Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the ...tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients.
In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular ...density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.
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Successful oral delivery of liposomes requires formulations designed to withstand harsh gastrointestinal conditions, e.g., by converting to solid-state followed by loading into ...gastro-resistant delivery devices. The hypothesis was that the use of dextran-trehalose mixtures for spray drying would improve the rehydration kinetics of dried liposomes. The objectives were to determine the protective capacity of trehalose-dextran dehydration precursors and to increase the concentration of liposomes in the dry formulation volume. The study successfully demonstrated that 8.5% dextran combined with 76.5% trehalose protected CAF®04 liposomes during drying, with the liposome content maintained at 15% of the dry powder. Accordingly, the rehydration kinetics were slightly improved in formulations containing up to 8.5% dextran in the dry powder volume. Additionally, a 2.4-fold increase in lipid concentration (3 mM vs 7.245 mM) was achieved for spray dried CAF®04 liposomes. Ultimately, this study demonstrates the significance of trehalose as a primary carrier during spray drying of CAF®04 liposomes and highlights the advantage of incorporating small amounts of dextran to tune rehydration kinetics of spray-dried liposomes.
The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ...ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.
Treatment options for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) are limited, with no standard of care; prognosis is poor, with 4- to 6-month median survival. Avadomide (CC-122) ...is a cereblon-modulating agent with immunomodulatory and direct antitumor activities. This phase 1 dose-expansion study assessed safety and clinical activity of avadomide monotherapy in patients with de novo R/R DLBCL and transformed lymphoma. Additionally, a novel gene expression classifier, which identifies tumors with a high immune cell infiltration, was shown to enrich for response to avadomide in R/R DLBCL. Ninety-seven patients with R/R DLBCL, including 12 patients with transformed lymphoma, received 3 to 5 mg avadomide administered on continuous or intermittent schedules until unacceptable toxicity, disease progression, or withdrawal. Eighty-two patients (85%) experienced ≥1 grade 3/4 treatment-emergent adverse events (AEs), most commonly neutropenia (51%), infections (24%), anemia (12%), and febrile neutropenia (10%). Discontinuations because of AEs occurred in 10% of patients. Introduction of an intermittent 5/7-day schedule improved tolerability and reduced frequency and severity of neutropenia, febrile neutropenia, and infections. Among 84 patients with de novo R/R DLBCL, overall response rate (ORR) was 29%, including 11% complete response (CR). Responses were cell-of-origin independent. Classifier-positive DLBCL patients (de novo) had an ORR of 44%, median progression-free survival (mPFS) of 6 months, and 16% CR vs an ORR of 19%, mPFS of 1.5 months, and 5% CR in classifier-negative patients (P = .0096). Avadomide is being evaluated in combination with other antilymphoma agents. This trial was registered at www.clinicaltrials.gov as #NCT01421524.
•Avadomide monotherapy is well tolerated and demonstrates preliminary clinical efficacy in the treatment of R/R DLBCL patients.•Avadomide monotherapy resulted in mPFS of 6 months in classifier-positive vs 1.5 months in classifier-negative R/R DLBCL patients.
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Large volumes of pesticides are applied every year to support agricultural production. The intensive use of pesticides affects soil quality and health, but soil surveys on pesticide residues are ...scarce, especially for northern Europe. We investigated the occurrence of 198 pesticide residues, including both banned and currently used substances in 148 field sites in Finland. Results highlight that pesticide residues are common in the agricultural soils of Finland. A least one residue was found in 82% of the soils, and of those 32% contained five or more residues. Maximum total residue concentration among the conventionally farmed soils was 3043 μg/kg, of which AMPA and glyphosate contributed the most. Pesticide residues were also found from organically farmed soils, although at 75–90% lower concentrations than in the conventionally farmed fields. Thus, despite the application rates of pesticides in Finland being generally much lower than in most parts of central and southern Europe, the total residue concentrations in the soils occurred at similar or at higher levels. We also established that AMPA and glyphosate residues in soil are significantly higher in fields with cereal dominated rotations than in grass dominated or cereal–grass rotations. However, risk analyses for individual substances indicated low ecological risk for most of the fields. Furthermore, the total ecological risk associated with the mixtures of residues was mostly low except for 21% of cereal dominated fields with medium risk. The results showed that the presence of mixtures of pesticide residues in soils is a rule rather than an exception also in boreal soils. In highly chemicalized modern agriculture, the follow-up of the residues of currently used pesticides in national and international soil monitoring programs is imperative to maintain soil quality and support sustainable environment policies.
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•Pesticide residues were frequently found in Finnish agricultural soils.•Glyphosate and AMPA were the most prevalent residues observed.•The residue contents were high in relation to the pesticides applied in Finland.•The risk assessment factors showed low ecological risk for most fields.
Cereblon (CRBN), a substrate receptor of the E3 ubiquitin ligase complex CRL4CRBN, is the target of the small molecules lenalidomide and avadomide. Upon binding of the drugs, Aiolos and Ikaros are ...recruited to the E3 ligase, ubiquitylated, and subsequently degraded. In diffuse large B-cell lymphoma (DLBCL) cells, Aiolos and Ikaros are direct transcriptional repressors of interferon-stimulated genes (ISG) and degradation of these substrates results in increased ISG protein levels resulting in decreased proliferation and apoptosis. Herein, we aimed to uncover the mechanism(s) Aiolos and Ikaros use to repress ISG transcription and provide a mechanistic rationale for a combination strategy to enhance cell autonomous activities of CRBN modulators (CELMoD).
We conducted paired RNA sequencing with histone modification and Aiolos/Ikaros chromatin immunoprecipitation sequencing to identify genes regulated by these transcription factors and to elucidate correlations to drug sensitivity. We confirmed Aiolos/Ikaros mediated transcriptional complex formation in DLBCL patient samples including those treated with avadomide.
In DLBCL, the repression of ISG transcription is accomplished in part through recruitment of large transcriptional complexes such as the nucleosome remodeling and deacetylase, which modify the chromatin landscape of these promoters. A rational combination approach of avadomide with a specific histone deacetylase inhibitor leads to a significant increase in ISG transcription compared with either single agent, and synergistic antiproliferative activity in DLBCL cell lines.
Our results provide a novel role for lineage factors Aiolos and Ikaros in DLBCL as well as further insight into the mechanism(s) of Aiolos and Ikaros-mediated transcriptional repression and unique therapeutic combination strategies.
Background
Farm‐derived dust samples have been screened for bacteria with potential allergo‐protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with ...regard to its protective capacities in murine models of allergic airway inflammation.
Methods
We employed two protocols of acute airway inflammation in mice administering either ovalbumin (OVA) or house dust mite extract (HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to S. sciuri W620 were carried out using human primary monocytic dendritic cells (moDC) and co‐culture with autologous T cells.
Results
The allergo‐protective properties of S. sciuri W620 were proven in a TH2‐driven OVA model as well as in a mixed TH1/TH2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell TH1/TH2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL‐12 production in moDC due to missing transcription of the IL‐12p35 mRNA. However, such DC selectively supported TH1 cytokine release by co‐cultured T cells.
Conclusion and clinical relevance
Our proof‐of‐concept experiments verify the screening system of farm‐derived dust samples as suitable to elucidate new candidates for allergo‐protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication.
Summary
Chemotherapy plus rituximab has been the mainstay of treatment for follicular lymphoma (FL) for two decades but is associated with immunosuppression and relapse. In phase 2 studies, ...lenalidomide combined with rituximab (R2) has shown clinical synergy in front‐line and relapsed/refractory FL. Here, we show that lenalidomide reactivated dysfunctional T and Natural Killer (NK) cells ex vivo from FL patients by enhancing proliferative capacity and T‐helper cell type 1 (Th1) cytokine release. In combination with rituximab, lenalidomide improved antibody‐dependent cellular cytotoxicity in sensitive and chemo‐resistant FL cells, via a cereblon‐dependent mechanism. While single‐agent lenalidomide and rituximab increased formation of lytic NK cell immunological synapses with primary FL tumour cells, the combination was superior and correlated with enhanced cytotoxicity. Immunophenotyping of FL patient samples from a phase 3 trial revealed that R2 treatment increased circulating T‐ and NK‐cell counts, while R‐chemotherapy was associated with reduced cell numbers. Finally, using an in vitro model of myeloid differentiation, we demonstrated that lenalidomide caused a reversible arrest in neutrophil maturation that was distinct from a cytotoxic chemotherapeutic agent, which may help explain the lower rates of neutropenia observed with R2 versus R‐chemotherapy. Taken together, we believe these data support a paradigm shift in the treatment of FL – moving from combination immunochemotherapy to chemotherapy‐free immunotherapy.
Summary
Lenalidomide is an immunomodulatory agent that has demonstrated clinical benefit for patients with relapsed or refractory mantle cell lymphoma (MCL); however, despite this observed clinical ...activity, the mechanism of action (MOA) of lenalidomide has not been characterized in this setting. We investigated the MOA of lenalidomide in clinical samples from patients enrolled in the CC‐5013‐MCL‐002 trial (NCT00875667) comparing single‐agent lenalidomide versus investigator's choice single‐agent therapy and validated our findings in pre‐clinical models of MCL. Our results revealed a significant increase in natural killer (NK) cells relative to total lymphocytes in lenalidomide responders compared to non‐responders that was associated with a trend towards prolonged progression‐free survival and overall survival. Clinical response to lenalidomide was independent of baseline tumour microenvironment expression of its molecular target, cereblon, as well as genetic mutations reported to impact clinical response to the Bruton tyrosine kinase inhibitor ibrutinib. Preclinical experiments revealed lenalidomide enhanced NK cell‐mediated cytotoxicity against MCL cells via increased lytic immunological synapse formation and secretion of granzyme B. In contrast, lenalidomide exhibited minimal direct cytotoxic effects against MCL cells. Taken together, these data provide the first insight into the clinical activity of lenalidomide against MCL, revealing a predominately immune‐mediated MOA.
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