traumatic brain injury (TBI), and can be achieved by thalidomide-like drugs albeit this approved drug class is compromised by potential teratogenicity. Tetrafluorobornylphthalimide (TFBP) and tetrafluoronorbornylphthalimide (TFNBP) were generated to retain the core phthalimide structure of thalidomide immunomodulatory imide drug (IMiD) class. However, the classical glutarimide ring was replaced by a bridged ring structure. TFBP/TFNBP were hence designed to retain beneficial anti-inflammatory properties of IMiDs but, importantly, hinder cereblon binding that underlies the adverse action of thalidomide-like drugs. METHODS: TFBP/TFNBP were synthesized and evaluated for cereblon binding and anti-inflammatory actions in human and rodent cell cultures. Teratogenic potential was assessed in chicken embryos, and in vivo anti-inflammatory actions in rodents challenged with either lipopolysaccharide (LPS) or controlled cortical impact (CCI) moderate traumatic brain injury (TBI). Molecular modeling was performed to provide insight into drug/cereblon binding interactions. RESULTS: TFBP/TFNBP reduced markers of inflammation in mouse macrophage-like RAW264.7 cell cultures and in rodents challenged with LPS, lowering proinflammatory cytokines. Binding studies demonstrated minimal interaction with cereblon, with no resulting degradation of teratogenicity-associated transcription factor SALL4 or of teratogenicity in chicken embryo assays. To evaluate the biological relevance of its anti-inflammatory actions, two doses of TFBP were administered to mice at 1 and 24 h post-injury following CCI TBI. Compared to vehicle treatment, TFBP reduced TBI lesion size together with TBI-induction of an activated microglial phenotype, as evaluated by immunohistochemistry 2-weeks post-injury. Behavioral evaluations at 1- and 2-weeks post-injury demonstrated TFBP provided more rapid recovery of TBI-induced motor coordination and balance impairments, versus vehicle treated mice. CONCLUSION: TFBP and TFNBP represent a new class of thalidomide-like IMiDs that lower proinflammatory cytokine generation but lack binding to cereblon, the main teratogenicity-associated mechanism. This aspect makes TFBP and TFNBP potentially safer than classic IMiDs for clinical use. TFBP provides a strategy to mitigate excessive neuroinflammation associated with moderate severity TBI to, thereby, improve behavioral outcome measures and warrants further investigation in neurological disorders involving a neuroinflammatory component.

Genetic Basis and Therapies for Vascular Anomalies.

Vascular and lymphatic malformations represent a challenge for clinicians. The identification of inherited and somatic mutations in important signaling pathways, including the PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin), RAS (rat sarcoma)/RAF (rapidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinases), HGF (hepatocyte growth factor)/c-Met (hepatocyte growth factor receptor), and VEGF (vascular endothelial growth factor) A/VEGFR (vascular endothelial growth factor receptor) 2 cascades has led to the evaluation of tailored strategies with preexisting cancer drugs that interfere with these signaling pathways. The era of theranostics has started for the treatment of vascular anomalies. Registration: URL: https://www.clinicaltrialsregister.eu; Unique identifier: 2015-001703-32.